Microbial Stress Adaptation and Food Safety

Microbial
Stress
Adaptation
and
Food Safety
Edited by
Ahmed E.Yousef
Vijay K. Juneja
CRC PR E S S
Boca Raton London New York Washington, D.C.
© 2003 by CRC Press LLC

Library of Congress Cataloging-in-Publication Data
Microbial stress adaptation and food safety / editors, Ahmed E. Yousef and Vijay K. Juneja.
p. cm.
Includes bibliographical references and index.
ISBN 1-56676-912-4
1. Food—Microbiology. 2. Adaptation (Biology 3. Stress (Physiology) I. Yousef,
Ahmed Elmeleigy. II. Juneja, Vijay K., 1956-
QR115 .M4585 2002

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Preface
Microorganisms, like all living beings, react when exposed to stress. When humans
are subjected to limited stress, their reaction varies from fatigue to endurance.
Interestingly, microorganisms behave similarly. When the stress cannot be tolerated,
both humans and microorganisms may suffer injury. Given time and rest, both
humans and microorganisms may recover from this injury. Severe injuries, however,
may lead to death. Mild stresses, on the other hand, may be beneficial to both
microorganisms and humans. These stresses activate the body’s natural defenses and
test its ability to protect itself against incoming danger. When living beings respond
appropriately to stress, they emerge from this experience better “adapted” and
prepared for future stressful situations. It appears that the saying, “what doesn’t kill
me only makes me stronger,” applies equally well to humans and microgorganisms.
The words “stress,” “adaptation,” “injury,” “recovery,” and “death” describe human
experiences and these expressions have been ported to the world of microbiology.
Keeping the analogies between human and microbial experience in mind, the reader
may quickly become familiar with the advanced terminologies used in this book.
This book presents essential and advanced knowledge about microbial adaptation
to stress, and relevance of this phenomenon to food safety. The book should serve
readers who have basic understanding of microbiology and prior knowledge of food
processing and preservation. The first chapter introduces the concept of stress adap-
tation in microorganisms. This contribution defines the terms, and briefly describes
the stress adaptive response phenomenon and its implications to the safety of food
processed by novel unconventional technologies. Responses of pathogens to physical
and chemical stresses encountered during food processing are addressed in
Chapters 2 and 3, respectively. Physical preservation factors discussed include heat,
pressure, electric pulses, dehydration, freezing and irradiation. Food preservatives
such as added organic acids and naturally occurring antimicrobials are also stresses
of interest. Adaptation of food microbiota to stress as a survival strategy is the topic
of Chapter 4. This covers important categories of foodborne microorganisms and
how these develop different schemes to combat deleterious factors in food and during
food processing.
The food-processing environment occasionally harbors pathogens that eventually
gain access to food. These environments may be hospitable enough to support not
only growth of these pathogens, but also building communities called biofilms.
Pathogens in food processing environments face a broad spectrum of stresses that
may increase their tenacity and resistance to processing. This topic is addressed in
detail in Chapter 5. The beneficial aspects of stress adaptation are the subject of
Chapter 6. Survival of lactic acid bacteria in food and the human intestine may have
a positive effect on food safety and human health. The authors of this chapter describe

in detail how these beneficial bacteria adapt to stresses such as heat, cold, acid, salt,
and oxidation. The physiological and molecular basis of stress adaptation response
in this group of bacteria is also presented.
Pathogens face stresses not only in food and processing environments, but also
during the infection process. Resistance or adaptation to these stresses is an essential
element of pathogens’ ability to cause diseases. Chapter 7 attends to this matter in
depth. There are many physiological and molecular mechanisms that microorganisms
use to sense the stress and respond to it. How foodborne microorganisms implement
these mechanisms to adapt to stress is discussed in Chapter 8. Finally, strategies to
overcome stress adaptation in foodborne pathogens are proposed in Chapter 9. The
authors suggest practical control measures and emphasize the need for future
research to counteract the stress adaptation phenomenon.
We hope this book raises awareness about the microbial stress adaptation phenom-
enon and its consequences for food safety and human health and welfare. We are also
hopeful that the topics covered in the book stimulate interest in research, leading to a
better characterization of stress adaptive responses in foodborne microorganisms.
Ahmed E. Yousef
Vijay K. Juneja

Acknowledgment
We would like to thank all the contributing authors for their fine chapters and the
staff members of CRC Press for their help and guidance. We also appreciate the
patience of the members of our families who endured with us the stress of compiling
this work. We are grateful to our co-workers who contributed significantly to our
knowledge on this subject.

Editors
Ahmed E. Yousef, a professor of food microbiology at the Ohio State University
(OSU), earned his Ph. D. degree from the University of Wisconsin-Madison in 1984.
He teaches food microbiology at the Department of Food Science and Technology
and the Department of Microbiology, OSU. His book, Food Microbiology, A Labo-
ratory Manual, represents the laboratory component of the course he teaches. Since
he joined OSU in 1991, Dr. Yousef has investigated methods to control foodborne
pathogens and to determine causes of resistance of these pathogens to preservation
factors. He is actively researching biopreservation, high pressure processing and
pulsed electric field technologies. Pathogens targeted include Listeria monocytogenes,
Salmonella spp., Escherichia coli O157:H7, and Clostridium botulinum. Adaptation
of these pathogens to environmental and processing stresses is an ongoing investi-
gation. He has published more than 70 research papers, review articles, books and
book chapters. Dr. Yousef served on the editorial boards of the Journal of Food
Protection and Journal of Food Science. He won several awards in recognition of
his accomplishments in teaching and research.
Vijay K. Juneja is supervisory microbiologist and lead scientist in the Microbial

Food Safety Research Unit at the Eastern Regional Research Center (ERRC) of the
Agricultural Research Service (ARS) branch of the United States Department of
Agriculture (USDA) in Wyndmoor, Pennsylvania. Dr. Juneja received his Ph.D.
degree in food technology and science from the University of Tennessee in 1991,
then was appointed as a microbiologist at the ERRC-USDA. Dr. Juneja has developed
a nationally and internationally recognized research program on foodborne patho-
gens, with emphasis on microbiological safety of minimally processed foods, and
predictive microbiology. He is a co-editor of the book Control of Foodborne Micro-
organisms and serves on the editorial boards of the Journal of Food Protection and
Journal of Food Science. Dr. Juneja is a recipient of several awards, including the
Agricultural Research Service Senior Research Scientist; North Atlantic Area Sci-
entist of the Year, 2000; Gold Medalist “Technical Accomplishment,” Federal Exec-
utive Board (FEB) 1998, 2000; ARS-FSIS Cooperative Research Award, 1998;
USDA-ARS Certificate of Merit for Outstanding Performance, 2002, among others.
His research interests include intervention strategies for control of foodborne patho-
gens and predictive modeling. Dr. Juneja’s research program has been highly pro-
ductive, generating more than 180 research articles, book chapters, and abstracts,
primarily in the area of food safety and predictive microbiology.

Contributors
Polly D. Courtney Vijay K. Juneja
Department of Food Science & U.S. Department of Agriculture
Technology Agricultural Research Service
The Ohio State University Eastern Regional Research Center
Columbus, Ohio Wyndmoor, Pennsylvania
P. Michael Davidson
Todd R. Klaenhammer
Department of Food Science and
Department of Food Science
Technology
Southeast Dairy Foods Research Center
University of Tennessee
North Carolina State University
Knoxville, Tennessee
Raleigh, North Carolina
Cormac G. M. Gahan
Department of Microbiology and John B. Luchansky
National Food Biotechnology Centre USDA Eastern Regional Research
University College Cork Center
Cork, Ireland Microbial Food Safety Research Unit
Wyndmoor, Pennsylvania
Hany S. Girgis
Department of Food Science
John S. Novak
Southeast Dairy Foods Research Center
U.S. Department of Agriculture
North Carolina State University
Agricultural Research Service
Raleigh, North Carolina
Eastern Regional Research Center
Mark A. Harrison Wyndmoor, Pennsylvania
Department of Food Science and
Technology Sadhana Ravishankar
University of Georgia National Center for Food Safety and
Athens, Georgia Technology
Summit-Argo, Illinois
Colin Hill
Department of Microbiology and
Robin J. Rowbury
National Food Biotechnology Centre
Biology Department
University College Cork
University College London
Cork, Ireland
London, United Kingdom
Eric A. Johnson
Food Research Institute John Samelis
Department of Food Microbiology National Agricultural Research
and Toxicology Foundation
University of Wisconsin Dairy Research Institute
Madison, Wisconsin Ioánnina, Greece

James Smith John N. Sofos
USDA Eastern Regional Research Center Department of Animal Sciences,
Microbial Food Safety Research Unit Colorado State University
Wyndmoor, Pennsylvania Fort Collins, Colorado
Ahmed E. Yousef
Department of Food Science & Technology
The Ohio State University
Columbus, Ohio

Table of Contents
Chapter 1
Basics of Stress Adaptation and Implications in New-Generation Foods
Ahmed E. Yousef and Polly D. Courtney
Chapter 2
Adaptation of Foodborne Pathogens to Stress from Exposure to Physical
Intervention Strategies
Vijay K. Juneja and John S. Novak
Chapter 3
Microbial Adaptation to Stresses by Food Preservatives
P. Michael Davidson and Mark A. Harrison
Chapter 4
Microbial Adaptation and Survival in Foods
Eric A. Johnson
Chapter 5
Adaptation or Resistance Responses of Microorganisms to Stresses in the
Food-Processing Environment
Sadhana Ravishankar and Vijay K. Juneja
Chapter 6
Stress Adaptations of Lactic Acid Bacteria
Hany S. Girgis, James Smith, John B. Luchansky, and Todd R. Klaenhammer
Chapter 7
Relationship between Stress Adaptation and Virulence in Foodborne
Pathogenic Bacteria
Cormac G. M. Gahan and Colin Hill
Chapter 8
Physiology and Molecular Basis of Stress Adaptation, with Particular
Reference to the Subversion of Stress Adaptation and to the Involvement
of Extracellular Components in Adaptation
Robin J. Rowbury

Chapter 9
Strategies to Control Stress-Adapted Pathogens
John Samelis and John N. Sofos

Basics of Stress Adaptation
1 and Implications in
New-Generation Foods
Ahmed E. Yousef and Polly D. Courtney
CONTENTS
Introduction
Definitions
Stress
Stress Response
Adaptation
Tolerance
Injury
Stress, Adaptation and Food Safety
Emerging Processing Technologies and Stress Adaptation
High Pressure Processing
Process
Mechanism
Potential Stress Adaptation
Radiation
Process
Mechanism
Pulsed Electric Field
Process
Mechanism
Mechanism of Stress Adaptive Response
Stress Sensing
Regulation of Stress-Related Protein Synthesis
General Stress Response
Specific Stress Responses
Heat
Cold

Acid
Osmotic Stress
Oxidative Stress
Monitoring Stress Response
Induction of Stress Adaptive Response: Practical Considerations
Heat
Acid
Acid Shock during Exponential Phase
Gradual Acid Stress
Detecting and Quantifying Stress Response
Detection of Stress Response Gene
mRNA Analysis
Detection of Stress Proteins
Biosensors
Measuring Increased Tolerance
Perspectives and Areas for Future Work
References
INTRODUCTION
For many decades, researchers have noticed that microorganisms that endure a
stressful environment subsequently survive conditions presumed lethal. Fay (1934),
for example, noticed that exposing bacteria to osmotic stress increases tolerance to
heat. Increase of an organism’s resistance to deleterious factors following exposure
to mild stress is commonly described as stress adaptation. Stress adaptation in
foodborne microorganisms was overlooked in the past, but now the significance of
this phenomenon is becoming recognized.
In 1987, Mackey and Derrick showed that heat shocking Salmonella enterica
serovar Thompson increased its thermal resistance in food. Enhanced thermal tol-
erance was also observed by Farber and Brown (1990) when they heat shocked
Listeria monocytogenes in sausage batter at 48°C for 120 min before the inoculated
mix was heated at 64°C. Leyer and Johnson (1992) inoculated acid-adapted (pH 5.8)
and non-adapted Salmonella typhimurium into fermenting milk. The researchers
noticed that acid adaptation of the pathogen enhanced its survival during milk
fermentation. Acid adaptation also enhanced survival in cheeses that were inoculated
with the pathogen. Subsequent studies provided additional evidence of the stress
adaptation phenomenon and its consequences during food processing. This chapter
covers the basic aspects of stress adaptation and the relevance of this phenomenon
to food safety, particularly products processed by emerging technologies.
DEFINITIONS
Some terms describing stress adaptation are used loosely in scientific literature, so
we will describe the way terms are used throughout this chapter. The interrelations
among some of these terms are depicted in Figure 1.1.

Stress-adapted
ry
Stress Adaptive Response

o ve
R ec
Relative Stress Tolerance
Healthy
(Steady state)
Mi
ld
St
res
Re s
co
ve
ry
Stressed Mod
St era
res te
Re s
co
ve
ry
Se
Injured Str vere
es
s
Dead
Physiological State
FIGURE 1.1 Proposed interrelations among physiological states of microbial cell subjected
to different stresses.
Stress
Stress has different meanings depending on the context of usage. In physics, for
example, stress is the force applied per unit area. When used in the field of biology,
stress refers to the imposition of detrimental nutritional conditions, toxic chemicals
and suboptimal physical conditions (Neidhardt and VanBogelen, 2000). Stress, as
used in this chapter, refers to any deleterious factor or condition that adversely affects
microbial growth or survival. According to this practical definition, many food
processing treatments are considered stresses.
Stresses encountered by microorganisms vary in magnitude and outcome. We
use the word “mild” to describe sublethal stress levels that do not result in viability
loss, but reduce or arrest growth rate. “Moderate” stress not only arrests microbial
growth but also causes some loss in cell viability. “Extreme” or “severe” describes
a stress level that is normally lethal to the cells, resulting in death of the majority
of the population. Stresses that food microbiota encounter include uncontrollable
pre-harvest environmental factors (e.g., radiation and dry air) and the deliberate post-
harvest application of preservation factors. Stresses to these microorganisms during
food production and processing include:
1. Physical treatments such as heat, pressure, electric pulses, ultrasonic

waves, light/radiation, and osmotic shock
2. Addition of chemicals such as acids, salts, and oxidants
3. Biological stresses, e.g., competition, microbial metabolites and antagonism

Foodborne microorganisms may experience stress gradually or abruptly, the
latter being referred to as shock. For example, a bacterium may experience a drastic
change in pH, or acid shock, when moving from the food medium into the stomach.
On the other hand, microorganisms experience a gradual pH decrease during food
fermentations.
Stress Response
Once microorganisms sense a stress, the cells respond in various ways. Bacteria sense
stresses that change membrane fluidity (e.g., cold shock), alter cell protein structure or
disrupt ribosomes (e.g., heat), or affect nucleic acids (e.g., γ radiation). At the molecular
level, stress response includes transcription leading to the synthesis of regulatory pro-
teins. The resulting regulation may lead to the synthesis of other proteins that cope
with the imposed stress. Microbial response to stress may produce these outcomes:
1. Production of proteins that repair damage, maintain the cell, or eliminate

the stress agent
2. Transient increase in resistance or tolerance to deleterious factors
3. Cell transformation to a dormant state, i.e., spore formation or passage to
the viable-but-not-culturable state
4. Evasion of host organism defenses
5. Adaptive mutations
Adaptation
When microorganisms are stressed, an adaptive or protective response may follow.

Response to stress, in this case, increases the organism’s tolerance to the same or
to a different type of stress. This phenomenon is occasionally described as adaptive
response, induced tolerance, habituation, acclimatization or stress hardening. Stress
adaptation and stress adaptive response will be used interchangeably in this chapter.
Tolerance
Tolerance to a deleterious factor (e.g., low pH) refers to a microorganism’s ability

to survive a stress. Each microorganism has an inherent tolerance level to a particular
stress, but a transient or adaptive tolerance may also be induced. For example, lactic
acid bacteria are inherently more acid tolerant than many other bacteria, yet they
can become even more acid tolerant after acid adaptation. Resistance and tolerance
have similar meanings; these terms will be used interchangeably in this chapter.
Injury
Damage to cellular components by stresses may impair the ability of microorganisms

to multiply or may sensitize the cells to mildly deleterious factors. These changes
are commonly described as injury. Injury is most noticeable when stress-exposed
cells become sensitive to selective agents that healthy cells readily survive. The
relationship between cell injury and stress adaptation has not been well characterized,

but injury may result from a cell’s inability to respond to stress or a delayed or
inadequate adaptive response. Injured cells may recover or die. Leistner (2000)
indicated that simultaneous exposure of bacteria to different stress factors requires
increased energy consumption and leads bacteria to cellular death through metabolic
exhaustion and disturbed homeostasis.
STRESS, ADAPTATION AND FOOD SAFETY

Bacteria are exposed to stress in all links of the food chain, from production to digestion
(Table 1.1). In the food production environment, sunlight, which contains ultraviolet
radiation, may stress, injure or kill bacteria. Heat generated by sunlight may lead to
microbial stress. Acidity of fermented vegetation, salinity of seawater, and dryness of
arid climates are examples of other stresses that bacteria may encounter in the envi-
ronment. Additionally, bacteria live in an environment that carries their own excretions
(metabolites). Some of these metabolites constitute unique stresses to bacteria. Lack
of essential nutrients for growth or survival (i.e., starvation) stresses, injures or kills
bacteria, depending on the severity and duration of starvation. In summary, bacteria
in the environment are frequently exposed to physical, chemical and nutritional stresses
of varying magnitudes. Bacteria in food also are exposed to stresses including heat,
acid, freezing, osmotic shocks, desiccation, oxidation, and starvation. Further infor-
mation about environmental and processing stresses may be found in Chapters 4 and 5.
Stress factors induce cellular responses that vary with the type, magnitude, and
method of stress application. Although there are multiple outcomes, microorganisms’
adaptive response to stress is of paramount significance in food safety (Figure 1.2).
Stress-adapted bacteria are capable of resisting similar (homologous) or different
(heterologous) stresses and, in many cases, survive normally injurious or lethal
conditions. For example, when bacteria are subjected to a heat shock, cells respond
by becoming resistant to lethal heat treatments (Bunning et al., 1990). When Listeria
monocytogenes was stressed by mild heat (45°C for 60 min), it became significantly
more resistant to lethal doses of ethanol, hydrogen peroxide, and sodium chloride
(Lou and Yousef, 1997). There are indications that adaptation of bacterial pathogens
to stress may increase their ability to cause diseases. Data about increased virulence
in stress-adapted cells are still limited, but if this relationship is confirmed in food
applications, these results will have far reaching implications (see Chapter 7).
During traditional food processing (e.g., pasteurization and retorting), bacterial
cells are more likely to be killed than injured or stressed. However, there are
processing conditions that constitute a mild stress and thus induce adaptive response
in bacteria. Adaptation of Salmonella to acid stress, for example, increased the
survival of this pathogen in cheese (Leyer and Johnson, 1992). Farber and Brown
(1990) noticed that when L. monocytogenes was heat-shocked at 48°C for 120 min,
the adapted cells exhibited increased tolerance to heat in sausage batter. Acid adap-
tation enhanced the survival of L. monocytogenes in acid foods such as yogurt,
orange juice and salad dressing (Gahan et al., 1996). One may similarly hypothesize
that certain processing conditions cause stress adaptation, which affects the safety
of numerous foods. For example, acidity developed during sausage fermentation and
the presence of salt in the formulation of this product may induce an acid adaptive

TABLE 1.1
Deleterious Factors Likely to Provoke Stress Response in Foodborne Microorganisms at Various Links of the Food Chain,
Including Production, Processing, Storage, Distribution, Consumption, and Digestion
Stage in the Food Chain Storage &
Factor Pre-Harvest (Environmental) Processing Distribution Consumption Site In Host
Heat shock Weather-related Mild processing Temperature control Cooking Fever

Composting failure Reheating
Cold shock Weather-related Refrigeration Refrigeration Refrigeration fluctuation —
Acidity Acid rain Food fermentations Spoilage by acid Acidic additives during food Stomach
Irrigation water Additives (e.g., producers preparation (e.g., vinegar Macrophages
Fermentation (e.g., silage production) acidulents, organic and lemon juice)
Spoilage and decay (vegetation or product) acids, acidic salts)
Muscle stress
Plant saps-fruit juices
Osmotic shock Soil salinity Additives (e.g., salt) Additives in food preparation
Irrigation water Concentration
Dehydration
Starvation Non-nutritious environment — — — Iron starvation in

macrophages
Oxidation Air exposure of anaerobic microbiota Exposure to air Exposure to air Exposure to air Macrophages
Oxidative sanitizers Oxidative sanitizers
Metal ions Irrigation water Equipment — Equipment —

Non-Stressed Process survivors
(relevant to preservation factors) *
Raw food Pre-processing Mildly-processed Processing Fully-processed

Food Food
Stressed Response Stress-adapted
FIGURE 1.2 Potential hazards associated with stress adaptation of pathogens during food
processing. *These cells may have been exposed to various environmental stresses during
food production, but not to stresses specific to food preservation, e.g., high pressure.
response and osmotic shock response in pathogenic bacteria. Pathogens, adapted to

acid and osmotic stress during sausage fermentation, may resist the heating and
smoking steps or persist during storage of the product. Similarly, bacteria in milk
that is heated at sub-pasteurization temperatures (e.g., for making certain varieties
of cheese) may only suffer a mild heat shock (i.e., heat stress). These bacteria may
become resistant to subsequent severe processing (e.g., cooking the product into
processed cheese). Minimally processed foods are produced using mild treatments
that may elicit stress adaptive responses in microbial contaminants including patho-
gens. Increasing use of alternative processing technologies (also referred to as “non-
thermal,” “novel,” or “emerging” technologies) is arousing curiosity about the poten-
tial stress adaptation of foodborne pathogens.
There are, however, some positive aspects to the adaptation of foodborne bacteria
to stress. Probiotic bacteria (e.g., Bifidobacterium spp. and Lactobacillus acidophilus)
are desirable supplements to some fermented products like yogurt. Viability of these
bacteria, however, may decline rapidly during storage of such an acid food. Pre-
adaptation to acid stress enhances survivability of probiotic bacteria in yogurt-like
products (Shah, 2000). Fermentation starter cultures must also endure the stress of
preservation by freezing or freeze-drying prior to use in food processing. Kim and
Dunn (1997) demonstrated that cold shocking various starter cultures prior to freez-
ing dramatically improved their cryotolerance compared to bacteria that were not
cold shocked. Readers are advised to review Chapter 6 for details about the impli-
cations of stress adaptation in beneficial bacteria.
In conclusion, microorganisms encounter a variety of sublethal stresses in food
and environment. These stresses may induce stress adaptive responses that make
foodborne pathogens resistant to subsequent lethal preservation factors (see
Figure 1.2). Adaptation of pathogens to these stresses, therefore, constitutes potential
health hazards to consumers.
EMERGING PROCESSING TECHNOLOGIES

AND STRESS ADAPTATION
Food processors currently rely on a variety of methods for preserving food. Con-
ventional methods include heating, drying, freezing, and the addition of approved

preservatives. Heat is the most commonly used preservation method and heat-treated
foods generally have a good safety record. When properly applied, heat can eliminate
bacteria, fungi, viruses, parasites, and enzymes, which are the biological agents that
spoil or compromise the safety of food. The applied dosage of conventional preser-
vation factors can be varied to accomplish almost any degree of microbial inactiva-
tion, ranging from limited reductions of microbial load to complete sterilization.
When heat is applied to milk, for example, at 71.6°C for 15 sec, a 5 to 6 log kill of
non-spore-forming bacterial pathogens occurs, and the resulting product is consid-
ered pasteurized. Heating milk at 145°C for a few seconds produces a commercially
sterile ultra high temperature-treated product, and the treatment is presumed to be
a 12-D process when targeting Clostridium botulinum spores.
Conventional technologies produce safe food but the product has lesser nutri-
tional and sensory quality and consumer acceptability compared with its fresh
counterpart (e.g., canned vegetables and fruits compared with fresh). Interest in
alternative food processing technologies has been driven by consumer demand for
food with fresh-like taste, crisp texture, high nutrient content, and natural color.
Alternative technologies have been advanced by both industry and academia in an
attempt to meet the challenge of producing safe processed food of a high quality.
These emerging technologies include high pressure processing (HPP), pulsed electric
field (PEF), pulsed light, and irradiation. The safety and microbiological quality of
food processed using these technologies, however, needs to be affirmed. Alternative
technologies cannot achieve the broad microbial lethalities that are currently attain-
able by conventional preservation factors, particularly heat. Current HPP and PEF
technologies can only accomplish the equivalent of pasteurization when applied at
their maximum lethal doses. The achievement of commercial sterility by these
alternative technologies is not currently feasible.
When food is treated with alternative processing technologies, the microbial
load may become stressed, injured, or killed. Response of foodborne pathogens to
the stress caused by these technologies is a concern and the adaptation of cells to
such stress may constitute a microbial hazard. Alternative processing technologies
introduce new challenges, and thus warrant the implementation of new safety strat-
egies. The following is an overview of selected alternative processing technologies,
structural and functional alterations in microbial cells by these technologies, and
adaptive responses to these stresses. For additional details about these technologies,
readers may seek relevant review articles, e.g., Barbosa-Canovas et al., 2000; Lado
and Yousef, 2002; Farkas and Hoover, 2000.
HIGH PRESSURE PROCESSING

Process
Processing food with high pressure involves applying hydrostatic pressure in the
range of 100 to 1000 MPa (equivalent to 14,500 to 145,000 psi). Equipment required
to apply this intense treatment includes a thick-walled pressure vessel and a pressure-
generating device (Figure 1.3). Food, in flexible packages, is loaded into the vessel
and the top is closed. The pressurizing medium, which is usually a water-based fluid,

Vessel
closure
Pressurization
fluid
Food in a
Flexible package
Vessel
Valve
Pressure
FIGURE 1.3 High pressure processing equipment: basic components.
is pumped into the vessel from the bottom. Since the applied pressure is uniform
throughout the pressure medium and the food, the product retains its original shape,
with minimal or no distortion. Once the desired pressure is attained, fluid pumping
is stopped and the product is kept “at pressure” for a predetermined treatment period.
Pressure is released after the treatment and the processed product is removed from
the vessel. A pressure treatment cycle is normally completed in 5 to 20 min, depending
on the pressure applied and equipment design. In lieu of this batch mode, semi-
continuous or continuous HPP systems are now being developed.
Mechanism
Timson and Short (1965) suggested that ultrahigh pressure destroys biological sys-
tems because of protein precipitation. According to these authors, high pressure
increases the solvation of ions and enhances the formation of ionic bonds. This
decreases the number of the hydrophilic groups on the protein molecules and thus
decreases the solubility of these proteins. On the contrary, Suzuki and Taniguchi
(1972) suggested that high pressure damages biological systems because the treat-
ment enhances protein–protein hydrophobic interactions. According to LeChatelier’s
principle, pressure enhances reactions which lead to a decrease in volume and
inhibits reactions which result in an increase in volume. Hydrophobic interactions
among protein molecules under high pressure cause a decrease in volume, thus these
reactions are favored during HPP. More recently, membrane damage was proposed
as a mechanism of cell death by high pressure. Benito et al. (1999) found that the
uptake of fluorescent stains (ethidium bromide and propidium iodide) was greater

in pressure-sensitive than in pressure-resistant strains of Escherichia coli O157.
Since these stains enter bacterial cells having damaged membranes, it follows that
membrane damage occurs during the high-pressure treatment. Change in ribosomal
conformation, as detected by differential scanning calorimetry, was proposed as a
mechanism of microbial inactivation by HPP (Niven et al., 1999).
Mild pressure treatments may induce a stress response. When Welch et al. (1993)
exposed exponentially growing E. coli to a pressure of 55 MPa, synthesis of several
proteins was induced, particularly a 15.6 kDa protein. Most of the induction occurred
after 60 to 90 min of pressure treatment. Many of these proteins were also induced
by heat shock or cold shock. Wemekamp-Kamphuis et al. (2002) used two-dimen-
sional gel electrophoresis, combined with western blotting, to demonstrate that cold
shock or HPP elevated the levels of cold shock proteins (CSPs) in L. monocytogenes.
When cold-shocked L. monocytogenes was pressure treated, the level of survival
was 100-fold higher than that of cells grown exponentially at 37°C before the
pressure treatment. The authors concluded that cold shock protects L. monocytogenes
against HPP. Lucore et al. (2002) provided evidence of pressure adaptive response
in E. coli O157:H7. When E. coli O157:H7 was subjected to sublethal pressure stress
at 100 MPa and 37°C for 30 min, cells developed resistance to lethal pressures (at
300 MPa) and heat (57°C). Heat shocking the pathogen at 46°C for 15 min protected
the cells against lethal heat and pressure treatments.
RADIATION
The spectrum of electromagnetic radiation includes regions that are useful in food
applications. Although some of these technologies were considered seriously by
mid-20th century, interest in use as alternative processing methods increased only
recently. Emerging radiation technologies in food preservation include gamma (γ),
x-ray, ultraviolet (UV), microwave and radio frequency. Pulsed light and pulsed UV
energy are beneficial technologies with great prospects in food applications. In this
chapter, γ and UV radiation technologies only will be addressed.
Process
Treatment with γ radiation involves placing the food in proximity of a radiation

source in a specially designed treatment chamber. The sources commonly used are
60Co and 137Cs. Ultraviolet radiation is generated from lamps that are placed in close
proximity to the treated food. Short-wave UV, particularly of wave lengths 250 to
260 nm, has strong microbicidal properties. These can be generated from mercury
lamps.
Mechanism
The short wavelengths of UV light inactivate microorganisms through alteration of

DNA structure (Bintsis et al., 2000). Interaction of UV with DNA results in dimer

formation, mainly cyclobutane-pyrimidine dimers, and DNA-protein cross linking.
These alterations interfere with the cell’s ability to multiply, and thus lead to micro-
bial demise. Pulsed light includes wavelengths that range from the ultraviolet (UV)
to the infrared regions (Clark, 1995). It is therefore plausible to assume that the UV
component of pulsed light contributes significantly to microbial lethality. Contrary
to this hypothesis, some researchers believe that the thermal effect of pulsed light
is the cause of microbial lethality (Corry et al., 1995).
Gamma radiation generates hydroxyl radicals, which interact with cellular com-
ponents and result in microbial inactivation. These radicals react with DNA and
cause base modifications, single-strand or double-strand breaks, and DNA protein
cross linkages (Von Sonntag, 1987). Kim and Thayer (1996) found that presence of
air increases the lethality of γ radiation.
Sinha and Hader (2002) reviewed strategies to repair damage caused by UV radiation
stress. Exposure of organisms to UV radiation induces mutagenic and cytotoxic
DNA lesions such as cyclobutane-pyrimidine dimers and 6-4 photoproducts. To
overcome this stress, cells have developed repair mechanisms to counteract this type
of DNA damage, regardless of the causative factor. One of the most common repair
mechanisms involves photoreactivation with the help of the enzyme photolyase.
Glycosylases and polymerases also help many organisms repair base and nucleotide
excisions, respectively. Activation of these repair mechanisms by sublethal UV
radiation likely protects cells against subsequent exposure to lethal doses of UV.
Gamma-radiation resistant E. coli mutants have been recovered and studied (Ver-
benko and Kalinin, 1995), illustrating the ability of bacteria to change genetically
to resist this stress.
PULSED ELECTRIC FIELD

Process
Pulsed electric field processing involves the application of pulses of high voltage
(typically 20 to 80 kV/cm) to foods placed between two electrodes (Figure 1.4). When
high electric voltage is applied, electrical current flows through liquid food materials.
Liquid foods are commonly electrical conductors due to the presence of electrically
charged ions. Because of the very short period of discharge time (i.e., microseconds
or nanoseconds), heating of foods is minimized. Food treated with PEF has a better
retention of natural flavor, color, taste, nutrients, and texture compared to that treated
with heat (Dunn and Pearlman, 1987; Jia et al., 1999; Knorr et al., 1994).
Mechanism
Loss of cell membrane function is believed to cause microbial death during the PEF
treatment (Tsong, 1991; Unal et al., 2002; Zimmermann, 1986). The cell membrane
may be considered as a capacitor filled with a dielectric substance, with free charges
accumulating on the inner and outer surfaces of the membrane. The normal resting

Liquid Food
Electricity Stepped-up Electric

source Voltage Pulses
(110 or 220 V) (ca. 10 kV) (µ s range ) Treatment
Chamber
(20-80 kV/cm)
Power Supply Pulser

PEF-treated
product
FIGURE 1.4 Pulsed electric field (PEF) processing equipment: basic components.
potential difference across the membrane is 10 mV. The application of an electric

field pulse causes an increase in the transmembrane potential. Since the charges at
the two membrane surfaces are opposite, attraction between these charges reduces
membrane thickness. This electric compressive force may reach a magnitude that
causes a local breakdown of membrane (Zimmermann, 1986). The breakdown and
pore formation occur when the PEF treatment induces a membrane potential greater
than 1.0 V. Tsong (1991) suggested that electroporation of the cell membrane is a
mechanism of microbial inactivation by PEF. When an external electric field is
applied, electroporation occurs at protein channels due to protein denaturation caused
by heating or electric modification of their functional groups. Electroporation leads
to an osmotic imbalance of the cell, which may lead to death. Recently, Unal et al.
(2002) stained PEF treated cells with fluorescent dyes and provided evidence of
membrane permeation at lethal and sublethal electric fields. The growth region of
yeast cells during budding was found particularly sensitive to PEF treatment (Castro
et al., 1993).
Russell et al. (2000) treated L. monocytogenes and Salmonella typhimurium with

PEF and plated the survivors on selective and nonselective agar media. These authors
observed that mildly lethal PEF treatments did not result in any detectable cell injury.
They concluded that PEF causes an “all or nothing” effect against foodborne patho-
gens. Unal et al. (2001) also observed no injury when foodborne bacteria were
processed with PEF and the treated cells were grown on selective and nonselective
media. Processes that result in no detectable cell injury usually do not induce a stress
adaptive response. However, when bacterial cells were processed with sublethal
levels of PEF and treated with fluorescent stains, leaky membranes were detected
indicating cell injury (Unal et al., 2002). The authors concluded that PEF causes
cell injury detectable only by the fluorescence staining technique. Evidence of stress
adaptation due to PEF treatment is yet to be investigated.

MECHANISM OF STRESS ADAPTIVE RESPONSE
Response of microorganisms to stress includes immediate emergency responses (e.g.,
those produced in response to shock) and longer-term adaptation. In some cases,
the same proteins are involved in both rapid and long-term responses. In addition
to a general stress response that helps protect cells from a variety of stresses, cells
have self-protective mechanisms against specific stresses. Overlap exists between
the proteins involved in the general stress response and some specific stress
responses.
This section will focus on molecular mechanisms of stress adaptation in bacteria.
Stress adaptation is a complex phenomenon that differs depending on the type of
stress and the bacterial species. Adaptation results from induction of various stress-
related proteins that protect the cell from stress. Many stress-induced proteins have
been identified. This chapter does not intend to provide a comprehensive review of
stress-induced proteins in bacteria, but will introduce the variety of molecular mech-
anisms by which cells respond to stress and provide a general overview of how those
mechanisms are regulated. Examples of a few well-characterized systems will be
provided. For reviews of the molecular basis for stress response, the reader is referred
to Chapter 8 of this book, the review by Abee and Wouters (1999), and the com-
prehensive book edited by Storz and Hengge-Aronis (2000).
STRESS SENSING
For the cell’s metabolism to respond to a stress, the stress must somehow be sensed.
In general, bacterial sensing of environmental changes is not well understood. Some
stresses may affect folding of mRNA or change a protein’s half-life, resulting in
changes in gene expression (Yura and Nakahigashi, 1999). Other stresses may affect
protein structure. For example, OxyR senses reactive oxygen species via cysteine
residues that are oxidized to form a disulphide bridge. The resulting oxidized protein
positively regulates oxidative stress response (Mongkolsuk and Helmann, 2002).
Levels of certain cellular metabolites, such as guanosine phosphate, guanosine tetra-
(ppGpp) and pentaphosphates (pppGpp) and phosphate, may also trigger the synthesis
of stress-related proteins (Chatterji and Ojha, 2001; Rallu et al., 2000; Rao and
Kornberg, 1999). Ribosomes were suggested as sensors for temperature shocks
because of the sensitivity of these cellular components to heat (Duncan and Hershey,
1989). In addition, changes in the membrane structure or fluidity may trigger a signal
to synthesize proteins to counteract a stress (Bremer and Krämer, 2000).
Two-component signal transduction systems, consisting of a membrane-associ-
ated sensor kinase and an intracellular response regulator, have been implicated in
the sensing of and response to some stresses. For example, in Bacillus subtilis, a
two-component system is involved in expression of cold-inducible genes. In this
system, a membrane-bound histidine kinase (DesK) that may sense changes in
membrane fluidity transduces the signal to a response regulator (DesR) that puta-
tively activates the transcription of fatty acid desaturase gene, des (Sakamoto and
Murata, 2002).

Molecular factors involved Methods
in sensing and controlling to measure
stress response stress response
DNA
• Alternative σ factors • Molecular probes to
Transcription • Anti σ factors detect genes involved
• Transcription repressors in stress response
• Northern blotting
mRNA • mRNA stability • Microarray
• RT-PCR
• Measurements of
Ribosome • mRNA secondary structure
Translation ribosome integrity
• Ribosome stability
(e.g., DSC methods)
Stress- related • 2-D gel

• Protein stability
protein electrophoresis
• Protein modifications
• Immunodetection
• Relative stress
Changes in cell physiology to
resistance
increase stress tolerance
FIGURE 1.5 A simplified representation of general cellular processes involved in stress

response, molecular factors involved in sensing and controlling stress response, and methods
used to measure some of these responses. The stress sensor is not depicted, but this includes
a lipid, protein, or nucleic acid component that senses the stress and ultimately causes a
change in transcription or translation. DSC: differential scanning calorimetry; RT-PCR:
reverse transcription-polymerase chain reaction.
REGULATION OF STRESS-RELATED PROTEIN SYNTHESIS

Regulation of stress response is essential for the synthesis of appropriate stress-
related proteins only when necessary for protection of the cell. Regulation of stress
responses occurs at different levels depending on the stress and the bacterium.
Control may occur at the transcriptional or translational levels or by adjusting the
stability of the mRNA or protein (Figure 1.5). Regulatory strategies vary consider-
ably among bacteria and stresses. To add to the complexity, one stress response
factor may be regulated at one or more levels.
Transcriptional control of stress-induced genes and operons is a frequently
encountered mechanism to control stress responses. One type of transcriptional
control employs alternative sigma factors. The sigma subunit of RNA polymerase
determines the specificity of promoter binding. Under non-stress conditions the
constitutive sigma factor (σ70 in E. coli and σA in B. subtilis) directs expression of
“housekeeping” genes. Binding of an alternative sigma subunit to the RNA poly-
merase core enzyme changes its specificity, directing it to transcribe a different group
of genes and operons. Several stress-related regulons (coordinately regulated oper-
ons) are positively controlled by the synthesis of an alternative sigma factor. For
example, the presence of active σS causes transcription of genes involved in the
general stress response and stationary phase in E. coli.

A strategy to negatively control transcription of stress-related genes involves
anti-sigma factors. Anti-sigma factors bind to a specific sigma factor forming a
complex that prevents the sigma factor from binding to the RNA polymerase core
enzyme (Hughes and Mathee, 1998). In E. coli, the RssB protein has anti-sigma
factor properties; it inhibits the expression of σS-dependent genes in the presence
of high σS levels (Becker et al., 2000). A stress sensor may trigger release of the
sigma factor from the anti-sigma factor complex, resulting in transcription of stress-
related genes. A sigma factor may be released from the anti-sigma factor by an anti-
anti-sigma factor that binds to the anti-sigma factor. For example, σB, required for
general stress response in B. subtilis, is bound by an anti-sigma factor. An anti-anti-
sigma factor is present in a phosphorylated form in the absence of stress. Stress
increases the level of non-phosphorylated anti-anti-sigma factor, which is then able
to bind to the anti-sigma factor, releasing σB (Hecker and Volker, 1998).
Other transcriptional control mechanisms utilize repressor proteins that bind to
the promoter region of a specific gene or operon, preventing transcription until
conditions are appropriate, at which time the repressor protein is released from the
DNA allowing transcription to proceed. The heat stress operons, dnaK and groE,
are controlled in this manner in B. subtilis. They are under the negative regulation
by the HrcA repressor protein binding to the CIRCE (controlling inverted repeat of
chaperone expression) operator (Narberhaus, 1999).
Synthesis of stress-related proteins can also be controlled at the translational
level. Messenger RNA secondary structure near the ribosome binding site or trans-
lation start site can inhibit ribosome binding and translation of mRNA until stress
conditions are experienced (Takayama and Kjelleberg, 2000). Translation of mRNA
for the heat shock sigma factor (σ32) is regulated in this manner. Heat disrupts the
hydrogen bonds holding the mRNA secondary structure together allowing the trans-
lation of the transcript under hot conditions (Yura and Nakahigashi, 1999).
Changes in mRNA and protein stability provide another method of controlling
the activity of stress-related proteins. The half-life of some molecules can be
increased or decreased in response to stress. For example, the CspA mRNA involved
in cold tolerance is extremely unstable at 37°C and dramatically stabilized at lower
temperatures (Phadtare et al., 1999). Proteolytic degradation of stress-related pro-
teins is also observed as a control mechanism. The ClpXP protease degrades σS
under non-stress conditions (Hengge-Aronis, 1999).
GENERAL STRESS RESPONSE

A general stress response system can be activated by several different stresses and
protects against multiple stresses. Activation of the general stress response usually
results in reduced growth rate or entry into stationary phase (Hengge-Aronis, 1999).
The best-characterized general stress response systems are controlled by alternative
sigma factors, σS, in E. coli and other Gram-negative bacteria and σB in B. subtilis
and other Gram-positive bacteria.
The general stress response induces multiple physiological changes in the cell
including “multiple stress resistance, the accumulation of storage compounds, changes
in cell envelope composition and altered overall morphology” (Hengge-Aronis, 1999).

Genes induced by σS and σB include those for catalase, DNA repair, and osmopro-
tectant importation, suggesting that the cell is preparing for oxidative and osmotic
stress (Hecker and Volker, 1998; Petersohn et al., 2001).
Stress adaptive response in E. coli is coordinated by σS. Very little if any σS is
detectable in non-stressed E. coli cells. When cells are exposed to stress, σS is
induced, activating the σs-controlled promoters. Expression of these genes is neces-
sary for survival under stress conditions. σS is regulated by transcriptional and
translational control as well as by proteolysis (by ClpXP protease) in E. coli
(Hengge-Aronis, 1999). Different stresses differentially affect these various levels
of control. In B. subtilis, the activity of σB is modulated by an anti-sigma factor and
an anti-anti-sigma factor as described in the previous section.
SPECIFIC STRESS RESPONSES

Heat
Foodborne bacteria commonly encounter heat stress during food preservation and
processing. Heat causes damage to macromolecular cell components; thus the main
function of heat-induced stress proteins is to repair or destroy these damaged com-
ponents so they do not disrupt cellular metabolism. Many heat-induced stress pro-
teins are protein chaperones that assist in folding and assembly of heat-damaged
proteins (e.g., GroEL and DnaK) or are ATP-dependent proteases that degrade
damaged proteins (e.g., Lon and ClpAP) (Arsène et al., 2000; Hecker et al., 1996).
In addition to these changes, some bacteria also alter their cell membrane in response
to heat by increasing the ratio of trans to cis fatty acids in the membrane. This
structural change is thought to decrease fluidity caused by increasing temperatures
(Cronan, 2002).
In E. coli, the major heat-induced genes are controlled by the alternative sigma
factor, σ32. Approximately 50 genes are induced by σ32 when denatured proteins are
detected in the cytoplasm (Yura and Nakahigashi, 1999). σ32 is present at low levels
under non-heat-stress conditions. This low level is governed by the short mRNA
half-life and the low translation rate resulting from secondary structure at the 5′ end
of the mRNA. After a temperature increase, the secondary structure is destabilized
allowing translation to proceed. The half-life of σ32 also increases dramatically upon
exposure to heat (Arsène et al., 2000; Yura and Nakahigashi, 1999).
Two other alternative sigma factors, σE and σ54, control other regulons induced
by heat. σE, an extracytoplasmic function (ECF) sigma factor, responds to the
appearance of non-native proteins within the periplasm by means of an inner mem-
brane-bound anti-sigma factor (Raivio and Silhavy, 2001). Release of σ E from the
anti-sigma factor activates transcription of about 10 genes involved in proper assem-
bly of outer membrane proteins (Raivio and Silhavy, 2001). How non-native proteins
are sensed resulting in release of σE is not understood. σ54 controls one operon and
is activated by disturbances in the cytoplasmic membrane by an unknown mechanism
(Kuczynska-Wisnik et al., 2001).
Gram-positive bacteria differ markedly in their regulation of heat shock response.
In B. subtilis, several classes of heat shock genes have been identified. Class I consists

of the chaperone-encoding dnaK and groE operons. These operons have σA-depen-
dent promoters that are under the negative regulation of the HrcA repressor protein
binding to the CIRCE operator. This regulatory system is widespread and conserved
within the bacterial kingdom and has been described in more than 40 different species
(Hecker et al., 1996). The σB regulon constitutes the Class II genes, the largest group
of heat-induced genes in B. subtilis. These genes are not only induced by heat, but
also by other stresses, as discussed above (Hecker and Volker 1998). Class III heat-
induced genes are negatively controlled at the transcriptional level by a repressor
protein, CtsR. CtsR binds to a specific sequence in the promoter region upstream
of clp genes, clpP, clpE and clpC. These three genes are components of the Clp
protease system which degrades damaged proteins (Derre et al., 1999). It is not clear
how CtsR activity is changed after an increase in temperature. Other heat-induced
genes, not controlled by the above mechanisms, are yet to be classified.
Cold
Physiological changes in response to cold include changes in the membrane fatty

acid composition to promote optimum membrane fluidity (Russell et al., 1995),
synthesis of DNA- and RNA-binding proteins that counteract the stabilizing effect
of cold temperatures on nucleic acid secondary structures (Phadtare et al., 1999),
and importation of compatible solutes (Ko et al., 1994; Angelidis et al., 2002).
Proteins synthesized in response to cold can be classified as Csps (cold shock
proteins) or Caps (cold-shock acclimation proteins). Csps are rapidly, but transiently
overexpressed in response to cold. Caps are synthesized during continuous growth
at cold temperatures; they are rapidly induced, but remain overexpressed several
hours after the temperature downshift. A slow temperature downshift results in
synthesis of some Csps and Caps (Phadtare et al., 1999).
Upon decrease in temperature, the phospholipid bilayer membranes of all cells
decrease in fluidity. To maintain optimum fluidity, cells increase the unsaturation or
decrease the chain length of the membrane fatty acids, resulting in increased fluidity
at lower temperatures (Russell et al., 1995). After cold shock in B. subtilis and
cyanobacteria, synthesis and stability of a fatty acid desaturase increase as controlled
by a two-component signaling system (Aguilar et al., 1998; Sakamoto and Murata,
2002).
Cold shock also causes stabilization of the hydrogen bonds in nucleic acid
secondary structures resulting in reduced efficiency of translation, transcription and
DNA replication. These deleterious effects are overcome by induction of cold-shock
proteins that serve as nucleic acid chaperones. CspA, the major cold-shock protein
of E. coli, is proposed to regulate gene expression by functioning as an RNA
chaperone at low temperatures. CspA-like proteins contain two conserved RNA
binding sequences. CspA is regulated at the transcriptional and translational levels
and by increased mRNA stability at low temperatures (Phadtare et al., 1999).
In E. coli, Csps have been grouped into two classes. Class I proteins consist of
RNA/DNA chaperones (including CspA), ribosome-associated proteins, a ribonu-
clease, and a protein involved in termination of transcription. Class I genes are barely
expressed at 37°C, but dramatically increase after a shift to lower temperatures.

Class II genes are involved in DNA stability and structure and include the DNA-
binding protein, H-NS, and a subunit of DNA gyrase. Class II proteins are present
at 37°C; after shift to colder temperatures, their transcription is only slightly higher
(<10-fold) (Phadtare et al., 1999).
Transport or synthesis of compatible solutes (see osmotic stress section) was
reported to confer cold shock tolerance. In E. coli, the σS-dependent synthesis of
trehalose by the otsAB gene products is cold-inducible. An additional level of
regulation is provided by the instability of otsAB mRNA at higher temperatures
(Kandror et al., 2002). Listeria monocytogenes transports the compatible solutes,
betaine (Ko et al., 1994) and carnitine (Angelidis et al., 2002), in response to cold
temperatures. Regulation of this system has not been reported.
Acid
Foodborne bacteria encounter organic and inorganic acids in foods or in the gas-
trointestinal tract and cells of the host. Bacteria respond to acid stress in many ways
including changes in membrane composition, increase in proton efflux, increase in
amino acid catabolism, and induction of DNA repair enzymes. Observed in most
bacteria, the acid tolerance response (ATR) is a phenomenon whereby exposure to
moderately low pH induces the synthesis of proteins that promote survival at
extremely low pHs. ATR differs in exponential and stationary phase cells. This
response also differs dramatically among different bacterial species. An overview
of strategies which bacteria employ to combat acid stress is described in this section.
The reader is referred to Chapter 8 of this book for more details.
The signal for induction of acid shock or adaptation proteins may be intracellular
or extracellular pH. External or periplasmic pH may be sensed by membrane bound
proteins (Foster, 1999). Internal pH may affect gene expression directly or may alter
a cellular component involved in gene expression.
Exponential phase ATR in Salmonella typhimurium involves several regulatory
proteins that each control a subset of acid-induced proteins. These regulatory proteins
include σS, the two-component signaling system PhoPQ, and the iron regulator, Fur
(Foster, 1999, 2000). The σS-dependent ATR genes that have been identified consist
of several proteins of unknown function and a superoxide dismutase. Most of the
PhoPQ-controlled genes are of unknown function, though Adams et al. (2001)
reported decreased flagellin expression and cell motility upon activation of the
PhoPQ pathway by acid. The authors suggest that “flagellar repression at low pH
conserves ATP for survival processes and helps to limit the influx of protons into
the cytosol.” The Fur-controlled acid-induced genes in Salmonella have not been
identified (Foster, 2000), but Fur modulates urease expression in enterohemorrhagic
E. coli, and thus, may be involved in acid tolerance of this organism (Heimer et al.
2002). Urease hydrolyzes urea into ammonia and carbon dioxide. The resulting
ammonium ions may accumulate and modify internal and/or external pH.
Stationary phase ATR in Salmonella involves stationary phase induction of σS
resulting in a general stress tolerance and induction of acid stress proteins by OmpA
(Foster, 2000). A deletion in the gene encoding σB in L. monocytogenes renders
stationary phase cells acid sensitive (Gahan and Hill, 1999).

Cyclopropane fatty acid (CFA) synthase catalyzes the synthesis of CFAs from
unsaturated fatty acids in the bacterial membrane. In E. coli, CFA synthase gene
expression increases with a decrease in pH to 5. Transcriptional activation is σS-
dependent. The increase in cfa gene expression results in increased survival to the
lethal challenge of pH 3 (Chang and Cronan, 1999). The investigators suggest that
the resulting changes may affect proton permeability through the membrane or the
activity of a membrane-bound protein involved in acid stress.
Limited information is available about the association of extracellular cell-to-
cell signaling and stress adaptation. Acid adapted E. coli is believed to secrete an
extracellular protein that causes unadapted cells to become acid tolerant without
acid adaptation (Rowbury and Goodson, 1999; Chapter 8 of this book).
Gram-positive bacteria, which regulate internal pH with an F0F1 ATPase, can
increase synthesis or activity of the ATPase upon pH decrease, providing the cell
with a higher capacity for proton efflux (Foster, 2000). The F0F1ATPase is acid-
inducible at the transcriptional level in Lactobacillus acidophilus (Kullen and Klaen-
hammer, 1999), whereas in Streptococcus spp. or Enterococcus spp., enzyme activity
is controlled at the subunit assembly stage (Foster, 2000).
Low cytoplasmic pH can cause DNA damage. An acid-inducible DNA repair
enzyme was identified in Streptococcus mutans (Hahn et al., 1999). The importance
of DNA repair in acid stressed cells is supported by data revealing that mutations
in the ada gene, involved in DNA repair, cause acid sensitivity in Salmonella (Foster,
2000).
Amino acid catabolism can also help cells to fight a proton influx. Some Gram-
positive bacteria use the arginine deiminase system to alkalinize the cytoplasm
(Foster, 1999). Arginine is broken down into ornithine, carbon dioxide and ammonia.
The glutamate decarboxylase/GadC antiporter system (E. coli, Shigella, Lactococ-
cus, [Foster, 2000], and Listeria [Gahan and Hill, 1999]) requires extracelluar
glutamate which is imported via the GadC antiporter and decarboxylated within the
cell, a reaction that consumes a proton. The resulting gamma amino butyric acid is
exported via GadC. This system is induced by stationary phase or by acid in the
exponential phase. A similar system involving arginine decarboxylase also protects
E. coli from pH 2 (Foster, 2000).
Osmotic Stress
Bacteria may encounter osmotic stresses in foods that are high in salt or sugar or
in a dried state. Under such conditions, it is essential for the cell to maintain turgor
pressure and hydration. The mechanisms described refer to bacteria that reside in
environments with moderate or occasional hyperosmotic conditions.
The best-characterized mechanism by which bacterial cells respond to hyperos-
motic conditions involves intracellular accumulation of compatible solutes. This
accumulation can be accomplished by synthesis or import from the environment.
Compatible solutes are polar, highly soluble compounds that counteract osmotic
pressure without affecting normal cellular functions, even at very high concentrations.
Glycine betaine, proline, ectoine, carnitine, choline, and trehalose, among others, are
common compatible solutes. Accumulation of these compounds is regulated at the

gene transcription level or by modifying enzyme activity directly (Bremer and
Krämer, 2000). σS (E. coli) and σB (B. subtilis) control synthesis of some proteins
required for osmoprotectant synthesis or transport. Sensing of osmotic stresses is
poorly understood (Culham et al., 2001; Mellies et al., 1995; von Blohn et al., 1997).
Additional changes in cell metabolism in response to osmotic stress involve the
cell membrane. An increase in the ratio of trans to cis unsaturated fatty acids is
observed in cells exposed to high salt concentrations (Cronan, 2002). In addition,
the proportion of anionic phospholipid and/or glycolipids is increased in salt-
stressed, compared with unstressed, cells (Russell et al., 1995). In addition to σS,
the σ32 and σE regulons are activated when E. coli experiences hyperosmotic condi-
tions. Both regulons encode protein chaperones and proteases that assure proper
assembly of proteins in the stressed cell (Bianchi and Baneyx, 1999). Hyperosmotic
stress not only activates the σB regulon in B. subtilis, but also induces the extracy-
toplasmic function (ECF) sigma factor σW (Petersohn et al., 2001). This sigma factor
controls expression of >30 genes, many encoding membrane proteins of unknown
function (Huang et al., 1999).
Oxidative Stress
In foods, bacteria may be exposed to increased levels of reactive oxygen species

such as hydrogen peroxide, hydroxyl radicals and superoxide. Such oxidants cause
damage to cellular proteins, lipids and nucleic acids. Many of the known proteins
induced by oxidative stress have antioxidant roles. Others are involved in repair of
oxidative damage, particularly damage to nucleic acids.
In E. coli, most oxidative stress-induced genes are part of the oxyR and soxRS
regulons induced by hydrogen peroxide and superoxide, respectively (Storz and
Zheng, 2000). OxyR senses oxidative damage via cysteine residues that are oxidized
to form a disulphide bridge, altering the protein structure into the active form
(Mongkolsuk and Helmann, 2002). There is significant overlap between the oxidative
stress-induced proteins and those induced by σS, suggesting that oxidative damage
is significant in stationary phase or stressed cells.
MONITORING STRESS RESPONSE

Microorganisms in food or environment are often exposed to stresses and some of
these evoke measurable responses (see Figure 1.5). The response varies mainly with
the type and magnitude of stress and the microorganism’s physiological state. Under
some stress conditions, microbial response is a protective effect, i.e., an adaptive
response. Food microbiologists and processors are interested in the stress adaptive
response since it alters the microorganism’s resistance to processing and preservation
factors. Higher levels of stress may injure the cells. Injured cells probably become
energy-exhausted by multiple responses which decrease their capacity to react to
additional insults. Additional stress usually kills injured cells (see Figure 1.1). Injury
is evident by the sensitization of treated cells to selective agents, antibiotics and
other deleterious factors, or the impairment of cells’ ability to multiply.

Detecting and measuring stress response have many beneficial applications. Food
processors may learn about the consequences of mild treatments and the causes of
resistance of pathogens to processes that are presumed lethal to these microorgan-
isms. On the contrary, stresses that sensitize pathogens to processing may have
beneficial applications in food preservation. Using stress response to sense undesir-
able agents (stressors) in the food processing environment is another area of potential
interest to food processors, but this has not been explored.
To determine the conditions likely to lead to adaptive responses, researchers
may vary stress level and apply stress at various physiological states of the targeted
microorganism. Based on experience and a large amount of published literature,
microbial adaptive response is most apparent at sublethal levels of stress and when
the microorganism is in an active metabolic state, i.e., the exponential phase of
growth. Many researchers, however, have demonstrated appreciable stationary-phase
inducible adaptive responses (e.g., Buchanan and Edelson, 1999). Similarly, lethal
doses of stress may trigger considerable adaptive responses in the fraction of the
population that survives the treatment. After applying the stress under investigation,
procedures to detect or quantify the response should be followed. Stress responses
measured include changes in gene expression products (RNA and proteins) and stress
tolerance (see Figure 1.5).
Although detection of stress adaptive response is generally laborious, distinction
of injury is relatively simple. Stress-sensitized cells (i.e., injured) demonstrate
reduced growth rate (e.g., reduced colony size on agar media), impaired growth in
the presence of selective agents such as NaCl and bile salts, increased sensitivity to
antibiotics, and loss of aerotolerance. Details about adaptive responses are included
in this contribution, but sensitization by stress will not be addressed.
INDUCTION OF STRESS ADAPTIVE RESPONSE: PRACTICAL CONSIDERATIONS

The following are examples of the most commonly investigated stresses, heat and
acid. Included is a brief description of methods of applying theses stresses for
inducing adaptive responses. Once the stress response is developed, cells should be
handled in a way to preserve the response. Active metabolism and multiplication of
stress-adapted cells deteriorate the adaptation and thus it becomes difficult to detect.
Heat
Heat induces a universal protective response that is relatively easy to detect. Tem-
peratures conducive to growth normally do not constitute stress to cells and thus are
not used commonly in developing a stress response. Severe thermal stress may
eliminate sizable proportion of the cell population and the adaptive response in the
small fraction of the population that survives the treatment may not be measurable.
Response to a mild heat shock is readily detectable when cells are treated at sublethal
or minimally lethal temperatures. According to our experience, heat shock response
is demonstrated best when L. monocytogenes exponential-phase culture is heated at
45°C for 1 h (Lou and Yousef, 1997). By comparison, injury of L. monocytogenes
is most apparent at 55 to 60°C (El-Shenawy et al., 1989) and neither stress response

nor injury can be reliably detected at 70°C. Heat shocking E. coli O157:H7 at 45 to
46°C for 15 to 30 min produces appreciable thermal adaptation (Juneja et al., 1998;
Lucore et al., 2002). Heat may be applied rapidly, i.e., as a heat shock (Lou and
Yousef, 1997) or gradually (Stephens et al., 1994), since both procedures produce
significant adaptive response.
Acid
Acid Shock during Exponential Phase

Actively growing microbial cells, in their mid-exponential phase, are treated with
sublethal levels of an acid, i.e., cells are acid shocked. Incubation is continued to
allow one to two doublings under the acid stress. During this additional incubation
period, cells normally develop an acid adaptive response. Since the adaptive response
is a transient phenomenon, further processing of these cells (e.g., centrifugation and
washing) should be done promptly and under refrigeration conditions in order to
preserve the developed response. This technique produces a strikingly different
response from that observed in the non-treated culture and thus the adaptation is
relatively easy to track. Response of these cells, however, is transient and the
adaptation may degrade quickly before it can be measured, particularly if treated
cells are mishandled. Additionally, collecting cells from mid-exponential phase can
be tricky since cell density at this stage is normally low. Phase of growth should be
determined in advance by plating the culture after different incubation periods and
constructing a growth curve. Correlation of microbial counts with culture turbidity
(measured spectrophotometrically) allows estimation of growth phase prior to the
experiment. Researchers who successfully applied acid stress to mid-exponential
phase cultures include Foster and Hall (1990), Leyer and Johnson (1992), and Lou
and Yousef (1997).
Gradual Acid Stress
Microorganisms that produce acid as a byproduct of carbohydrate metabolism expe-
rience a gradual decrease in pH during culturing. This gradual acidification induces
a stationary-phase acid resistance response (Buchanan and Edelson, 1999). Gradual
acid exposure is a simple and practical method of producing acid-adapted cells.
Most of the adaptation, however, occurs during the stationary phase when cells
generally develop resistance to various deleterious factors (Watson, 1990). Conse-
quently, the intrinsic stationary phase acid resistance may overshadow induction of
acid resistance by carbohydrate fermentation. The non-acid adapted cells (control
culture) are grown in the absence of a fermentable carbohydrate and thus produce
energy through alternative metabolic ways. Unfortunately, these control cells may
inadvertently be sensitized to acid or develop a starvation response during growth
in the carbohydrate-free medium. Gradual application of acid stress may also be
accomplished by manual incremental addition of acid to a growing culture. Alter-
natively, a chemostat may be used to gradually apply acid stress to a growing culture
in a controlled manner. This latter procedure is most useful when the test microor-
ganism does not produce acid during growth.

DETECTING AND QUANTIFYING STRESS RESPONSE
Methods to detect and measure stress response vary depending on the response
measured (see Figure 1.5). Evidence of stress response includes presence of genes
involved in stress response mechanisms, elevated level of gene products such as
mRNA, de novo protein synthesis in response to stress, and increased tolerance to
lethal levels of the stress.
Detection of Stress Response Genes
Presence of genes encoding stress response proteins may indicate that the microor-
ganism is capable of responding to a stress in a predictable fashion. Comparing the
genomes of resistant and sensitive strains may reveal these genes involved in stress
response (Koonin et al., 2000). Researchers have developed probes for detecting
genes that contribute to stress response; these are useful tools to determine potential
response to stress by an isolate.
mRNA Analysis
While presence of the gene is a prerequisite for a response, expression of this gene
is needed for the ultimate manifestation of the response. Therefore, interest in
detecting stress response at the transcriptional level is increasing. Synthesis of
proteins that protect cells against stress is sometimes preceded by increased tran-
scription of the relevant mRNA. Measuring these mRNAs demonstrates, or even
quantifies, the stress response. Methods to measure mRNA include Northern anal-
ysis, microarray-genome-wide expression monitoring (also known as microarray
analysis) and reverse transcription polymerase chain reaction (RT-PCR).
Detection of Stress Proteins
Synthesis of stress proteins provides yet more direct evidence of the microorganism’s
response to stress. Proteins synthesized in response to stress include regulatory pro-
teins (e.g., σ32 in E. coli and σB in L. monocytogenes), chaperones (e.g., GroEL),
ATP-dependent proteases (e.g., Lon), and DNA repair proteins (e.g., UspA) (Duncan
et al., 2000; Diez et al., 2000; Rosen et al., 2002). Many of these proteins have been
successfully detected using a two-dimensional electrophoresis (e.g., Rince et al.,
2002). Antibodies specific to some of the well-characterized stress proteins are
commercially available to detect a stress response by immunodetection methods
such as Western blotting (Duncan et al., 2000). If the corresponding antibodies are
not commercially available, the gene of a specific stress protein can be cloned. The
recombinant protein is then amplified, purified and used to generate the correspond-
ing specific antibodies (Jayaraman and Burne, 1995).
Biosensors
Microorganisms have been genetically engineered for easy detection of stress

response (LaRossa and Van Dyk, 2000). Reporter genes (e.g., lacZ which encodes

for β-galactosidase) were fused to promoters of genes involved in adaptive response.
Other useful reporter genes include luxAB, which encodes bacterial luciferase, luc,
encoding insect luciferase, and gfp, for green fluorescence protein. When these
fusion strains respond to stress, the reporter gene is expressed and fluorescent or
luminescent products are produced. Gene fusion strains (biosensors) for detecting
DNA damage, heat shock, oxidative stress, and starvation have been developed for
basic research and are potentially useful in the field of food microbiology.
Measuring Increased Tolerance
Adaptive responses may be measured by comparing stress tolerance of cells that

have been pre-exposed to sublethal stress to those that have not. Measurement of
inactivation by stress uses simple plating techniques. A greater degree of survivabil-
ity of the cells exposed to sublethal stress may indicate that the stress induced an
adaptive response. Quantifying the stress by the cultural technique may require
measuring changes in death rates as a result of pre-exposure to stress. Determining
D-value (time required to decrease the population under stress by one log CFU unit)
is a useful quantitative measure of resistance. Culture techniques provide direct
evidence of stress adaptive response and the results of the analysis have great
practical value to food processors. These techniques, however, are time-consuming
and the results may be compromised by experimental artifacts.
PERSPECTIVES AND AREAS FOR FUTURE WORK

Some researchers question the relevance of stress adaptation to food safety. This
argument is based on these observations:
• Stress adaptation is best demonstrated at the exponential, rather than at

the stationary, phase of growth. Since pathogens in food are rarely in the
exponential phase, significant adaptation to stress under most processing
and production practices may be unlikely.
• Direct determination of the degree of adaptation of microbiota in food is
not currently feasible. Therefore, there is no knowledge on how much of
processing resistance that these microorganisms experience is attributed
to stress adaptive response.
• Although the number of reports linking stress adaptation and virulence is
rising (see Chapter 7), there is no evidence that directly links stress adap-
tation of pathogens to foodborne disease outbreaks.
While these arguments have some merits, we believe that the stress adaptation
phenomenon has a profound effect on the safety of food:
• Although stress adaptation is remarkable in actively metabolizing cultures,

microorganisms at all phases of growth do adapt to stress. Induction of
stress adaptive response in stationary-phase cultures is well documented.

Nevertheless, demonstration and quantification of these adaptive responses,
under real processing conditions, need to be carefully investigated.
• Lack of direct evidence is not a proof of the absence of the relationship
between stress adaptation and food safety. With the continuous improve-
ments in analytical tools and protocols, researchers may soon be able to
verify these associations. Rapid methods to differentiate between transient
and inherent resistance, and to quantify these traits in the food microbiota,
are urgently needed. Availability of these methods will not only reveal
the risks associated with stress adaptation, but processors may also use
these techniques to gauge processing severity with the anticipated toler-
ance of the microbiota in food.
Many researchers agree that there is a considerable potential risk of disease as

a result of stress adaptation, particularly in food produced by minimal-processing
or novel, alternative processing technologies (Abee and Wouters, 1999; Archer,
1996; Rowan, 1999; Yousef, 2000). Interest in these technologies has increased
appreciably in the past decade. These technologies promise to maintain the critical
balance between safety and marketability of a new generation of foods. It is of
concern that processing conditions may be conducive to stress adaptive response in
foodborne pathogens. Currently, stress adaptive responses of microorganisms in food
processed by these technologies are poorly understood. As these novel food pro-
cessing technologies become commercialized or used more widely, it is essential
that researchers understand the adaptive responses that are induced by these treat-
ments.
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Adaptation of Foodborne
2 Pathogens to Stress from
Exposure to Physical
Intervention Strategies
Vijay K. Juneja and John S. Novak
CONTENTS
Introduction
Sublethal Heat Stress
Heat-Shock Response
Synthesis of Heat-Shock Proteins
Factors Affecting Heat-Shock Response
Cell Membrane Adaptations
Cross Protection
Management Strategies
High Hydrostatic Pressure
Dehydration
Restricting Water Activity
Freezing
Pulsed Electric Field
Irradiation
Ultraviolet Irradiation
Gamma Irradiation
Concluding Remarks
References
INTRODUCTION
The growth or survival of potentially life-threatening pathogens is a significant food
safety hazard. The ability of low numbers of these pathogens to survive or proliferate
Note: Mention of a brand or firm name does not constitute an endorsement by the U.S. Department of
Agriculture over other products or companies of a similar nature.

even when stored under refrigeration or in reduced oxygen atmospheres constitutes
a potential public health hazard. It is estimated that foodborne diseases cause approx-
imately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United
States each year (Mead et al., 1999). Known pathogens cause 14 million illnesses,
60,000 hospitalizations, and 1,800 deaths (Mead et al., 1999). Only a small portion
of the foodborne illness episodes are reported and investigated annually, and the
pathogens identified comprise an even smaller portion (Mead et al., 1999).
Annual economic losses attributed to foodborne diseases associated with medical
costs, productivity losses, and business losses due to legal problems may be as large
as $5 billion to $6 billion (CAST, 1996). These food safety concerns are magnified
because of consumer preference for convenient meals, processed using mild heat
treatment, that require minimal preparation time prior to consumption. Accordingly,
it is important to emphasize development and application of physical control pro-
cesses for microorganisms with the objective of improving the safety of our food
supply.
A variety of established physical intervention strategies for control of foodborne
pathogens include heat treatment, refrigeration, modified gaseous atmosphere, and
ionizing irradiation. Microorganisms can also be controlled by novel nonthermal
treatment methods, such as application of high hydrostatic pressure, high intensity
pulsed electric field, oscillating magnetic field, or combinations of physical pro-
cesses. These physical treatments used in food processing are designed to kill or
decrease the number of pathogenic and spoilage microorganisms. If the treatment
is not severe enough to ensure lethality, the surviving organisms are injured and may
recover under the proper conditions (Iandolo and Ordal, 1966).
SUBLETHAL HEAT STRESS

HEAT-SHOCK RESPONSE
The microbial safety of thermally processed foods depends upon the assurance that
foodborne pathogens, likely to be present in foods, are killed during heating. While
thermal processing guidelines are generally adequate for destruction of pathogens
in foods, there may be conditions when the microorganisms become more heat
resistant. Sublethal heat stress (heat shock) or prior exposure to low heat renders
the organism more resistant to subsequent heat treatment which would otherwise be
lethal (Murano and Pierson, 1992; Lou and Yousef, 1996; Juneja et al., 1997).
Microbial cells synthesize heat-shock proteins, coincident with sublethal heat stress
acquired, which appear to render the cell resistant to a second elevated challenge
normally considered to be lethal. Typically, “heat-shocked” cells need to be heated
twice as long as “nonheat-shocked” cells in order to achieve the same extent of
lethality (Farber and Brown, 1990).
This phenomenon of a heat shock response and induced thermotolerance is of
substantial practical importance to food processors for products normally heated at
temperatures below 65°C. Thermotolerance may become a concern in meat products
kept on warming trays before a final heating or reheating step, or when there is an
interrupted cooking cycle during processing due to equipment failure. Thus,

increased heat resistance due to prior heat shock must be considered while designing
thermal processes to assure the microbiological safety of thermally processed foods.
The heat treatment should be sufficient to inactivate the thermotolerant foodborne
pathogens and spoilage organisms. Potential known pathogens which have been
adapted to stress should be preferentially chosen for challenge tests as opposed to
general foodborne pathogens during the simulation of process conditions.
The heat shock response and induced thermotolerance has been reported in a
wide range of bacteria, including Actinobacillus actinomycetemcomitans (Paju et al.,
2000), Agrobacterium tumefaciens (Nakahigashi et al., 1999), Escherichia coli
(Tsuchido et al., 1984), Salmonella typhimurium (Mackey and Derrick, 1986, 1990),
Salmonella thompson (Mackey and Derrick, 1987), Salmonella enteritidis phage
type 4 (Humphrey et al., 1993), Staphylococcus aureus (Hurst et al., 1974), Strep-
tococcus thermophilus (Auffray et al., 1995), Lactococcus lactis (Kilstrup et al.,
1997), Listeria monocytogenes Scott A (Fedio and Jackson, 1989; Linton et al.,
1990), Leuconostoc eonos (Guzzo et al., 1997), E. coli O157:H7 (Murano and Pier-
son, 1992; Jorgensen et al., 1996; Juneja et al., 1997), and Yersinia enterocolitica
(Shenoy and Murano, 1996).
An increase in heat resistance of spores following heat shock has also been
reported in spore-forming organisms such as Bacillus anthracis, B. cereus, B. mega-
terium, B. subtilis (Streips and Polio, 1985), B. stearothermophilus (Etoa and
Michiels, 1988; Beaman et al., 1988), Clostridium acetobutylicum (Bahl et al.,
1995), C. botulinum (Appleyard and Gaze, 1993), C. perfringens (Heredia et al.,
1997, 1998), and C. sporogenes (Alcock, 1994). A direct relationship has been shown
to exist between C. perfringens spore heat resistance and the temperature at which
the spores are produced (Garcia-Alvarado et al., 1992).
In addition to being studied extensively in broth systems, heat shock response
and induced thermal tolerance have been shown to have significance in foods
(Shenoy and Murano, 1996; Farber and Brown, 1990; Mackey and Derrick, 1987;
Juneja et al., 1997). The extent to which cells become more thermotolerant after a
heat shock has been found to depend on the physiological state of the cell (Lindquist,
1986; Linton et al., 1992), the time/temperature combination used in the sublethal
heat treatment (Bunning et al., 1990; Farber and Brown, 1990), the media (Mackey
and Derrick, 1990) and the method of recovery (Knabel et al., 1990; Linton et al.,
1990).
Synthesis of Heat-Shock Proteins
Heat shock triggers a physiological response that leads to the synthesis of a specific
set of proteins known as heat-shock proteins (HSPs) (Schlesinger, 1990; Lindquist,
1986). The increased synthesis of these HSPs usually occurs 5 to 60 min after heat
shock and declines with the onset of normal protein synthesis 60 to 90 min after
return to normal temperatures (Watson, 1990). These HSPs are highly conserved
among prokaryotic and eukaryotic organisms (Lindquist, 1986) and increase the
potential of bacteria to withstand severe subsequent stresses. HSPs may enhance the
survival of pathogens in foods during exposure to high temperatures. Although the
scientific literature provides some evidence regarding the cause and effect relationship

between the synthesis of HSPs and the induced thermotolerance response, this
evidence is largely indirect and insufficient.
Researchers have suggested that HSPs are not necessarily the major contributory
agents in the development of thermotolerance but are required for recovery from
heat stress (Susek and Lindquist, 1989; Barnes et al., 1990; Smith and Yaffe, 1991).
Their apparent role is to protect the cells against heat damage and to help the cells
return to their normal physiological state following the stressful event. Schlesinger
(1990) and Sanchez and Lindquist (1990) suggested that the role of HSPs in ther-
motolerance may be to act as chaperones to remove denatured proteins. Molecular
chaperones constitute 15 to 20% of the total cellular protein in response to elevated
(46°C) temperatures (Arsene et al., 2000). The primary function of classic chaper-
ones, E. coli DnaK (HSP 70), DnaJ, GrpE, GroEL (HSP60), and GroES is to bind
to and stabilize polypeptides already present in cells, modulate protein folding
pathways to prevent misfolding or aggregation of proteins, and promote refolding
and proper assembly (Georgopoulos and Welch, 1993).
Some E. coli HSPs are proteases that are able to digest irreversibly damaged
polypeptides for removal and assist in nucleic acid synthesis, cell division, and
motility (Morris, 1993). In E. coli, regulation of stress responses through the tran-
scriptional control of alternate sigma factors encoded by rpoS and rpoH in response
to general stress and heat, respectively, has been studied in greater detail (Hengge-
Aronis, 1993; Yura et al., 1984). The sigma factors direct RNA polymerase in the
transcription of select subsets of genes including those associated with virulence
(Aldsworth et al., 1998). In E. coli, about 17 heat-shock proteins are found which
are diverse with respect to size, net charge, and levels or extent of induction in
response to heat shock (Neidhardt et al., 1984). Ten of these Hsps are the products
of known genes and have been characterized (Neidhardt and VanBogelen, 1987).
It has been reported that two stress proteins of 60 and 69 kDa correspond to the
GroEL and DnaK proteins of E. coli (Georgopoulos et al., 1990). The 60 kDa
(GroEL) protein is involved in the morphogenesis of coliphage and is also essential
for E. coli growth (Friedman et al., 1984; Fayet et al., 1989). GroEL has been shown
to protect RNA polymerase (RNAP) from heat inactivation and “resurrect” heat-
inactivated, aggregated RNAP (Georgopoulos et al., 1994). Juneja et al. (1997)
observed that the level of the 60 kDa GroEL protein in E. coli O157:H7 increased
significantly following heat shock (46C/15 min), whereas the increase in the level
of the 69-kDa DnaK protein was not as high.
Murano and Pierson (1992) described stress proteins with molecular masses of
71 and 84 kDa in E. coli O157:H7 and found that the concentration of these proteins
in heat-shocked cells depended upon the gaseous growth atmosphere prior to heat
shock. Xavier and Ingham (1997) found an overexpression of seven proteins in
S. enteritidis with apparent molecular weights of 14, 16, 21, 23, 60, 75, and 89 kDa,
respectively, after application of a heat-shock treatment of 42°C for 60 min.
Mackey and Derrick (1990) reported the induction of four major S. typhimurium
heat-shock proteins with approximate molecular weights of 25, 64, 72, and 83 kDa.
The authors thought that these heat-shock proteins observed in Salmonella spp. may
be homologous with highly conserved heat-shock proteins such as DnaK and GroEL

of E. coli. These proteins are examples of the extensively studied Hsp70 and Cpn60
groups (Landry et al., 1992).
Morgan et al. (1986) observed that S. typhimurium produces certain stress pro-
teins during anaerobic growth, which are similar in size to proteins produced as a
result of heat shock. One of these proteins has a molecular weight of 85,000 Da and
is produced in S. typhimurium also as a result of heat stress, as well as anaerobic
stress.
Anaerobic growth of E. coli as well as heat shocking of aerobically grown cells
leads to the production of proteins of 84,000 and 71,000 Da (Murano and Pierson,
1992). Western blot analysis using monoclonal antibodies specific for the sigma
subunit indicated that this protein was immunologically similar to the sigma 32
subunit of RNA polymerase, which is considered to be a stress protein (Grossman
et al., 1987).
Factors Affecting Heat-Shock Response
According to Lindquist (1986), the persistence of heat shock-induced thermotoler-

ance appears to be a function of many factors including the temperature at which
heat shocking is done, previous incubation temperature of the cell, and the metabolic
state of the cell. As temperature fluctuations are a common occurrence in food
processing environments as well as during transportation, distribution, and storage
or handling in supermarkets or by consumers, bacterial cells in meats are likely to
encounter temperature shifts. Therefore, temperature plays a significant role and
must be considered when determining the persistence of heat shock-induced ther-
motolerance. Juneja et al. (1997) suggested that guidelines be established so that
prophylactic measures are adopted and environmental stresses (such as heat) do not
render bacteria able to survive thermal processing procedures better than normally
would be considered adequate.
The time–temperature combination that produces the maximum thermotolerance
and the persistence of the induced thermotolerance response after heat shock has
been investigated. Lindquist (1986) has pointed out that for mesophilic bacteria,
temperatures between 45 and 50°C are optimum for development of the heat-shock
response and the demonstration of the increased thermotolerance. In a study by
Farber and Brown (1990), where a sausage mix inoculated with 7 log10 CFU/g of
L. monocytogenes was heat shocked at 48°C for 30, 60, and 120 min before being
heated to a final temperature of 64°C, cells heat shocked for 30 or 60 min did not
show a significant increase in thermotolerance. However, cells preheated for 120 min
exhibited increased thermal tolerance when compared to non-heat-shocked cells. A
2.4-fold increase in D-value at 64°C was observed for heat-shocked compared to
non-heat-shocked cells. After heat shocking, inoculated meat samples were held at
4°C for 24 h and then the bacteria were tested for heat resistance at 64°C. The
authors reported that the heat-shocked cells retained their increased heat resistance.
Mackey and Derrick (1986) increased the heat resistance of S. typhimurium
grown at 37°C in tryptone soya broth by exposing the cultures to sublethal heat
shock at 42, 45, or 48°C before exposing the organism to 55°C for 25 min. In that

study, the heat resistance increased rapidly following the temperature increase and
the degree of resistance as well as the rapidity of its onset increased with increasing
temperature of heat shock, reaching near maximum levels within 30 min and per-
sisting for 10 h. Pre-incubating cells at 48°C for 30 min increased their resistance
to a range of lethal temperatures (52 to 59°C). The authors demonstrated a similar
effect with S. thompson when the organism was preheated at 48°C and then heated
to 54 or 60°C in tryptone soya broth, liquid whole egg, 10% (w/v) or 40% (w/v)
reconstituted dried milk, or minced beef (Mackey and Derrick, 1987).
Shenoy and Murano (1996) heat shocked Yersinia enterocolitica in brain heart
infusion broth at 45°C for 5, 10, 15, 30, 45, or 60 min and reported that 60 min
consistently resulted in an increased number of survivors following a subsequent
treatment at 55 or 60°C when compared with non-heat-shocked cells. In a study by
Pagan et al. (1997), the thermotolerance of L. monocytogenes at 65°C increased with
the increase in the duration of heat shock for up to 120 min, regardless of the heat
shock temperature of 40, 43, or 46°C.
In contrast to these studies demonstrating a parallel increase in heat resistance
with the increase in the time of heat shocking, Murano and Pierson (1992) heat
shocked E. coli O157:H7 in trypticase soy broth (TSB) at 30, 34, 42, or 45°C for
0, 5, 10, or 15 min and reported that heat shocking at 42°C for 5 min resulted in
the greatest log number of survivors to the subsequent heat treatment at 55°C
compared to non-heat-shocked controls, regardless of the growth atmosphere.
In another study, Linton et al. (1990) heat shocked log phase cells of L. mono-
cytogenes Scott A in trypticase soy broth supplemented with 0.6% yeast extract
(TSYE) at 40, 44, and 48°C for 3, 10, and 20 min, followed by heating at 55°C for
50 min. The optimum heat shock condition for increasing the heat resistance was
48°C for 10 min where D-values at 55°C increased 2.3-fold in nonselective agar
(TSYE) and 1.6-fold in selective agar (McBride Listeria). Cells which were heat
shocked at 48°C for 10 min were consistently more resistant to heating at 50, 55,
60, and 65°C as compared with non-heat-shocked cells. Although D-values increased
due to heat shocking, z-values were not significantly affected regardless of the plating
medium or heat shock.
Juneja et al. (1997), used the submerged coil heating apparatus to determine the
effect of heat shocking E. coli O157:H7, inoculated in a model beef gravy, on the
persistence of the thermotolerance at 4, 15, and 28°C after heat shock. When beef
gravy samples inoculated with a cell culture suspension of a four strain cocktail of
E. coli O157:H7 were subjected to sublethal heating at 46°C for 15 to 30 min,
followed by cooking to a final internal temperature of 60°C, the organism survived
longer than non-heat-shocked cells and the “time to a 4-D (time to inactivate 99.99%
of the population) inactivation” value at 60°C increased 1.56-fold. In this study, a
linear decline in the log number of survivors with time was observed. The induction
of thermotolerance by heat shock was maintained for at least 48 h at 4, 15, or 28°C
(Table 2.1). However, when a similar study was conducted in bags of ground beef
heated using a water bath, the primary thermotolerance response of E. coli O157:H7
switched to non-linear inactivation kinetics resulting in the presence of a shoulder
(Juneja et al. 1997). The T4D values at 60°C increased 1.50-fold in ground beef.

TABLE 2.1
The Effect of Prior Exposure of Escherichia coli
0157:H7 in Beef Gravy to 46°C Followed by Storage
at 4, 15, or 28°C for 6 to 48 h on the Microorganism’s
Time to a 4-D Inactivation (T4D)ab at 60°C
Storage Time after Heating T4D (min) at Temperatures (°C)c

(h) 4 15 28
6 3.91 3.44 3.78

14 3.14 3.11 3.39
24 3.06 3.17 4.07
30 3.48 3.01 3.67
48 3.15 3.29 3.24
a The T4D of non-heat-shocked cells was 2.38 ± 0.2 and was not signifi-
cantly altered (p < 0.05) after storage at 4, 15, or 28°C for up to 48 h.
b The T
4D of cells immediately after heat shocking was 3.73 ± 0.22 min.
c Mean of two replications, each performed in duplicate.
(Adapted from Juneja, V.K. et al., J. Appl. Microbiol., 84, 677, 1997.)
Unlike the beef gravy, it was interesting to note that E. coli O157:H7 cells in beef
lost their thermotolerance after 14 h at 4°C and after 24 h in beef held at 15 or 28°C.
Bunning et al. (1990) heat shocked stationary phase cells of L. monocytogenes
grown at 35°C (control), at 42, 48, and 52°C for 5 to 60 min prior to heating at
57.8°C. Although heat shocking at 42 to 48°C for 5 to 60 min increased D-values
at 57.8°C by 1.1- to1.4-fold, these data were not statistically different from non-
heat-shocked cells. When similar experiments were conducted with S. typhimurium,
D-values increased by 1.1- to 3.0-fold and were significantly different from those
of non-heat-shocked cells. When L. monocytogenes cells were held at 42°C, ther-
motolerance remained at a maximum level for at least 4 h. However, in preheated
cells incubated at 35°C the increased thermal tolerance lasted less than 1 h.
Heat stress interacts with growth atmosphere in increasing the heat resistance
of E. coli O157:H7. In the previously mentioned study by Murano and Pierson
(1992), when log phase cells of E. coli O157:H7 grown either aerobically or anaer-
obically in trypticase soy broth (TSB) at 30°C were subjected to a heat shocking at
42°C for 5, 10, or 15 min before final heating at 55°C, D-values increased by more
than 2-fold for aerobically grown cells, and 1.5-fold when grown under anaerobic
conditions. Interestingly, the D-values at 55°C of anaerobically grown non-heat-
shocked control were significantly higher than those of aerobically grown controls.
It has been reported that anaerobiosis is considered a form of stress to bacterial cells.
Jorgensen et al. (1996) used the submerged coil apparatus, set at 58°C, to assess
the effect of growth temperature and post heat shock incubation temperature on heat-
shock-induced thermotolerance and the persistence of this thermotolerance in
L. monocytogenes. The authors reported that cells grown at 10 or 30°C showed no

TABLE 2.2
Influence of Incubation Temperature and Time after Heat Shock on the
Inactivation of Listeria monocytogenes Cells Grown at 4, 10, or 30°C
Growth Temperature of Time after Time to
Temperature Heat Shock Post-Heat Shock Heat Shock 4 log cfu/ml
of Cells, °C (30 min at 46°C) Incubation (°C) (h) Reductions (min)
4 No — — 3.4
10 No — — 5.4
30 No — — 5.1
4 Yes — 0 19.8
10 Yes — 0 15.0
30 Yes — 0 14.8
4 Yes 4 48 7.1
10 Yes 10 24 6.6
4 Yes 30 4 7.6
10 Yes 30 4 6.5
30 Yes 30 4 7.1
(Adapted from Jorgensen, F. et al., J. Appl. Bacteriol., 79, 274, 1996.)
differences in thermotolerance but were significantly (p < 0.001) more heat resistant
(1.5-fold) than cells grown at 4°C (Table 2.2). In this study, exposing cells grown
at 10 and 30°C to a heat shock resulted in similar increases in thermotolerance, but
this increase was significantly (p < 0.001) higher when cells were grown at 4°C
prior to the heat shock. The effect of growth temperature prior to inactivation had
negligible effects on the persistence of heat-shock-induced thermotolerance. For
example, cells grown at 4, 10, or 30°C showed the same amount of reduction when
held at 30°C after the heat shock.
The degree to which E. coli O157:H7 heat-shocked and non-heat-shocked cells
are injured following a heat process and the ability of injured cells to repair them-
selves under aerobic and anaerobic conditions have been described by Murano and
Pierson (1993). It is known that bacteria encounter stress due to both excess oxygen
and oxygen deprivation (Potter et al., 2000). In the prior study, not only was the
D-value at 55°C of heat shocked cells (42°C/5 min) significantly increased, but the
number of injured cells was also higher in heat-shocked cells than in controls
(Murano and Pierson, 1993). Furthermore, when cells were recovered under anaer-
obic conditions, a higher recovery of injured cells was observed and thus a signifi-
cantly higher D-value as compared with cells recovered aerobically. Interestingly,
this phenomenon was observed regardless of whether the cells were previously heat
shocked or not. This is probably attributable to the spontaneous formation of toxic
oxygen radicals in aerobic media, which heated cells are unable to deactivate due
to the heat inactivation of detoxifying enzymes like catalase and superoxide dismu-
tase. Since anaerobic storage is a practice which is prevalent in the food industry
for shelf-life extension of processed meats, the microbiological safety of such foods
should be of concern because of the enhanced recovery of injured pathogens following

heat treatment. The concern is further magnified if the cells are exposed to heat
stress before heat treatment as the recovery can be enhanced even more.
Linton et al. (1992) assessed the effect of recovery medium in conjunction with
atmosphere (aerobic vs. anaerobic) during recovery on the heat resistance of
L. monocytogenes. These researchers heat shocked log phase cells of L. monocyto-
genes Scott A in TSB supplemented with 0.6% (w/v) yeast extract (TSYE) at 48°C
for 10 min, followed by heating at 55°C for up to 50 min. D-values at 55°C for
heat-shocked cells were 2.1- fold higher than for non-heat-shocked cells on nonse-
lective agar (TSYE) incubated aerobically and similarly 2.2- fold higher for cells
enumerated anaerobically on TSYE agar. On selective medium (McBride Listeria-
ML), the values were 1.4-fold higher as compared with non-heat-shocked cells.
Interestingly, no growth was observed on ML agar incubated anaerobically. Fedio
and Jackson (1989) exposed stationary-phase cells of L. monocytogenes Scott A to
a preheating treatment of 48°C for 1 h in TSYE broth followed by heating at 60°C
for 20 min. Preheating rendered the pathogen more resistant, and a 4 log10 higher
number of cells were recovered as compared to non-heat-shocked cells regardless
of the recovery medium (selective or nonselective).
Increases in D-values (up to 22% compared to the control) for S. enteritidis fol-
lowing heat shock (42°C for 60 min) were reported by Xavier and Ingham (1997). This
study suggested that: (1) short-term temperature abuse of foods containing S. enteritidis
may render the cells more resistant to subsequent heat treatments; (2) anaerobic
microenvironments may enhance survival of heat-stressed cells (i.e., increases in
D-values up to 28% compared to the aerobic value); and (3) heat shock results in the
overexpression of proteins that may be related to increased thermotolerance.
Heat-shock-induced thermal resistance conditions may be encountered in min-
imally processed, cook-chill processed foods of extended durability such as sous-
vide foods, in which there is a current increased interest. Slow heating rate/long
come-up times and low heating temperatures employed in the production of sous-
vide cooked foods expose the microbial cells to conditions similar to heat shock,
with the possibility of rendering these cells more thermal resistant. Stephens et al.
(1994) and Kim et al. (1994) have shown that slowly raising the cooking temperature
enhanced the heat resistance of L. monocytogenes in broth and pork, respectively.
Hansen and Knochel (1996) found no significant difference between slow (0.3 to
0.6°C/min) and rapid (>10°C/min) heating and the heat resistance of L. monocyto-
genes in low pH (<5.8) sous-vide cooked beef prepared at a mild processing tem-
perature. However, the latter authors did observe an increase in the heat resistance
of L. monocytogenes in higher pH (6.2) sous-vide beef.
Tsuchido et al. (1982 and 1984) increased the thermotolerance of E. coli by
raising the temperature of the cell suspension from 0 to 50°C at various rates prior
to holding at 50°C. Also, Thompson et al. (1979) increased the thermotolerance of
S. typhimurium in beef under realistic conditions of constantly rising temperature.
Subsequently, Mackey and Derrick (1987) reported that the heat resistance of
S. typhimurium, measured as survival following a final heating at 55°C for 25 min,
increased progressively as cells were heated during linearly rising temperatures. In
that study, cells were heated at a rate of 0.6 or 10°C per min from 20 to 55°C, and
then subjected to a heat challenge at 55°C for 25 min. The authors reported that the

extent of induced thermotolerance was inversely related to the rate of heating, i.e.,
the slower the temperature rise, the greater the increase in resistance. Quintavala
and Campanini (1991) determined the heat resistance of L. monocytogenes 5S heated
at 60, 63, and 66°C in a meat emulsion at a rate of 5°C/min compared to instanta-
neous heating. D-values of cells heated slowly were two-fold higher than the cells
heated instantaneously at all heating temperatures.
Cell Membrane Adaptations
Cellular targets for heat damage are ribosomes, nucleic acids, enzymes, and proteins
(Abee and Wouters, 1999). Mild heat treatment can lead to modification of the cell
membrane by increasing the saturation and length of the fatty acids in order to
maintain optimal fluidity of the membrane as well as activity of intrinsic proteins
(Russell and Fukanaga, 1990).
In a study on the physiological state of cell membranes from Gram-negative
bacteria, the total saturated fatty acids (SFA) and total unsaturated fatty acids (UFA)
were highly influenced by temperature (Dubois-Brissonnet et al., 2000). When the
temperature was increased from 15 to 40°C, SFA increased from 25 to 39%, whereas
UFA decreased from 66.5 to 51% (Dubois-Brissonnet et al., 2000). An increase in
unsaturation would be expected to contribute to membrane fluidity, especially at
lower temperatures (Russell et al., 1995).
Cross Protection
Stress-adapted bacteria are capable of resisting similar (homologous) or different

(heterologous) stresses. Termed “cross protection,” exposure to one stress alters
resistance to another. This cross protection has been shown to be mediated by the
rpoS gene, alluded to earlier. For example, heat shocking renders bacteria more
resistant to treatments other than heat. Wang and Doyle (1998) reported that sublethal
heat treatment of E. coli O157:H7 cells substantially increased their tolerance to
acidity. Lou and Yousef (1996) examined the effect of sublethal heat (45°C/1 h) on
the resistance of exponential phase cultures of L. monocytogenes to certain environ-
mental stresses and found that this greatly increased resistance of the pathogen to
normally lethal doses of hydrogen peroxide, ethanol and NaCl, etc. As a consequence
of stress-induced cross-protection, Lou and Yousef (1996) introduced the “stress
hardening” concept as a component of the “hurdle” concept, stating that stress
hardening may counterbalance the benefits of the hurdle approach. In L. monocyto-
genes, stress induced with heat does not lead to acid tolerance, but cells induced
with low pH do become more tolerant of heat, salt concentration, and antimicrobial
peptide (Hill and Gahan, 2000).
All of the different cross protection adaptations of specific pathogens of concern
must be taken into account when assessing the microbial safety of different combi-
nations of minimal food processing technologies. Komatsu et al. (1990) showed that
exposure of yeast cells to a heat shock conferred protection against freezing in liquid
nitrogen. Additionally, it was found that carbon starvation in E. coli elicits an
essential need for dnaK expression in order to acquire heat and oxidation resistance

(Rockabrand et al., 1998). Further research is needed to determine whether stresses
elicit universal or specific adaptations in the different foodborne pathogens.
Management Strategies
Strategies for control of microorganisms are effective when they overcome, tempo-
rarily or permanently, the various homeostatic reactions that microorganisms have
evolved to resist stress (Gould et al., 1995). Researchers have reported that food
handling conditions should be optimized for maximum effectiveness of cooking
treatments. For example, storage of foods at low temperatures may affect the
response of pathogens such as E. coli O157:H7 to sublethal stresses. E. coli O157:H7
has been reported to be resistant to freezing in ground beef (Pandhye and Doyle,
1992) and chicken meat (Conner and Hall, 1996).
Jackson et al. (1996) reported that the heat resistance of E coli O157:H7 in a
nutrient medium and in ground beef patties was influenced by storage and holding
temperatures. Cultures stored frozen (–18°C) without holding at elevated tempera-
tures had greater heat resistance than those stored under refrigeration (3°C) or at
15°C, perhaps due to physiological changes within the bacterial cell as a result of
freezing (Jackson et al., 1996). Another study (Katsui et al., 1982) showed that the
exposure of non-heat-shocked E. coli to 0°C before heating significantly increased
the heat sensitivity of the exposed cells. Juneja et al. (1997) reported that the heat
resistance of non-heat-shocked cells of E. coli O157:H7 inoculated in ground beef
was not altered after storage at 4°C for 48 h. The heat-shock response and exhibition
of increased thermotolerance is rapidly lost upon chilling and rewarming of cells.
Williams and Ingham (1997) refuted the hypothesis that short-term temperature
abuse significantly increased the heat resistance of E. coli O157:H7 in ground beef.
HIGH HYDROSTATIC PRESSURE

Elevated pressure manifests a variety of detrimental effects on microbial physiology
and viability, including inhibition of protein and DNA synthesis, membrane-associ-
ated processes, and disruption of macromolecular quaternary structure (Somero,
1992; Yayanos and Pollard, 1969). Growth of microorganisms is generally inhibited
at pressures in the range of 20 to 130 MPa, whereas higher pressures between 130
and 800 MPa could result in cell death (Abee and Wouters, 1999).
Welch et al. (1993) found that the stress responses of E. coli to high hydrostatic
pressure and cold temperature share some common features. In that study, the authors
monitored the proteins induced by high pressure in E. coli by analyzing the biochem-
ical and physiological responses to elevated pressure. They reported that exposure of
E. coli to high hydrostatsic pressure induces a unique stress response which results
in high levels of cold-shock proteins (CSPs) and heat-shock proteins (HSPs), as well
as other proteins which appear only in response to high pressure. Increased pressure
has been shown to result in enhancement of proteolytic activity by extracellular
proteases and fatty acid desaturases in membrane systems (Lanciotti et al., 1997).
Pressure-induced rates of elevated protein synthesis may result from the induction
of ribosomes and the state of the ribosome at the time of exposure to high pressure.

Both high pressure and high temperature can destabilize the quarternary structure of
proteins (Jaenicke, 1981). It is presumed that an increased proportion of dissociated
subunits could induce a δ32 factor-dependent heat-shock response (Craig and Gross,
1991). Alternatively, high pressure may affect the phosphorylation state or ATPase
activity of the HSP, DnaK, which in turn could also modulate the heat shock response
(McCarty and Walker, 1991). It would be interesting to examine cross tolerances to
determine if a bacterial stress response to high pressure or cold temperature confers
resistance to the other, and whether such cross-protective resistance would be relevant
to the cold temperature storage or the hydrostatic pressure processing of foods.
The food industry requires a better understanding of the kinetics and mechanism
of pressure inactivation before adoption of pressure-based preservation processes.
E. coli can acquire high levels of resistance to pressure killing by spontaneous muta-
tion (Hauben et al., 1997). The authors used alternating cycles of exposure to high
pressure and outgrowth of surviving populations to select for highly pressure-resistant
mutants of E. coli MG1655. Three barotolerant mutants (LMM1010, LMM1020 and
LMM1030) were isolated independently by using outgrowth temperatures of 30, 37,
and 42°C. Survival of these mutants after pressure treatment for 15 min at ambient
temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival
of the parent strain decreased from 15% at 220 MPa to <0.1% at 700 MPa. Two of
the three mutants (LMM1020 and LMM1030) also exhibited higher heat resistance,
expressed as increased D-values at 58 and 60°C, and lower z-values compared to
those for the parent strain. Interestingly, the ability of the mutants to grow at mod-
erately elevated pressure (50 MPa) was reduced at temperatures above 37°C, sug-
gesting that resistance to pressure inactivation in these mutants is unrelated to baro-
tolerant growth. The generation of increased pressure-resistant mutants questions the
safety of high pressure food processing, and may have significant implications for
the successful application of high pressure processing in food preservation.
Another investigation which examined the potential for high pressure-resistant
mutants of E. coli to survive high pressure treatment (500 MPa) of fruit juices and
low-pH buffers also indicated increased pressure resistance in mutants when com-
pared to the parent strain (Garcia-Graells et al., 1998). However, surviving popula-
tion densities declined considerably during subsequent storage of the pressure-treated
juices at 8°C. This finding suggests exposure to high pressure sublethally injures
the bacterial cells, thereby increasing sensitivity to low pH. It can be argued that
ultrahigh pressure can inactivate microorganisms without compromising the quality
of the food (Bower and Daeschel, 1999). However, sporeformers are known to exhibit
enhanced pressure resistance; therefore it is recommended that high pressure tech-
nologies be used in combination with other treatments to be truly effective (Bower
and Daeschel, 1999).
DEHYDRATION
RESTRICTING WATER ACTIVITY
Water activity (aw) is a measure of the free unbound water molecules available for
metabolic reactions. Microorganisms are capable of growth within a very limited

TABLE 2.3
Effect of NaCl Treatment during Growth and Heating on
the Heat Resistance of Listeria monocytogenes at 60°C
Treatment NaCl Content (moles/ml) Time to 4-D

to NaCl Growth Medium Heating Menstruum Inactivation
Control 0.09 0.09 1.6

Up-shock 0.09 0.50 2.0
0.09 1.00 4.6
0.09 1.50 13.2
Adaptation 0.50 0.50 2.5
1.00 1.00 7.4
1.00 1.00 7.4
1.50 1.50 38.1
1.50 0.09 3.8
(Adapted from Jorgensen, F. et al. J. Appl. Bacteriol., 79, 274, 1995.)
range of aw values specific for that microbe. Solutes such as NaCl or sugars are
frequently used to lower or control aw levels to prevent pathogen growth in processed
foods. Another available method is the physical freeze-drying of foods in combina-
tion with the use of preservatives.
Jorgensen et al. (1995) used the submerged coil heating apparatus to determine
the effect of osmotic up-shock and down-shock, and osmotic adaptation using
different levels of NaCl on the corresponding changes in thermotolerance of
L. monocytogenes. In this study, subjecting cells to an osmotic down-shift (1.5 to
0.09 mol/ml) caused a rapid loss of thermotolerance rendering cells ten-fold more
heat sensitive than cells grown and heated in TPB containing 1.5 mol/ml NaCl
(Table 2.3). Subjecting cells grown in media containing 0.9 mol/ml NaCl to a short
osmotic up-shock in media containing 0.5, 1.0 or 1.5 mol/ml NaCl resulted in 1.3,
2.5 and 8-fold increases in thermotolerance, respectively. When cells were allowed
to adapt to high salinities, an additional two- to three-fold increase in thermotolerance
occurred compared to cells subjected to an osmotic up-shock at the equivalent level
of NaCl. Thus, varying degrees of physical dehydration would lead to enhanced
thermotolerance of the foodborne pathogen. The increased thermotolerance observed
during the extended exposure to high salinities might be associated with the degree
to which the cells have undergone deplasmolysis and accumulated compatible sol-
utes, i.e., the concentration and composition of intracellular solutes. According to
Piper (1993), increased thermotolerance could be a result of increased structurization
of the intracellular water. This mechanism could be linked with the enhanced ther-
mostability of ribosomal contents known to occur by both osmotic dehydration and
heat shock in L. monocytogenes (Stephens and Jones, 1993).
Foods contain a broad range of osmoprotectants, such as glycine betaine and
carnitine, which L. monocytogenes can scavenge and use to regulate osmotic stress

(Gutierrez et al., 1995; Smith, 1996). It may very well be possible to control growth
of pathogens in foods by creating environments free from osmoprotectants provided
that the physical act of dehydration does not cross-protect the microorganism against
other types of food processing physical stresses.
FREEZING
The increased use of freezing temperatures to minimize growth of spoilage organ-
isms and to control pathogens has resulted in increased recent attention to microbial
adaptations to freeze conditions. Bacterial adaptation to low temperature (cold shock)
is thought to involve modification of membrane lipid composition for the purpose
of maintaining optimum membrane fluidity in a process called homeoviscous adap-
tation (Hazel and Williams, 1990). Survival of the foodborne pathogen L. monocy-
togenes in low temperature environments and high salt concentrations is attributed
to the accumulation of the osmoprotectants glycine betaine and carnitine (Sleator
et al., 2001). Low temperature growth requires, in addition to membrane fluidity,
mechanisms for regulating the uptake or synthesis of solutes and the maintenance
of macromolecular structural integrity of ribosomes and other components important
for gene expression and metabolism (Wouters et al., 2000). Proteins (7 kDa) pro-
duced in response to temperature downshock or sudden decrease in temperature are
known as cold-shock proteins (CSPs). These proteins share greater than 45% amino
acid similarity in a variety of foodborne pathogens including E. coli (Goldstein et al.,
1990), B. subtilis (Willimsky et al., 1992), B. cereus (Mayr et al., 1996), S. enteritidis
(Jeffreys et al., 1998), and S. typhimurium (Craig et al., 1998).
In order to examine the adaptive response to cold shock in Vibrio vulnificus, a
culture of the microorganism was shifted from 35 to 6°C with the resultant transition
of the bacterium to a viable, but non-culturable state (Bryan et al., 1999). Cultures
which were first adapted to 15°C prior to the 6°C downshift cold shock remained
viable and culturable (Bryan et al., 1999). The exposure to 15°C was found to be
necessary for the cold adaptation. Additionally, iron was found to be necessary for
the stress adaptation to chill temperatures as addition of the iron chelator, 2,2′-
dipyridyl, to the culture prior to cold stress resulted in a decrease in culture viability
by 2 log10 following cold adaptation (Bryan et al., 1999).
Goldstein et al. (1990) reported that when E. coli cells grown at 37°C were
frozen and thawed following preincubation at 10°C for 6 h, there was a 70-fold
increase in survival compared to frozen and thawed cells that were not preincubated
at 10°C. Willimsky et al. (1992) replaced a functional cspB gene on the B. subtilis
chromosome with an interrupted copy of the gene, and then compared the effects
of freezing on cell viability of the parent and mutant strain. In this study, freezing
at –80°C for 24 h after incubation at 37°C resulted in the survival of 27% of the
parent cells and only 2% of the mutant cells. However, preincubation at 10°C for
2 h prior to freezing increased the survival of both the parent and mutant, thus
partially compensating for the lack of CspB in the mutant strain. These results
suggest that bacteria that are cold shocked, or adapted to cold temperatures, prior
to freezing, are more likely to survive.

TABLE 2.4
Cold Shock Effects on Thermal Inactivation at 60°C of Listeria
Strains Grown to Stationary Phase at 37°C
Strain
L. monocytogenes L. monocytogenes
D-values (min) L. innocua (Scott A) (V7)
Control 1.44 ± 0.06 1.27 ± 0.09 1.31 ± 0.03

Cold shock (0°C/3 h) 1.08 ± 0.03 0.76 ± 0.05 0.88 ± 0.02
% D-value decrease 25 40 33
(Adapted from Miller, A.J. and Eblen, B.S., Proc. 43rd Int. Congr. Meat Sci. Technol.,
Auckland, NZ, 1997.)
TABLE 2.5
Post-Cold-Shock Thermotolerance, Expressed
as D-Values, of Listeria monocytogenes Grown
at 37°C to Different Growth Phases
Growth Phase Cold Shock Temperature

of 37°C Cells Control 15°C 0°C
Lag 0.83 ± 0.05 0.60 ± 0.08 0.58 ± 0.09

Exponential 0.79 ± 0.043 0.74 ± 0.04 0.75 ± 0.05
Stationary 1.27 ± 0.09 0.75 ± 0.05 0.74 ± 0.00
(Adapted from Miller, A.J. and Eblen, B.S., Proc. 43rd Int. Congr.
Meat Sci. Technol., Auckland, NZ, 1997.)
In a study by Miller and Eblen (1997), the submerged coil heating apparatus was
used to determine if L. monocytogenes is more vulnerable to heating after a cold shock.
In the model system, cultures were cold shocked by a temperature down-shift from
37 to 15 or 0°C for 0, 1, and 3 h. Cold-shocked and control samples were then evaluated
for thermal resistance at 60°C. Heated samples were collected in 1 ml portions, plated
onto a non-selective medium (brain–heart infusion agar) to allow recovery of both
heat-injured and non-injured cells, then enumerated after a 36 h incubation at 37°C.
The results indicated that the cells grown at 37°C to stationary phase, cold shocked
at 0°C for 3 h, then heated at 60°C, exhibited lower D-values as compared to control
cells that were not cold shocked (Table 2.4). The decrease in D-values at 60°C ranged
from 25 to 40% for two L. monocytogenes strains and a strain of L. innocua.
In a second experimental series by Miller and Eblen (1997), the effect of cold
shock on thermal resistance (D60-values) of cells grown at 37°C to lag, exponential,
or stationary phase was determined (Table 2.5). Stationary cells were over 50% more
thermally resistant (D60 = 1.27 min), compared to lag and exponential cells, which
had D60-values of 0.83 and 0.79, respectively. When these cells were cold shocked

at 15 or 0°C prior to heating at 60°C, D-values were lowered by 42, 30, and 8%,
compared to non-shocked controls for stationary, lag, and exponential cells, respec-
tively. The authors pointed out that the maximum effect was in stationary phase
cells, which most closely simulates cells contaminating food.
PULSED ELECTRIC FIELD

Pulsed electric field is a technique that uses short electric field pulses (ns–ms) to
permeabilize microbial membranes and can lead to death from an inability to main-
tain pH gradients necessary for cellular functions (Sale and Hamilton, 1967). This
process is effective in creating pores in the cytoplasmic membrane dependent upon
the intensity of the electric field and the number of pulses applied (Castro et al.,
1993).
The ability to inactivate L. monocytogenes has been measured, along with loss
of cellular membrane integrity by leakage of UV absorbing materials into culture
supernatants (Russell et al., 2000). Pulsed electric field may have limited application
with regard to spore-formers; it may be effective against non-spore-formers when
combined with other physical or chemical treatments. Cross protection is still being
evaluated with this novel technology, as are the detrimental effects on food products.
IRRADIATION
ULTRAVIOLET IRRADIATION
Some physical preservation treatments may be best when applied in combination
with other technologies. Ultraviolet irradiation (254 nm) can cause cumulative dam-
age to microbial DNA (Bower and Daeschel, 1999). Sublethal UV irradiation leads
to the induction of numerous proteins as well as increased protection against heat
(Duwat et al., 2000). Strains of E. coli have been found to become UV resistant.
Although the methodology is effective in decreasing cell numbers, it does not result
in complete sterilization and therefore cannot be recommended as a definitive process
to sanitize foods by itself (Bower and Daeschel, 1999).
GAMMA IRRADIATION
It is known that Gram-negative bacteria are more sensitive to gamma irradiation
than Gram-positive bacteria, such as lactobacilli (Tiwari and Maxcy, 1971). Lacto-
bacillus sake exhibits gamma irradiation resistance with enhanced effects under
nitrogen packaging (Bower and Daeschel, 1999). Medium doses of gamma irradia-
tion (1.0 to 10.0 kGy or 100 to 1000 krad) reduced or eliminated non-spore-forming
pathogens (Tarkowski et al., 1984).
Ionizing radiation is known to damage pathogen DNA. At temperatures above
freezing, cellular inactivation by DNA disruption and production of hydroxyl radicals
occurs (Buchanan et al., 1999). At freezing temperatures, DNA damage was the
cause of irradiation inactivation and not cellular membrane disruption (Kim and
Thayer, 1996). Detrimental effects of ionizing radiation on food products include

oxidative rancidity of lipids, which can be prevented by vacuum packaging, and a
loss of some minor vitamin components (Farkas, 1987).
Although bacterial spores are more resistant, synergistic effects of gamma irra-
diation and heat may be used to control spore-formers since the heat sensitization
of irradiated spores is not readily repaired (Gombas and Gomez, 1978). Unfortu-
nately, as with other food preservation methods, there is some indication of an acid
(low pH)-induced cross protection against gamma radiation sensitivity in enterohe-
morrhagic E. coli (Buchanan et al., 1999). It is important to be mindful that the
limitations of radiation that may be applied to a particular product are determined
by the organoleptic changes that occur (Grant and Patterson, 1991).
CONCLUDING REMARKS
Strategies for control of foodborne pathogens include physical microbiocidal treat-
ments such as heat, ionizing radiation, cold, dehydration, high hydrostatic pressure,
and pulsed electric field. Heat inactivation of pathogens is the oldest and most
effective food-processing technology in use today. Due to the negative impact on
the quality of certain foods, such as fruits and vegetables, or minimally processed,
refrigerated, ready-to-eat foods, these are subjected to non-thermal cold pasteuriza-
tion alternatives. As these techniques must be applied in intensities and durations
that retain the organoleptic attributes of foods, there is the likelihood that sublethal
doses are used on contaminating pathogenic microorganisms. Consequently, the
pathogens may be stressed and acquire resistance and increased tolerance to subse-
quent homologous or heterologous treatments, thereby compromising the microbi-
ological safety of foods. Researchers have provided sufficient evidence to document
the stress adaptations and increased survival capabilities of the foodborne pathogens,
even though they are transient in duration.
Microorganisms have evolved various homeostatic mechanisms in order to with-
stand the stress imposed by a variety of physically based strategies used for their
control. Interference with homeostasis by selective and logical application of phys-
ical preservation factors remains an attractive area of future research. Combinations
of successive physical food processing technologies may provide the most effective
solutions for processing high quality, microbiologically safe foods. Studies aimed
at providing insight into the physiological and molecular basis of stress response
mechanisms that underlie the physically based control strategies are expected to
shed light on the phenomenon termed “stress hardening.” This area continues to
challenge the efficiency and efficacy of emerging techniques in ensuring the micro-
biological safety of refrigerated foods. Researchers can use the available information
from gene sequence analyses to identify target genes or encoded proteins perceived
to play a role in microbial stress responses. Certainly, an increased understanding
of mechanism and regulation of the way in which the “stress adaptation” protective
mechanisms are triggered and proceed in response to physical control strategies and
other preservation regimes will offer methodical solutions to pathogen control and
increase effective design of novel physical intervention technologies. Thus, the
knowledge will provide information and leads that are essential in guarding against
the pathogens and in the development of microbiologically safe foods.

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Microbial Adaptation
3 to Stresses by
Food Preservatives
P. Michael Davidson and Mark A. Harrison
CONTENTS
Introduction
Resistance Mechanisms
Traditional Antimicrobials
Short Chain Organic Acids
Benzoic Acid
Sorbic Acid
Alkyl Esters of p-Hydroxybenzoic Acid (Parabens)
Resistance to Naturally Occurring Antimicrobials
Summary
References
INTRODUCTION
Food preservatives are chemical compounds added directly to food for the purpose
of extending shelf life and improving food safety. The group of compounds known
as food preservatives includes antioxidants and antibrowning agents in addition to
antimicrobials. Therefore, a more precise term for those compounds used to control
microorganisms is food antimicrobials. Food antimicrobials may be arbitrarily clas-
sified into two groups: traditional or “regulatory approved” and naturally occurring
(Davidson, 2001). The former includes acetic acid and acetates, alkyl esters of
p-hydroxybenzoic acids (parabens), benzoic acid and benzoates, dimethyl dicarbon-
ate, lactic acid and lactates, nitrites and nitrates, sorbic acid and sorbates and sulfites.
The latter includes compounds from microbial, plant and animal sources that are,
for the most part, only proposed for use in foods as antimicrobials. A few, including
lactoferrin (FDA, 2001), lysozyme (Federal Register, 1998), nisin (21CFR
184.1538), and natamycin (21CFR 172.155) are approved in the United States and
certain other countries for use in selected foods.

Throughout the ages, food antimicrobials have been used primarily to prolong
shelf life and preserve quality of foods through inhibition of spoilage microorgan-
isms. In the past 10 to 15 years, antimicrobials have been increasingly relied upon
to inhibit or inactivate pathogenic microorganisms in foods.
While food antimicrobials have been in use since ancient times, few are used
exclusively to control the growth of specific foodborne pathogens. An exception is
nitrite, which has been used for hundreds of years to inhibit growth and toxin
production of Clostridium botulinum in cured meats in association with salt, ascor-
bate and erythorbate, and low pH. More recently, other antimicrobials have been
applied to foods against foodborne pathogens. For instance, organic acids (e.g., lactic
acid, acetic acid) have been employed as spray sanitizers against pathogens on beef
carcasses. Organic acid salts (e.g., sodium lactate, sodium diacetate) have been added
to processed meats to inactivate pathogens (primarily Listeria monocytogenes)
(9CFR 424.21). Finally, nisin and lysozyme are approved for use in pasteurized
process cheese as a safeguard against growth and toxin production by C. botulinum.
In most instances, the antimicrobial is part of a multiple intervention system that
involves the chemical along with environmental (extrinsic) and food related (intrin-
sic) stresses and processing steps. This has been termed “hurdle technology” or
multiple interventions (Leistner, 2000; Leistner and Gorris, 1995).
Some food antimicrobials have been used for thousands of years (e.g., sulfites,
some organic acids) while most have been added to foods for the purpose of
preservation for 100 years or less. Surprisingly, there are few data in the literature
concerning developed resistance by microorganisms to these chemical compounds.
Considering the length of time these compounds have been in use, that could indicate
that resistance development has not been (and therefore is not) a major problem.
However, there is concern about use of food antimicrobials for two major reasons.
First is the increasing development and spread of therapeutic antibiotic-resistant
microorganisms in the environment. Second is the new paradigm of using food
antimicrobials as exclusive or primary methods for protection of foods against
growth of, or presence of, foodborne pathogens. If traditional and natural food
antimicrobials are to be a major part of the control system for inhibiting foodborne
pathogens, we need to be well aware of the resistance factors that these microor-
ganisms might possess.
RESISTANCE MECHANISMS
Resistance responses of microorganisms to antimicrobials may be classified as either
innate or acquired (Russell, 1991). Innate resistance is a chromosomally controlled
property associated with the microorganism. Innate resistance is demonstrated by
differences in resistance among related genera, species or strains of microorganisms
under identical conditions of exposure. Because food antimicrobials are generally
broad spectrum, they do not trigger specific microbial responses and, therefore,
resistance is most likely due to unspecified reduced uptake controlled primarily by
innate characteristics (Russell et al., 1997). Mechanisms may include barriers such
as outer membrane of Gram-negative bacteria, teichoic acids of Gram-positive bac-
teria, efflux or pumping of the compounds and inactivation via enzymes. In food

application of antimicrobials, innate resistance may be influenced by environment,
food component interactions, processing interactions or presence of antagonistic
inhibitors. All of these factors may contribute to a microorganism’s resistance profile.
Resistance caused by these factors may be more properly termed “apparent.”
Acquired resistance results from genetic changes in the microbial cell through
mutation or acquisition of genetic material. For example, resistance to therapeutic
antibiotics can include enzymatic inactivation or modification, absence of enzyme
or metabolic step, impaired uptake, efflux, modification of target site, bypass of a
sensitive step, or overproduction of the target (Russell et al., 1997). Acquired resis-
tance is of greatest concern for use of food antimicrobials. While acquired resistance
to antimicrobials is rare (Russell et al., 1997), investigations into the potential for
such resistance are of utmost importance to the future use of traditional and naturally
occurring antimicrobials
TRADITIONAL ANTIMICROBIALS
SHORT CHAIN ORGANIC ACIDS
Short chain organic acids, including acetic acid and its salts (acetates and diacetates),
lactic acid and lactates, propionic acid and propionates and, to a lesser extent, citric
acid and citrates, are commonly utilized in a variety of foods as antimicrobial
preservatives or acidulants (Doores, 1993). They may be added directly to foods or,
in the case of acetic and lactic acids, as sprays or dips for surface decontamination
of fresh meat and poultry. In the undissociated or protonated form, organic acids,
which are weak acids, can diffuse across the cell membrane lipid bilayer. Once
inside the cell, the acid dissociates because the cell interior (pHi) has a higher pH
than the exterior (pHo). Microorganisms maintain pHi near neutrality to prevent
conformational changes to the cell structural proteins, enzymes, nucleic acids and
phospholipids. Protons generated from intracellular dissociation of the organic acid
acidify the cytoplasm and must be extruded to the exterior using energy in the form
of ATP. The lower the pHo, the greater the influx of organic acids. This constant
influx of protons will eventually deplete cellular energy (Bearson et al., 1997).
Resistance by microorganisms to organic acids and/or low pH must be a response
to this mechanism.
Some foodborne pathogens, when exposed to low pH via short chain organic
acids or inorganic acids (e.g., HCl) may undergo changes that provide them with
varying degrees of resistance to subsequent exposure to normally lethal acidic
conditions. This increased resistance to low pH and/or organic acids through pre-
exposure to acidic conditions has no universally accepted terminology but has been
called acid habituation, tolerance and shock. Reviews on the mechanisms of acid
stress and stationary phase responses of foodborne bacterial pathogens were pub-
lished by Rees et al. (1995), Bearson et al. (1997), Abee and Wouters (1999) and
Foster (1999).
Acid habituation may be defined as extended exposure to moderately acid
conditions (pH 4.5-6.0) leading to resistance at low pH (ð 2.5) (Buchanan and
Edelson, 1999; Rowbury, 1995). Acid tolerance response (ATR) is generally defined

as enhanced survival of a microorganism to pH of 2.5 to 4.0 following brief exposure
to moderate (ca. pH 5.0 to 5.5) conditions (Buchanan and Edelson, 1999). ATR may
be divided into stationary phase ATR and log phase ATR (Rowbury, 1995; Bearson
et al., 1997; Foster, 1999). Stationary phase ATR has been attributed to rpoS-regu-
lated and inducible pH-dependent acid resistance responses (Abee and Wouters,
1999; Rowbury, 1995). rpoS controls expression of several genes involved in the
general stress response of Escherichia coli, Salmonella and Shigella, including acid
stress (Abee and Wouters, 1999).
Log phase ATR in Salmonella has two phases, pre-acid-shock and post-acid-
shock. Pre-acid shock involves an “emergency” pH homeostasis system in which
amino acid decarboxylases are induced to consume intracellular protons (Bearson
et al., 1997). Post-acid-shock is controlled by rpoS and includes production of a
number of acid shock proteins that protect and repair cellular macromolecules
(Bearson et al., 1997). Other regulators, including PhoPQ, a two component signal
transduction system, and Fur, a ferric uptake regulator, may also control expression
of several acid shock proteins (Foster, 1999; Bearson et al., 1997). Escherichia coli
stationary phase ATR is apparently distinct from Salmonella and has three inducible
acid resistance systems (Foster, 1999). The glutamate-dependent system which uti-
lizes glutamate decarboxylase to neutralize protons is the dominant system. It is
important to note that Foster (1999) also reports a difference in resistance responses
of Salmonella to inorganic and organic acids. For example, the PhoP system affected
tolerance to inorganic — but not organic — acids in Salmonella Typhimurium.
Therefore, resistance characteristics may differ depending upon whether inorganic
or organic acids are used to induce tolerance responses.
There is no question that tolerance or adaptation to short chain organic acids
exists among foodborne pathogens (Tables 3.1 through 3.3). However, is this impor-
tant to food safety? To have an impact on food safety, acid adaptation or tolerance
has to be induced in foodborne pathogens by conditions present in the current food
processing system; any acid adapted or tolerant foodborne pathogens would have to
possess enhanced survival in foods or food processing systems in which they are
normally inactivated. As to the first point, there are a number of processing systems
that could cause acid adaptation among foodborne pathogens. Direct acidification
of a food or food ingredient may shock microflora so they become more acid
resistant. For example, lactic acid is added to improve flavor and quality in cheese
curd, unsalted butter, egg whites, egg yolks, beer, bread dough, olives, pickles,
relishes, and infant foods containing dried milk (Shelef, 1994). Many other organic
acids are utilized for improved sensory properties and as antimicrobials in a variety
of foods (Doores, 1993). In fermented foods, the situation can be somewhat different.
Lactic acid bacteria lower the pH of a substrate gradually over time so a pH gradient
is more likely, rather than a sharp alteration in the pH as would be expected with
direct acidification. The use of organic acid sprays as a sanitizer for meat carcasses
(Dickson, 1995) could cause acid shock or tolerance of the meat microflora.
Several research studies have demonstrated that acid adaptation or tolerance may
produce pathogens with enhanced survival in fermented foods or foods to which
organic acids have been added. Leyer et al. (1995) found that acid adapted (pH 5.0)

TABLE 3.1
Conditions of Organic Acid or Mineral Acid Adaptation, Tolerance or Shock
Applied to Escherichia coli O157:H7 and Their Subsequent Organic Acid, Food
Antimicrobial or Acid Food Tolerance Responses
Acid Adaptation Conditions Exposure Conditions and Response Reference
pH 5.0 (HCl adjusted), 37°C, 4–5 h Increased resistance to pH 3.85, 125 mM lactic Leyer et al.
acid, 60 min of three strains (1995)
Extent of resistance increase varied by strain
from 1 to 5 logs
Enhanced survival in acid foods, including
sausage fermentation (+2 log at 16 h), salami
(pH 5.0) and apple cider (+4 log at 30 h)
3 strains plus Salmonella, 3 serovars Increased survival of all strains in ketchup at Tsai and
4 h at pH 5.0, 37°C pH 3.6 Ingham
No difference in mustard (pH 3.1) or sweet relish (1997)
(pH 2.8)
1 pathogenic and 1 nonpathogenic Increased resistance to acetic acid, pH 3.5 or 4.0 Brudzinski
strain Extent of resistance dependent upon temperature and
(1) acetic acid, pH 5.0, 1 doubling of exposure, time and strain Harrison
time (1998)
(2) acetic acid, pH 2.5
2 strains and 1 nonpathogenic strain Resistance to sodium lactate up to 30% (w/w) at Garren et al.
lactic acid, pH 5.5, stationary phase pH 4.0, depending on strain (1998)
cells Increased resistance to sodium chloride,
lactic acid, pH 4.0, stationary phase depending upon strain
cells
Stationary phase, acid resistance Increased resistance to 0.5% acetic, lactic, malic, Buchanan
response through growth in TSB + and citric acids at pH 3.0 and Edelson
1% glucose Extent variable depending upon strain (1999)
Lactic acid caused greatest decrease for all
treatments
TSB + glucose (1%) for 18 h Little difference in resistance to lactic, malic, Ryu et al.
acetic, or citric acids at pH 3.9 to 5.4 (1999)
3 strains Little or no difference in resistance to acetic, Deng et al.,
TSB + glucose (1 or 1.25%) for 18 h citric or malic acids at pH 3.9 to 7.2 (1999)
Strains: ATCC 43895, ATCC 43889, 12 strains tested: 6 strains acid resistant (ATCC Berry and
ATCC 43890 strains: 43895, 43894, 35150, 2886-75; 86-24; Cutter
TSB + glucose (1%) for 18 h NADC 5570) to TSB, pH 2.5 (HCl), 6 h, i.e., (2000)
non-adapted cells resistant; 2 strains adaptable
to acid resistance (ATCC 43889, NADC 4477),
to TSB, pH 2.5 (HCl), 6 h, i.e., non-adapted
cells sensitive
Acid resistant strain (43895) and acid adaptable
strain (43889) showed increased resistance to
2% acetic acid spray treatment on pre-rigor beef
carcass tissue compared to unadapted cells

TABLE 3.1 (continued)
Applied to Escherichia coli O157:H7 and Their Subsequent Organic Acid, Food
Acid Adaptation Conditions Exposure Conditions and Response Reference
Strains: ATCC 43889, ATCC 43895 Increased resistance in mango juice (pH 3.2) at Cheng and
pH 5.0 (HCl), 4 h at 37°C 25°C but not at 7°C Chou
Increased resistance in asparagus juice (pH 3.6) (2001)
at end of 25°C storage period (6–14 d) for both
strains, and at end of storage period (12–20 d)
at 7°C for strain 43889 only
Adapted cells less susceptible than unadapted
cells inoculated into yakult (fermented milk
drink, pH 3.6) or low-fat yogurt (pH 3.9)
E. coli O157:H7 had greater survival than unadapted cells in acid foods including
salami and apple cider (Table 3.1). Similarly, Tsai and Ingham found enhanced
survival of E. coli O157:H7 and three Salmonella serovars in ketchup, but not in
mustard or sweet relish. Interestingly, Cheng and Chou (2001) found that, while
acid adapted cells of E. coli O157:H7 (pH 5.0) ATCC 43889 and 43895 were more
tolerant to acidic conditions of mango juice and asparagus juice, both were actually
less tolerant than unadapted cells in yakult (fermented milk drink) and yogurt. They
also found that resistance was dependent upon temperature (lower storage temper-
ature, less difference in resistance) and strain.
Salmonella serovars Typhimurium, Enteritidis, Heidelberg and Javiana that were
pre-exposed to pH 5.8 (HCl) demonstrated increased resistance to the food antimi-
crobials lactic, propionic and acetic acid (Leyer and Johnson, 1992) (Table 3.2). In
addition, they had greater survival than unadapted strains in a milk fermentation and
cheddar, Swiss and mozzarella cheeses. Gahan et al. (1996) demonstrated that
L. monocytogenes LO28 acid adapted by exposing the microorganism to lactic acid
at pH 5.5 for 60 min had enhanced survival in yogurt, cottage cheese, orange juice
and salad dressing (Table 3.3). Little or no enhancement was found with higher pH
foods such as cheddar cheese (pH 5.16) or mozzarella cheese (pH 5.6). Ravishankar
and Harrison (1999) found that L. monocytogenes exhibited an acid tolerance
response (ATR) when it was acid adapted to pH of 5.5 with lactic acid, then
challenged in acidified skim milk at pH 3.5 and 4.0 (Table 3.3). However, when the
challenge pH of 4.5 was used, there was no adaptive ATR. Since pH 4.5 is more
closely related to pH levels that might be attained in fermented products made from
skim milk, results based on this pH should be the most meaningful. In summary,
induced acid tolerance may theoretically cause some pathogens to have enhanced
survival in certain fermented or acidified foods.
In food, numerous product and antimicrobial combinations are possible. Under
certain scenarios, there may be reason for concern when considering whether or not

TABLE 3.2
Applied to Salmonella and Shigella and Their Subsequent Organic Acid,
Food Antimicrobial or Acid Food Tolerance Responses
Exposure Conditions
Microorganism Acid Adaptation Conditions and Responses Reference
Salmonella Typhimurium HCl, pH 5.8, 1–2 doublings Increased resistance to Leyer and
Salmonella Enteritidis lactic, propionic, acetic Johnson
Salmonella Heidelberg acids (1992)
Salmonella Javiana Increased survival in milk
fermentation, and cheddar,
Swiss, and mozzarella
cheeses
Salmonella Typhimurium pH 5.8 (HCl), 1–2 doublings Increased resistance to Leyer and
activated lactoperoxidase Johnson
and NaCl (1993)
Increased cell surface
hydrophobicity
Salmonella Typhimurium, Growth in TSB acidified with Inoculated onto beef and Dickson
2 strains lactic acid at pH 5.0 for 24 h treated with 1.5 or 3.0% and
Salmonella Dublin lactic acid Kunduru
Salmonella Heidelberg No difference in (1995)
susceptibility to lactic acid
by acid tolerant or
unexposed cells
Acid adapted cells more heat
sensitive than unadapted
cells
Salmonella Typhimurium S1 4 transfers, 48–72 h at 30°C, No significant increase in Van Netten
Staphylococcus aureus 147A reduced pH from 6.4 to 5.8 resistance to 2% lactic acid et al.
Escherichia coli O157:H7 with 10% lactic acid decontamination step for (1998)
Campylobacter jejuni C186 2 min on pork skin
Salmonella Typhimurium (1) Short chain fatty acid Mixture (2) and propionate Kwon et al.
mixture 1: acetate 8mM, (3) significantly increased (2000)
butyrate 3 mM, lactate resistance (>3–4 logs) to
14 mM, propionate 2 mM, pH 3.0 for up to 3 h at 37°C
succinate 9 mM in TSB medium
(2) Short chain fatty acid Mixture (1) increased
mixture 2: acetate 70 mM, resistance to pH 3.0
butyrate 26 mM, lactate (ca. 2 logs) for 1 h only
5 mM, propionate 25 mM, in TSB medium
succinate 1 mM, valeate No acid specified for pH
5 mM adjustment
(3) 100 mM propionate
All at pH 7.0, exposure for 1 h
at 37°C

Applied to Salmonella and Shigella and Their Subsequent Organic Acid,
Food Antimicrobial or Acid Food Tolerance Responses
Exposure Conditions
Microorganism Acid Adaptation Conditions and Responses Reference
Shigella flexneri, 3 strains TSB + 1% glucose, 37°C, 18 h Increased resistance to lactic Tetteh and
Exposed to pH 5.05 lactic acid and acetic acid at pH 3.5 for Beuchat
after growth for 16 h in TSB 2 h and 30 min, respectively (2001)
without glucose at 37°C No increase in resistance to
propionic acid at pH 3.5
Very slight increase in
resistance to propionic acid
at pH 4.5
Acid adapted slightly more
resistant than acid shocked
bacteria can acquire some degree of resistance toward a particular antimicrobial. For
example, Kwon et al. (2000) found that S. Typhimurium cells adapted by exposure
to short chain fatty acid mixtures or propionic acid at pH 7.0 had significantly
increased resistance to low pH, compared to unadapted cells (Table 3.2). Pickett and
Murano (1996) exposed L. monocytogenes to sublethal levels of lactic acid, citric
acid, and propionic acid before challenging the cells to minimum inhibitory con-
centrations of each compound under various conditions (Table 3.3). There was no
difference in susceptibility with cells pre-exposed to sublethal levels. While citric
acid did not produce resistant cells at the test pH of 2.8, when the pre-exposure pH
was raised to 5.1, the dissociated form of the acid yielded cells that were able to
survive exposure to lethal levels. Pickett and Murano (1996) suggested that pre-
exposure of the cells to the dissociated form of the acid enabled the cells to survive
a lethal dose. This is supported by the reported conditions required to induce the
acid tolerance response as outlined by Buchanan and Edelson (1999).
Use of organic acid sprays as a sanitizing treatment of meat carcasses has become
very common (Dickson, 1995). Could pathogens on the meat surface become more
acid resistant when exposed to weak acid (e.g., <3%) solutions? One study by Van
Netten et al. (1998) demonstrated a lack of increased resistance to lactic acid for
E. coli O157:H7, S. Typhimurium, Staphylococcus aureus, Campylobacter jejuni
when acid-adapted cells were inoculated on pork bellies and treated with 2% lactic
acid as a sanitizer (Table 3.1). Similarly, Dickson and Kunduru (1995) found no
difference in acid resistance between unadapted cells of S. Typhimurium, Dublin or
Heidelberg and those adapted by exposure to pH 5.0 (lactic acid) on the surface of
beef treated with 1.5 or 3.0% lactic acid (Table 3.2). They also found a lower heat
resistance of adapted cells compared to unadapted cells. In contrast, Berry and Cutter

TABLE 3.3
Applied to Listeria monocytogenes and their Subsequent Organic Acid, Food
Acid Adaptation Exposure Conditions
Strain(s) Conditions and Response Reference
Scott A Incubation at pH 5.4 Significant resistance to pH 3.35 (no Okereke and

(citric acid) acid specified) for 4 h at 35°C Thompson
Acid tolerance No resistance increase to pH 4.35, for (1996)
response not 4 h at 35°C; constitutive acid
inducible at 5.0, 5.8 resistance proposed
or 6.0 Little or no cross protection for
0.3–1.5 µg/ml (12-60 IU/ml) nisin at
pH 6.0, 35°C for 90 min
Scott A (1) Sublethal citric (1) No increased resistance in cells Pickett and
(0.2%), lactic (0.2%), exposed to sublethal concentrations Murano
propionic (0.1%) of organic acids at pH 2.8 (1996)
acids at pH 2.8 for up (2) Increased resistance to lethal
to 60 min at 37°C concentration (0.3%) of citric acid at
(2) Sublethal citric pH 2.8
acid (0.2%) at pH 5.0
LO28 (serotype 1/2c) Lactic acid, pH 5.5, Increased resistance to lactic acid at O’Driscoll
37°C for 60 min pH 3.5, 2 h at 37°C et al. (1996)
(early log phase Hydrochloric acid less effective at
cells) inducing ATR
Growth to stationary phase induced
lactic acid tolerance
LO28 (serotype 1/2c) Lactic acid, pH 5.5, Enhanced survival in cottage cheese Gahan et al.
37°C for 60 min (pH 4.71) and yogurt (pH 3.90) (1996)
(early log phase Enhanced survival during milk
cells) fermentation by lactic acid bacteria
Enhanced survival in orange juice
(pH 3.76) and salad dressing
(pH 3.0)
Marginal or no enhanced survival in
whole-fat cheddar cheese (pH 5.16),
low-fat cheddar (pH 5.25) and
mozzarella cheese (pH 5.6)
Scott A pH 5.5, lactic acid Increased resistance to lactic acid, pH Van Schaik
3.5 for 2 h at 37°C et al. (1999)
Increased resistance to 100-200 IU/ml
nisin and 650 AU/ml lactacin 3147,
pH 5.5 (lactic acid)
Increased C14:0, C16:0 and decreased
C18:0 fatty acids

Applied to Listeria monocytogenes and their Subsequent Organic Acid, Food
Acid Adaptation Exposure Conditions
Strain(s) Conditions and Response Reference
V7, V37, CA (1) Lactic acid in skim Skim milk with lactic acid at: Ravishankar
milk at pH 3.5 or 4.0 pH 3.0 — acid-adapted had greater and
(2) Skim milk survival (0.5–1.0 log); pH 4.0 — Harrison
acidified to 5.5, acid-adapted had greater survival (1999)
transfer to skim milk (3–4 logs); pH 4.5 — no difference
with lactic acid at Lactoperoxidase system at pH 4.5 —
pH 3.5 or 4.0 no difference
N-7155 (1/2b) Stationary phase Exposure to acetic or lactic acid at pH Samelis et al.
N-7144 (1/2b) growth, 24 h at 35°C 2.5 or 3.5 (2001)
No resistance to lactic or acetic acid
at pH 2.5 or acetic acid at pH 3.5
Increased resistance of all strains to
lactic acid at pH 3.5
Increased resistance of most acid
tolerant strains (N-7155,
N-7144Sm+) to lactic acid (pH 3.5),
and 2% acetic acid spray wash water
(pH 3.2) and lactic acid spray wash
water (pH 2.5) from beef
decontamination
Increased acid tolerance by Listeria
monocytogenes in the presence of
viable natural meat microflora was
transient
(2000) showed increased resistance to a 2% acetic acid spray treatment on beef

carcasses by acid-resistant (ATCC 43895) and acid-adapted (ATCC 43889) E. coli
O157:H7 strains (Table 3.1). The differences in these studies are most likely due to
differences in experimental conditions, especially those used to create acid tolerant
cells.
BENZOIC ACID
Benzoic acid and its salts were the first antimicrobials approved for use in foods,
about 1900. Their primary use is as antifungal agents in high acid foods. There are
differences in resistance to benzoates among microorganisms due to innate tolerance.
Since these are antifungal agents, innate resistance to benzoates would be more of
a concern with the target microorganisms, yeasts and molds. Warth (1985) identified
a number of yeasts that grew in the presence of ca. 500 µg/ml benzoic acid including
Schizosaccharomyces pombe and Zygosaccharomyces bailii. Other yeasts, including

Pichia membranefaciens and Byssochlamys nivea, have also been shown to be
naturally resistant to benzoates (Chipley, 1993). Warth (1988) suggested that the
resistance mechanism of yeasts to weak-acid type preservatives, including benzoic
and propionic acids, was related to membrane permeability and the ability of the
cells to continuously remove the preservative. Some microorganisms, including the
bacteria Bacillus, Pseudomonas, Corynebacterium, Micrococcus, and the mold
Aspergillus, are able to metabolize benzoic acid utilizing the β-ketoadipate pathway
to succinic acid and acetyl CoA (Chipley, 1993).
A few studies have examined the potential for acquired resistance to benzoic
acid. Warth (1988) incubated strains of yeast, including Candida krusei, Hansenula
anomala, Kluyveromyces fragilis, Kloeckera apiculata, Saccharomyces cerevisiae,
Saccharomycodes ludwigii, Schizosaccharomyces pombe, and Z. bailii, overnight in
the presence of either 0.25 mM (31 µg/ml) or 2 mM (244 µg/ml) of benzoic acid.
The minimum inhibitory concentration for unexposed cells was significantly lower
than cells exposed to subinhibitory levels of benzoic acid. A 1.4 to 2.2-fold increase
in minimum inhibitory concentration was achieved with pre-exposure. Zygosaccha-
romyces bailii and S. pombe showed the greatest increases. The resistance mecha-
nism proposed was an increased efflux by the adapted cells. There is little or no
evidence of acquired resistance to benzoic acid by bacteria, including acid adapted
cells.
SORBIC ACID
Sorbic acid has been used as an antimicrobial in foods since the 1940s. Innate
resistance to the compound has been demonstrated by bacteria, including catalase-
negative lactic acid bacteria, Sporolactobacillus and some Pseudomonas, yeasts,
including Z. bailii, Saccharomyces, Torulopsis, Brettanomyces and Candida, and
molds, including Aspergillus, Penicillium, Fusarium, Geotrichum, and Mucor (Sofos
and Busta, 1993). As with benzoic acid, some microorganisms can metabolize sorbic
acid. Molds isolated from cheese, including seven Penicillium species, were shown
to grow in the presence of and degrade 0.3 to 1.2% sorbate (Finol et al., 1982).
Penicillium puberulum and P. cyclopium were the most resistant species evaluated.
Marth et al. (1966) demonstrated that Penicillium species isolated from cheese
degraded sorbic acid and produced 1,3 pentadiene, which was volatile and had a
kerosene off-odor. Sorbic acid has been shown to be degraded by Mucor species to
4-hexenol and Geotrichum species to 4-hexenoic acid and ethyl sorbate (Liewen and
Marth, 1985). High numbers of lactic acid bacteria can produce compounds such as
ethyl sorbate, 2,4-hexadien-1-ol, 1-ethoxyhexa-2,4 diene, 5-hexadien-1-ol, and
2-ethoxyhexa-3,5 diene in sorbic acid-treated red wine (Liewen and Marth, 1985).
This can result in geranium off-odors in wines and fermented vegetables which have
been attributed to the 2,4 hexadien-1-ol (Liewen and Marth, 1985; Sofos and Busta,
1993).
There is evidence that certain yeasts may acquire resistance to sorbic acid. Warth
(1977) found that Z. bailii grown in the presence of 224.2 µg/ml acquired resistance
to sorbic acid. Bills et al. (1982) investigated acquired resistance with an osmotol-
erant yeast, Saccharomyces rouxii. The yeast was pre-conditioned by growth in the

presence of 0.1% sorbic acid for four transfers. Pre-exposure significantly increased
resistance (shorter lag times, shorter time to stationary phase) of the cells to subse-
quent exposures of 0.1% sorbic acid in microbiological medium and chocolate sauce.
The mechanism proposed for the resistance acquired by the yeasts is an inducible,
energy-requiring system that increases efflux of the sorbic acid (Warth, 1977; Bills
et al., 1982). Yeast resistance to sorbic acid and other weak acids probably involves
several components (Brul and Coote, 1999). First, a H+-ATPase utilizes energy to
remove excess protons from the cell. A membrane protein may also be induced,
which can decrease the activity of the ATPase to conserve cellular energy pools.
Finally, Piper et al. (1998) found that exposure of Saccharomyces cerevisiae to sorbic
acid strongly induces the membrane protein ATP-binding cassette transporter Pdr12,
a multi-drug resistance pump. Mutants without the transporter are hypersensitive to
sorbic, benzoic, and propionic acids. Further, Piper et al. (1998) stated that Pdr12
conferred resistance by mediating energy-dependent extrusion of anions. To prevent
a futile cycle of allowing the acid back into the cell, adapted yeasts reduce diffusion
of weak acids, most likely, by altering cell membrane structure to reduce passage
of the acids into the cell (Brul and Coote, 1999).
There is little information on acquired resistance to sorbic acid among bacteria
or molds. Schroeder and Bullerman (1985) found little or no increase in the resistance
of Penicillium digitatum or P. italicum when exposed to increasing levels of sorbic
acid. Considering the length of time that sorbic acids (and benzoic acid) have been
applied to food products would indicate that the development of acquired resistance
by spoilage or pathogenic microorganisms is virtually non-existent.
ALKYL ESTERS OF P-HYDROXYBENZOIC ACID (PARABENS)

Moir and Eyles (1992) compared the effectiveness of methyl paraben and potassium
sorbate on the growth of four psychrotrophic foodborne bacteria, Aeromonas hydro-
phila, L. monocytogenes, Pseudomonas putida and Yersinia enterocolitica. Little or
no adaptation was found to occur when cells were exposed to subinhibitory concen-
trations of antimicrobials. Bargiota et al. (1987) examined the relationship between
lipid composition of S. aureus and resistance to parabens. Differences were found
for total lipid, phospholipids and fatty acids between S. aureus strains which were
relatively resistant and sensitive to parabens. The paraben-resistant strain was shown
to have a higher percentage total lipid, higher relative percentage of phosphatidyl
glycerol and decreased cyclopropane fatty acids than sensitive strains. It was sug-
gested that these changes could influence membrane fluidity and, therefore, adsorp-
tion of the parabens to the membrane.
Juneja and Davidson (1993) altered the lipid composition of L. monocytogenes
by growth in the presence of added fatty acids (C14:0, C18:0 or C18:1). Growth of
L. monocytogenes in the presence of exogenously added C14:0 or C18:0 fatty acids
increased the resistance of the cells to parabens. However, growth in the presence
of C18:1 led to increased sensitivity to the antimicrobial agents. Results indicated
that, for L. monocytogenes, a correlation existed between lipid composition of the
cell membrane and susceptibility to antimicrobial compounds.

RESISTANCE TO NATURALLY
OCCURRING ANTIMICROBIALS
Naturally occurring antimicrobials are being studied extensively as alternatives to
the so-called “synthetic” traditional antimicrobials. Naturally occurring antimicro-
bials come primarily from three sources: animals, plants and microorganisms. Ani-
mal sources include milk (lactoperoxidase system and lactoferrin) and eggs
(lysozyme). Plant sources include herbs and spices (extracts and essential oils),
onions and garlic (sulfur compounds), and the mustard family (isothiocyanates).
Microbial products with antimicrobial activity include bacteriocins from Lactococ-
cus, Pediococcus, Lactobacillus, Leuconostoc, Carnobacterium, Propionibacterium
and fermentation products.
There are innate differences in resistance among microorganisms to plant and
animal antimicrobials. For example, lysozyme (1,4-β-N-acetylmuramidase) is an
enzyme present in biological secretions that catalyzes hydrolysis of the β-1,4 gly-
cosidic bonds between N-acetylmuramic acid and N-acetylglucosamine of the pep-
tidoglycan of bacterial cell walls. Lysozyme is most active against Gram-positive
bacteria, most likely because of the exposed peptidoglycan in the cell wall. Lysozyme
is less effective against Gram-negative bacteria due to a reduced peptidoglycan
content and presence of outer membrane of lipopolysaccharide and lipoprotein.
However, there seem to be few studies on directly acquired resistance to natural
antimicrobials from animal and plant sources.
Some researchers have investigated the potential for cross-protection to natural
antimicrobials afforded foodborne pathogens through acid adaptation or acid shock.
Ravishankar and Harrison (1999) conducted experiments to determine if acid adap-
tation of L. monocytogenes enhanced survival in the presence of an activated lacto-
peroxidase system by means of cross-protection (Table 3.3). The lactoperoxidase
system occurs in raw milk and involves the enzyme lactoperoxidase, thiocyanate and
hydrogen peroxide. The survival rates were similar for the acid adapted and non-
adapted cells at pH 4.5, both in the presence and absence of an activated lactoperox-
idase system, indicating no cross-protection. In contrast, Leyer and Johnson (1993)
reported cross-protection against an activated lactoperoxidase system with acid
adapted S. Typhimurium when tested in a laboratory culture medium (Table 3.2).
Since the activity of the lactoperoxidase system can vary depending on the medium,
this may explain the contrasting result. Another factor could be related to the greater
degree of acid tolerance exhibited by Salmonella compared to Listeria. The lactop-
eroxidase system may also be less effective as an antimicrobial toward Salmonella.
In contrast to plant and animal sources, there has been significant research on
microbially derived antimicrobials. The probable reason for this is that these com-
pounds have distant similarities to therapeutic antibiotics, about which there is much
concern due to resistance development. However, they generally have a much nar-
rower spectrum and often have different mechanisms, reducing chances for resistance
development.
Two microbially derived antimicrobials that have been evaluated for develop-
ment of acquired resistance are natamycin and nisin. Natamycin (formerly called

pimaricin) is a polyene macrolide antibiotic produced by Streptomyces natalensis
that is effective against nearly all molds and yeasts but has little or no effect on
bacteria. Its primary use is as an antifungal agent on cheese. De Boer and Stolk-
Horsthuis (1977) investigated the potential for development of resistance to nata-
mycin among fungi. They found no evidence of resistant fungi in cheese warehouses
in which natamycin had been used for various periods of time, up to several years.
They also attempted to induce tolerance in 26 strains of fungi by transferring each
culture 25 to 31 times in media containing concentrations of natamycin equal to and
higher than the minimum inhibitory concentration (MIC). The MIC following mul-
tiple transfers increased in only 8 of 26 strains and by a maximum of 4 µg/ml. They
concluded that the lack of increased resistance was due to strongly fungicidal activity
of the compound along with its environmental instability.
In contrast to natamycin, nisin resistance is known. Nisin is a peptide produced
by a Lactococcus lactis ssp. lactis strain. Nisin has a narrow spectrum affecting
primarily only Gram-positive bacteria and their spores, including lactic acid bacteria,
Bacillus, Clostridium, Listeria, and Streptococcus. The compound alone generally
does not inhibit Gram-negative bacteria, yeasts, or molds. Streptococcus thermophi-
lus, Lactobacillus plantarum, other lactic acid bacteria, and certain Bacillus species
produce the enzyme, nisinase, which neutralizes the antimicrobial activity of the
peptide. More important, however, is the fact that spontaneous nisin resistant
mutants, including L. monocytogenes and C. botulinum, could occur due to exposure
of wild-type strains to high concentrations of nisin or transfer in increasing concen-
trations of nisin (Harris et al., 1991; Ming and Daeschel, 1993; Mazzotta et al.,
1997). Listeria monocytogenes resistant mutants may occur at a rate of 1 in 106 to
108 (Harris et al., 1991; Ming and Daeschel, 1993) or even lower (Schillinger et al.,
1998). These have been shown to be stable mutants. The mechanism of action of
nisin against vegetative cells includes binding to the anionic phospholipids of the
cell membrane, insertion into the membrane and pore formation. This disruption of
cytoplasmic membrane causes efflux of intracellular components and eventual deple-
tion of the proton motive force (PMF; Crandall and Montville, 1998). As might be
expected, resistance of the cell involves adjusting to prevent these actions.
Crandall and Montville (1998) did an extensive study on the potential mechanism
of nisin resistance. They found that nisin resistant strains of L. monocytogenes (NisR)
had altered phospholipid composition, including decreased anionic phospholipid
(cardiolipin and phosphatidylglycerol) and increased phosphatidylethanolamine.
This resulted in a decreased net negative charge, which would hinder binding of
cationic compounds such as nisin. In addition, NisR strains have increased long chain
fatty acids and reduced ratios of C15/C17 fatty acids, resulting in reduced fluidity
and stabilization due to reduced effect on PMF (Ming and Daeschel, 1993; Mazzotta
and Montville, 1997). Other cellular changes include cell wall alterations as evidenced
by increased lysozyme resistance, decreased resistance to ampicillin and benzylpen-
icillin and increased resistance to Gramicidin S and Gentamicin. These changes
suggested an alteration of cytoplasmic membrane to prevent access by nisin. NisR
cells required divalent cations for resistance. This might be due to reduced binding
or stabilization of the cytoplasmic membrane.

Cross-protection of foodborne pathogens to bacteriocins may be induced by acid
adaptation or acid tolerance. Van Schaik et al. (1999) demonstrated that acid adapted
(pH 5.5, lactic acid) cells of L. monocytogenes Scott A had increased resistance to
nisin and lacticin in addition to lactic acid at pH 3.5. These cells had altered fatty
acid composition with increased C14:0, C16:0 and decreased C18:0. In contrast,
Okereke and Thompson (1996) found no increase in resistance to nisin with acid
adapted cells of L. monocytogenes. Van Schaik et al. (1999) suggested this was
caused by a lower concentration of nisin used by the latter researchers.
The obvious implication of the emergence of bacteriocin-resistant pathogenic
microorganisms would be potential hazards in foods preserved exclusively by the
compounds. To overcome the potential hazard, researchers have suggested use of
combinations of bacteriocins or combinations of bacteriocins and other preservation
methods or antimicrobials (Mulet-Powell et al., 1998; Schillinger et al., 1998). Cross
resistance between various classes of bacteriocins has been demonstrated (Crandall
and Montville, 1998). However, this cross resistance appears to be variable as Rasch
and Knøchel (1998) found none between nisin- and pediocin-resistant strains of L.
monocytogenes. Dykes and Hastings (1998) demonstrated that leucocin- and saka-
cin-resistant L. monocytogenes had a reduced growth rate in BHI broth without
bacteriocin compared to bacteriocin-sensitive strains. In addition, resistant strains
failed to compete with sensitive strains when grown in mixed populations even at
frequencies of 1:1. The researchers concluded that the bacteriocin-resistant pheno-
type of L. monocytogenes was not likely to become stable in natural populations. In
addition, nisin resistant C. botulinum spores have similar heat resistance to wild type
spores, and nisin-resistant strains of L. monocytogenes and C. botulinum were not
as resistant as wild-type strains to other traditional food antimicrobials including
sodium chloride, sodium nitrite or potassium sorbate (Mazzotta and Montville, 1999;
Mazzotta et al., 2000). All of these studies suggest that acquired resistance to bac-
teriocins may not confer resistance to other antimicrobials or preservative treatments
or any natural advantage over susceptible populations in the absence of the inhibitor.
SUMMARY
There is little evidence that microorganisms can directly acquire resistance or tol-
erance to most traditional food antimicrobials. The possible exception is microbial
tolerance to short chain organic acids through acid adaptation. Little research has
been done on acquired resistance to naturally occurring antimicrobials with the
exception of microbially derived bacteriocins. The latter have definitively been
shown to induce acquired resistance to microorganisms exposed to the compounds.
Acquired resistance may be important in the microbiological safety of food products
if the resistance is induced in foodborne pathogens by conditions present in the
current food processing system and if that resistance enhances survival of those
pathogens in foods or food processing systems in which they are normally inacti-
vated. Both acid adaptation and acquired resistance to bacteriocins have the potential
to induce resistance in foodborne pathogens under conditions currently present in
the food processing system. Bacteriocin resistance development is of less concern

because foodborne pathogens are not often repeatedly exposed to such compounds.
In contrast, there are multiple steps in the food production and processing system
in which a microorganism may be exposed to organic acids. While resistance the-
oretically enhances survival of foodborne pathogens to exposure to lethal treatments,
cells that are resistant to bacteriocins have been found to be less able to compete
than non-resistant cells in the environment. There is limited evidence that acid
tolerance may increase resistance of pathogens to lethal organic acid treatments or
conditions in foods. More evidence is needed to confirm that this is a real problem.
If antimicrobials are to be used for exclusive control of foodborne pathogens,
then potential for development of resistant cells should be evaluated. Development
of resistance to processing and handling treatments could occur at numerous points
in the food processing system and could influence the treatment efficacy. Evaluation
of this type of response is probably more meaningful when conditions most like
those in actual products or situations are included in the experimental plan. As was
noted by Leyer and Johnson (1993), the physiological state of foodborne pathogens
used in challenge studies in food and in evaluating hazard analysis critical control
point (HACCP) programs is an important consideration.
REFERENCES
Abee, T. and Wouters, J.A. 1999. Microbial stress response in minimal processing. Intl. J.
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Microbial Adaptation and
4 Survival in Foods
Eric A. Johnson
CONTENTS
Introduction
The Spectrum of Stress Responses in Microorganisms
Importance of Microbial Stress Responses to the Safety and Quality of Foods
Impact of Stress Responses on Preharvest Survival of Foodborne Pathogens
Stress Responses and Their Impact on Survival on Gram-Negative Foodborne
Pathogens and Spoilage Bacteria
Salmonella
Escherichia coli O157:H7
Shigella spp.
Yersinia enterocolitica
Campylobacter jejuni
Vibrio parahaemolyticus and Vibrio cholerae
Pseudomonas aeruginosa
Gram-Positive Bacteria
Staphylococcus aureus
Listeria monocytogenes
Bacillus spp.
Clostridium spp.
Impact of Stress Adaptation on the Performance of Beneficial
Microorganisms in Food Fermentations
Cross-Protection among Microbial Stress Responses
Conclusions and Perspectives
References
INTRODUCTION
Microorganisms can induce adaptation responses to environmental stresses by
expressing specific sets of genes on exposure to acid, salt, heat, cold, reactive oxygen
species (ROS), nutrient starvation, and other stresses (reviewed in Abee and Wouters,
1999; Costa and Moradas-Ferreira, 2001; Hecker and Völker, 2001; Hohmann and
Mager, 1997; Jennings, 1993; Lin and Lynch, 1996; Storz and Hengge-Aronis, 2000;
Welch, 1993; Young and Elliott, 1989).

Adaptation enhances tolerance to environmental, chemical, and biological
stresses and may promote survival or growth in adverse environments. Adaptation
to stresses is mediated by changes in physiology of the organism, including alter-
ations in metabolism and structural changes. These changes can have profound
effects on the ability of food-spoilage and pathogenic organisms to survive food
processing operations and to survive or even grow in normally adverse or harsh food
environments. Adaptation of stress responses also affects the performance of bene-
ficial organisms in carrying out food fermentations and other desired transformations
of foods. The stressed cellular state probably reflects the actual physiology of many
bacteria and fungi in foods and in food processing environments since microorgan-
isms in these conditions are often periodically or continually exposed to adverse
stresses. Exposure to stresses has been widely shown to induce a spectrum of adaptive
responses, ranging from relatively minor physiological adaptations to extreme
changes in cellular structure such as alterations in the cell surface or formation of
endospores, as well as changes in population structure, including entry into stationary
phase and the formation of macrocellular structures such as biofilms. It is surprising
that microbial stress responses have not, until recently, been integrally considered
in various disciplines of food microbiology including resistance to unit food pro-
cessing operations, formulation design of foods, challenge studies and shelf-life
evaluations, contributions to virulence, formation of endospores and biofilms, and
inactivation by sanitizing procedures.
This chapter focuses on practical aspects and industrial relevance of stress
responses of various microorganisms of importance in the food industry, with an
emphasis on foodborne bacterial pathogens and beneficial fermentation organisms.
This discussion makes a unique contribution since the majority of research on stress
responses has focused on the molecular aspects of the various responses, particularly
in Salmonella enterica serovar Typhimurium and Escherichia coli, and in the yeast
Saccharomyces cerevisiae and a few other fungal species (reviews are cited above).
Many of the practical consequences of stress that are of importance to the food
industry have only begun to be studied in depth. The investigation of stress responses
as applied to food-related microorganisms and food systems would appear to provide
a plethora of opportunities and is anticipated to yield valuable information to enhance
the safety and quality of many foods.
In this chapter, various stages in food production are considered, including
preharvest survival, resistance to food processing operations and sanitation proce-
dures, and survival and growth during a shelf-life of a food. The impact of stress
responses on beneficial aspects of microorganisms in food fermentations is also
discussed, followed by newer approaches to study stress responses, and conclusions
and perspectives.
THE SPECTRUM OF STRESS RESPONSES

IN MICROORGANISMS
The myriad of stress-associated phenomena in microorganisms can be classified into
distinct and yet interrelated classes, as shown in Table 4.1 (Storz and Hengge-Aronis,

TABLE 4.1
Classes of Microbial Stress Responses
Specific Stress Responses and Causative Phenotypic Triggers
Heat shock
Cold shock
Sensing envelope stress
Acids and acidity
Reactive oxygen species (ROS)
Reduced osmolarity
Sodium
Metals, possibly other ions
DNA damage
General Stress Responses
Bacterial sporulation and resistance
Survival in the stationary phase
Pathogenic responses
Stress-induced mutations
Drug resistance
Stress-induced competence in Bacillus and possibly other organisms
Cell-to-cell communication and quorum sensing
Stress-induced biofilm formation
(Adapted from Storz, G. and Hengge-Aronis, R., Eds., Bacterial Stress

Responses, ASM Press, Washington, D.C., 2000.)
2000). In addition to these categories, it seems plausible to include population-

induced stress responses including quorum sensing (Hardman et al., 1998), pro-
grammed cell death and cell aging (Beckman and Ames, 1998; Jazwinski, 1999;
Lewis, 2000; Nature Insight, 2000), and formation of multicellular or multispecies
biofilms (Costerton et al., 1999).
Of interest and potential importance in food microbiology is the finding that
Gram-negative bacteria (e.g., E. coli, Salmonella serovars), Gram-positive eubacteria
(e.g., Bacillus, Staphylococcus, Listeria, Clostridium) and fungi (S. cerevisiae)
appear to have evolved different physiological, genetic, and structural mechanisms
to cope with various stresses (reviewed in Hecker and Völker, 2001; Hohmann and
Mager, 1997; Jennings, 1993; Storz and Hengge-Aronis, 2000).
It is beyond the scope here to describe the molecular mechanisms of the different
responses among the various microbial groups; these are described elsewhere in this
book. In the literature on microbial stress, various descriptors have been used to
describe stress responses, including “adaptation,” “tolerance,” “habituation,”
“shock,” and other terms. These descriptors are used in various laboratories and may
very well reflect different physiological responses. As the overall theme of this
chapter is to describe the practical impact of stress responses on microorganisms of
importance in foods, the terms are collectively grouped as “stress responses” and
the reader is referred to the original papers for the specific experimental details and
interpretations of the responses.

IMPORTANCE OF MICROBIAL STRESS RESPONSES TO
THE SAFETY AND QUALITY OF FOODS
Stress responses would be expected to affect the resistance and survival of pathogens
and spoilage organisms through the entire food production chain, from preharvest
activities, processing operations, and storage of foods during their shelf life. The
stress responses elicited by food-related microorganisms would also vary according
to the food commodity, its processing steps, and its respective microbial ecology
and species associated with the different commodities (Roberts et al., 1998). It is
useful to consider stress responses of food-related bacteria and fungi in the context
of environmental factors that have systematically been demonstrated to affect growth
and survival in food systems. For most species of food-related bacteria and fungi,
survival has long been known to be affected by various chemical, processing and
environmental factors, commonly categorized as intrinsic factors, processing factors,
extrinsic factors, and implicit factors (Table 4.2) (Cole, 2001; Gould, 2000; Mossell
and Ingram, 1955). These preservative factors and processing technologies impose
stresses upon microorganisms in foods, potentially eliciting stress responses that
would affect growth and survival in many instances. The application of multiple
factors (hurdle technology) involving exposure to combinations of sublethal condi-
tions in foods could also result in the promulgation of adaptive stress responses
(Archer, 1996; Knøchel and Gould, 1995; Leistner, 1995; Rowan, 1999). Stress
responses elicited in response to traditional food preservatives such as sorbate,
benzoate, lactate, sulfite, nitrite, nisin, smoke, and other preservatives have not yet
been established. Newer processing technologies such as treatment of foods with
high pressure, pulsed electric fields, light, sound, and others (reviewed in Rahman,
1999) would also be expected to induce stress responses, including novel or unex-
pected responses, but more research is needed in this area.
As with most foodborne pathogens and spoilage organisms discussed in this
chapter, the vast majority of studies on stress responses have been conducted in vitro
in media or in buffer systems, and very few studies have investigated the impacts
of stress responses on growth or survival in food products. Secondly, many investi-
gations have demonstrated that considerable variation in resistance to environmental
conditions exists among different serovars and strains of pathogens, e.g., Salmonella
(Humphrey et al., 1995; reviewed in Roberts et al., 1996). Although less well doc-
umented for other species, strain variation is also observed for other bacterial and
fungal species, and the selection of standard universally accepted strains should be
used to enable interlaboratory comparisons. Care must be exercised in extrapolating
the resistance data and ability to induce stress responses among different species,
serovars, and strains of related organisms. Lastly, several reports have been published
in recent years on stress responses of microbes and their potential significance in
foods, and it is not possible in this chapter to discuss each of these studies. To
develop a comprehensive understanding for a single organism, the reader can refer
to references in recent publications or conduct literature searches using available
electronic databases.
The interactions of factors affecting survival (Table 4.2) have long been known
to affect the growth and survival of food-related organisms and their resistance to

TABLE 4.2
Classification of Preservation Factors Affecting
the Microbiological Safety and Quality of Foods
Intrinsic Factors
Chemical Physical
Nutrients Water activity, relative humidity
Acidity and organic acids Ice and freeze concentration
Oxidation-reduction potential Structure of food
Antimicrobial substances Microstructure (e.g., emulsification)
Processing Factors
Thermal heating and cooling
Cold storage and freezing
High pressure
Irradiation
Pulsed electric field treatment
Ohmic heating
Light treatment
Sonic treatment
Drying, reduction in water activity
Modified atmosphere packaging
Extrinsic Factors
Change in relative humidity during storage
Temperature during storage
Gas levels (oxygen, carbon dioxide, other gases)
Implicit Factors
Microbial growth rates
Synergistic effects derived from food components and intereactions
among microorganisms
Antagonistic effects derived from food components and intereactions
among microorganisms
(Modified from Gould, G.W., in The Microbiological Safety and Quality of

Foods, Lund, B.M. et al., Eds., Aspen Publishers, Gaithersburg, MD, 2000).
processing procedures (Cole, 2001; Gould, 2000; Mossel and Ingram, 1955). Some
early studies showed that exposure of salmonellae to low pH increased their acid
tolerance (Baird-Parker et al., 1970; Huhtanen, 1975). It was also known that
“injured” cells could be more readily recovered on rich media lacking antimicrobials
(Baird-Parker and Davenport, 1965; reviewed in Hurst, 1977; Ray, 1986). Further-
more, cross protection induced by one antimicrobial resulting in resistance against
other agents was also shown quite early (Szybalski and Bryson, 1952).
Surprisingly, the concept of stress responses and their impact on virulence and
survival of pathogens was not articulated until much later, particularly during the
past two decades. The origin in understanding of stress response systems emanated

from an increased understanding of fundamental microbial metabolism and physi-
ology (Gottschalk, 1979; Ingraham et al., 1983), emerging to studies of basic phys-
iological responses to specific stresses (Booth, 1985; Csonka, 1989). Experimental
strategies and techniques that enabled the elucidation of multigene systems and
regulons included the advent of two-dimensional polyacrylamide gel electrophoresis
(2-D gels) for analysis of the proteome (Neidhardt, 1987; Neidhardt and Van Bogelen,
2000; O’Farrell, 1975), advances in molecular genetics (Beckwith and Zipser, 1970;
Stent, 1978), and development of the fields of genomics and metabolomics.
The importance of stress responses for survival of pathogens and spoilage micro-
organisms in foods and during food processing was not widely studied until recently,
mainly during the past decade. Although studies of specific stress responses have
only recently been applied to evaluation of food safety and quality, survival studies
of pathogens in foods containing a variety of “barriers” have long suggested that
microbial stress responses are important in food safety and quality. The barrier or
“hurdle” (Leistner, 1995) approach has become popular for design of preservation
and safety systems for foods. However, sublethal exposure of microorganisms to
multiple stresses could also lead to adaptive responses and increased survival, as
well as possible induced adaptive mutations conferring resistance to the barriers
(Archer, 1996; Cairns et al., 1988; Rosche and Foster, 2000). These adaptive
responses could permit growth under adverse conditions and actually be detrimental
to the safety and quality of certain foods.
IMPACT OF STRESS RESPONSES ON PREHARVEST

SURVIVAL OF FOODBORNE PATHOGENS
Very little information is available regarding the impact of microbial stress responses
on survival of microbes in preharvest plant and animal tissues. Plants are well known
to induce responses to surface infections by bacterial and fungal pathogens and
saprophytes (Hammerschmidt, 1999; Kuc, 1995). These responses include the
induced synthesis of various classes of small molecular weight microbial inhibitors,
including organic acids, phenolic compounds, terpenoids, flavonoids, alkaloids, and
other classes of compounds (Billing and Sherman, 1998; Cowan, 1998; Hammer-
schmidt, 1999).
In response to microbial infection, plants also induce the synthesis of peptides
and enzymes, particularly cell wall hydrolases (Asselin, 1993; Lebeda et al., 2001),
enzymes that generate reactive oxygen species (ROS) such as peroxidases (Baker
and Orlandi, 1995), and defense peptides (Fritig et al., 1998). Pathogen virulence
and ability to colonize plant tissues correlates with phytoalexin tolerance, mediated
by detoxification of plant inhibitors and other mechanisms (Hammerschmidt, 1999).
Bacteria and fungi could adapt to other stresses including organic acids such as
salicylic and jasmonic acids (Verberne et al., 2000), small molecular weight phy-
toalexins (Hammerschmidt, 1999), lytic enzymes and defense peptides (Fritig et al.,
1998; Lebeda et al., 2001), and ROS produced during the plant oxidative burst (Low
and Merida, 1996), but little is known regarding microbial stress responses on
preharvest tissues. Biological control by bacterial or fungal antagonistic organisms

has been evaluated for prevention of growth of E.coli O157:H7 in apple wounds
(Janisiewicz et al., 1999; Riordan et al., 2000), but this research is in its early phases
and substantive conclusions on the efficacy of this intriguing approach cannot be
made at this time.
Mammals and their secretions (such as milk) also possess a diverse array of
inhibitory peptides and enzymes, including defensins, lysozyme, lactoferrin, and
peroxidases (Johnson et al., 1990; Kolb, 2001; Khush and Lemaitre, 2000; Low and
Merida, 1996). The ability of food-related organisms to induce resistance to these
agents has not been well studied, although some foodborne pathogens, including
certain species of staphylococci and bacilli, are known to be naturally resistant to
lysozymes (Hughey and Johnson, 1987). Salmonella enterica serovar Typhimurium
has been shown to acquire resistance to organic acids during passage in the rumen
(Kwon et al., 2000). These authors suggested that short chain fatty acids (SCFA) in
the gastrointestinal tract (particularly the large intestine) of animals may increase
the persistence of S. enterica serovar Typhimurium in the food animal and during
pre- and post-harvesting handling of the animal. S. enterica serovar Typhimurium
exposed to SCFA mixtures were more resistant to various stresses including acid
(pH 3.0), increased osmolarity (2.5 M NaCl), and ROS (20 mM H2O2) than were
cells exposed to SCFA from the small intestine. In contrast, hay-fed or grain-fed
steers yielded E. coli O157:H7 populations with equal acid resistances (Hovde et al.,
1999).
Bacteria and fungi have also been demonstrated to induce resistance responses
to ROS, antimicrobial peptides, and other antimicrobials associated with plants and
animals in preharvest or during harvesting or slaughter (Brul and Coote, 1999). Very
little is known about resistance to most naturally occurring antimicrobial compounds
(Brul and Coote, 1999). More research is needed to elucidate the frequency and
mechanisms of adaptation and resistance to natural antimicrobial agents.
STRESS RESPONSES AND THEIR IMPACT ON

SURVIVAL OF GRAM-NEGATIVE FOODBORNE
PATHOGENS AND SPOILAGE BACTERIA
As discussed above, a variety of stress responses have been demonstrated or postu-
lated to affect the resistance of foodborne pathogens to processing operations and
growth and survival during food storage. Since stress responses have been shown
to differ in various genera and species of pathogens, the responses of different species
are discussed separately.
SALMONELLA
Most studies of the influence of stress responses on survival of salmonellae and
other pathogens have been conducted in media. Salmonella enterica serovar Enter-
itidis were more resistant to heat and acid when grown to stationary phase cells in
the presence of glucose compared to cells grown in the absence of an added carbon
source (Wilde et al., 2000). The presence of the sugar promoted production of acid

with consequent acid habituation. The habituated cells were not more resistant to
inactivation during air drying. Bunning et al. (1990) showed that S. enterica serovar
Typhimurium subjected to heat-shock had substantially increased heat resistance in
trypticase-soy/yeast extract broth compared to non-shocked cell populations, and
they noted a complementary effect of anaerobiosis and heat shock on thermal
resistance. Mattick et al. (2000) showed that S. enterica serovars Enteritidis PT4 and
Typhimurium DT104 survived at low aw for long periods, and that rpoS mutants
were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol.
Incubation at an increased temperature of 37°C led to a greater rate of inactivation
by the solutes. Interestingly, when subjected to osmotic stress, the two serovars
formed long filaments, supporting that structural changes are involved in the stress
response. The formation of filaments would have significant implications for meth-
ods of enumeration and microbiological monitoring. The authors concluded that the
variable survival of Salmonella strains should be considered in predictive growth/sur-
vival modeling and in conducting risk assessments.
In addition to studies in media, the induction of adaptive responses has been
demonstrated to enhance the survival of Salmonella in adverse food environments.
Acid adaptation of S. enterica serovar Typhimurium at a pH of 5.0 to 5.8 for one
to two doublings enhanced survival compared to non-adapted cells during a milk
fermentation and on incubation in various cheeses including cheddar, Swiss, and
mozzarella cheeses kept at 5°C (Leyer and Johnson, 1992). These results supported
the theory that acid adaptation is an important survival mechanism enabling Salmo-
nella spp. to persist in fermented food products.
Acid-adapted salmonellae were reported to have equal or greater acid and ther-
mal sensitivity than control cells on lean beef tissue, suggesting that acid-adaptation
did not result in bacteria resistant to organic acid rinses on beef (Dickson and
Kunduru, 1995). Acid-shocked cells of multidrug-resistant S. enterica serovar Typh-
imurium DT104 did not show markedly different rates of thermal inactivation in
various egg products (Jung and Beuchat, 2000). Other studies indicated that resis-
tance to acid of salmonellae was not influenced by the pH of the food (Weissinger
et al., 2000). It was concluded that the acid stress may affect certain strains of
salmonellae to a greater extent than others, but the small differences in heat resistance
could also be due to differentially expressed stress responses in the various strains.
The variations in these studies suggest that the food matrix and composition influ-
ences stress responses and subsequent resistance properties of adapted cells. The
studies also indicate the need to develop appropriate biomarkers such as expression
of proteins or structural changes to clearly demonstrate the expression of a stress
response and to distinguish stressed cells from injured cells. Lastly, it is apparent
that the use of standard strains is necessary to accurately compare results from
different laboratories.
In addition to acid, other stress responses have been shown to affect resistance
properties of Salmonella spp in vitro and in foods. Exposure of various Salmonella
spp. to reduced aw increased subsequent heat tolerance (Bunning et al., 1990; Mattick
et al., 2000). Reduced water activity has long been known to increase the thermal
tolerance of various pathogens (Baird-Parker et al., 1970; Fay, 1934), although the
mechanisms were not known at the time of these early reports. Recent work indicates

that induction of stress response proteins (heat-shock proteins), including chaperones
and ATP-dependent proteases, contributes to increased heat tolerance (Yura et al.,
2000). In an interesting study, it was demonstrated that S. enterica serovar Enteriditis
strains with greater thermal or hydrogen peroxide tolerance also survived longer on
surfaces (Humphrey et al., 1995). In the same study, growth of the culture to the
stationary phase increased resistance to heat, acid and hydrogen peroxide.
Our laboratory made the unexpected observation that acid adaptation resulted
in marked sensitization of S. enterica serovar Typhimurium to halogen-based sani-
tizers including chlorine (hypochlorous acid) and iodine (Leyer and Johnson, 1997).
On the other hand, acid-adapted S. enterica serovar Typhimurium was more resistant
to certain other classes of sanitizers. The sensitization of S. typhimurium to chlorine
was attributed to alteration in structure and permeabilization of the outer membrane,
reaction with essential sulfhydryl groups in proteins, and disruption of energy metab-
olism (Leyer and Johnson, 1997), but other mechanisms such as activation of signal
transduction pathways by oxidation could also have contributed to sensitization. This
study on enhancement of sanitizer action could provide a basis for innovative
intervention strategies and technologies to inactivate Salmonella, and implies that
acid pretreatment of food plant environments may increase the efficacy of halogen
sanitizers.
ESCHERICHIA COLI O157:H7

Stress responses and cross-protection have been studied extensively in nonpatho-
genic E. coli (reviewed in Finkel et al., 2000; Lin and Lynch, 1996; Storz and
Hengge-Aronis, 2000; Matin et al., 1989; Rowbury, 1995). These studies with tra-
ditional laboratory strains showed that acid “habituation,” nutrient starvation, and
growth into the stationary phase yielded populations of cells that were more resistant
to various stresses than were control populations. Although studies of nonpathogenic
E. coli provided many basic insights into the physiology of stress responses, the
discussion in this chapter focuses on E. coli O157:H7 because of its importance as
a foodborne pathogen. Due to its significance as a pathogen, many studies of stress
responses in E. coli O157:H7 and their potential impact on survival in foods have
been performed, and representative studies are presented here.
As with other tested foodborne pathogens, different isolates of enterohemor-
rhagic E. coli (EHEC) vary in their resistance to acid (Benjamin and Datta, 1995;
Duffy et al., 2000). In general, this group of E. coli has relatively high acid tolerance
similar to that of Shigella flexneri (Benjamin and Datta, 1995; Gorden and Small,
1993). Outbreak strains of EHEC were reported to have greater acid resistance than
natural isolates (McKellar and Knight, 1999). Natural isolates of E. coli O157 also
varied in resistance to hydrostatic pressure, heat, salt, hydrogen peroxide, and com-
pounds causing membrane damage (Benito et al., 1999). As is the case with most
other foodborne pathogens, EHEC cells grown to stationary phase had higher resis-
tance to heat and acid than cells harvested during exponential phase. These variations
in resistance properties depended on the strain and growth conditions. These results
reinforce the importance of using resistant strains in evaluating the efficacy of food
preservation treatments and in developing process criteria.

Several researchers have evaluated the impact of stress responses on the resis-
tance of E. coli O157:H7 in buffer and media systems. Exposure to acidic environ-
ments significantly increased the heat tolerance of various strains of E. coli O157:H7
(Ryu and Beuchat, 1999). D-values of acid-adapted cells were significantly higher
than were those of acid-shocked or nonadapted cells. In concordance with these
results, E. coli O157:H7 exhibited a pH-dependent, stationary phase acid resistance
that increased the pathogen’s tolerance to heat (Buchanan and Edelson, 1999a). In
adapted cells, the time needed to obtain a five-log reduction of viable cells in
brain–heart infusion broth was increased two- to four-fold compared to nonadapted
cells, depending on the pH. Increased heat-resistance was also observed in milk and
chicken broth, but not with apple juice, indicating that the intrinsic parameters of a
food affected the resistance properties of the adapted cells. The authors indicated
that stress responses must be considered to accurately determine thermal tolerance
of E. coli O157:H7 in various foods.
The authors also evaluated the pH-dependent stationary-phase resistance of
E. coli O157:H7 to various acidulants (Buchanan and Edelson, 1999b). Nine strains
of E. coli O157:H7 differed markedly in their resistance to hydrochloric acid or
various organic acids. The variation in resistance differed with the medium for
recovery, and fewer numbers of cells were recovered on MacConkey agar compared
to BHI agar. Hydrochloric acid was the least damaging to the cells, and lactic acid
was most damaging, while acetic, malic and citric acids showed intermediate effec-
tiveness. These results are surprising, as acetic acid has been shown in many other
studies to be more effective than lactic acid against many foodborne pathogens (e.g.,
Roberts et al., 1996). The authors concluded that the accurate determination of
survival of enterohemorrhagic E. coli in acidic foods must consider the strain and
its ability to induce stress responses. The strain, type of organic acid, and acid
adaptation or acid shocking were also shown to affect the acid resistance of E. coli
O157:H7 in trypticase soy broth and in orange juice and apple cider (Ryu and
Beuchat, 1998). Combinations of lactate, ethanol, and low-pH conditions were
demonstrated to enhance the killing of E. coli O157:H7 (Jordan et al., 1999).
In addition to enhancing acid and heat resistance, acid adaptation also increased
resistance to other detrimental treatments. Radiation resistance of E. coli O157:H7
was dependent upon strain and the induction of acid-resistance (Buchanan et al.,
1999). While pH during exposure had little effect on survival during irradiation by
Cs-137, acid-resistance consistently enhanced radiation resistance. The results indi-
cated that induction of a pH-dependent stationary phase response afforded cross-
protection against irradiation, and such a response must be considered in determining
irradiation D values in foods.
Various studies have demonstrated that acid adaptation of E. coli O157:H7
enhanced survival in acidic foods including fermented dairy products, fermented
meats such as shredded hard salami (Leyer et al., 1995), and in acidic fruit juices,
particularly apple cider (Leyer et al., 1995; Miller and Kaspar, 1994). In contrast,
acid adaptation was reported to decrease resistance of E. coli O157:H7 to 2% acetic
acid spray in washing of carcasses (Berry and Cutter, 2000). Survival of E. coli
O157:H7 in dried beef powder was also not significantly enhanced by acid adapta-
tion, suggesting that this stress response did not afford cross protection against

dehydration or osmotic stresses (Ryu et al., 1999a). As with most foods, dried beef
powder has a complex composition and it is possible that other factors present in
the food also significantly affected survival. This study supports that it is difficult
to predict survival in many foods based on in vitro growth and survival responses,
and that challenge studies in the food systems are required to adequately assess
growth and survival of pathogens.
Collectively, these various studies indicate that it is very important to precisely
define the strain, its growth conditions, and the food environment to determine
survival. It would be valuable for food microbiologists to reach a consensus on the
use of strains, and to show their uniformity among labs by discriminating techniques
such as pulsed field gel electrophoresis (Swaminathan et al., 2001) or other com-
parative typing methods.
One aspect of stress adaptation that has received limited attention is the mea-
surement of in vivo expression of stress-related genes in food systems. A method of
RNA isolation and RT-PCR to detect E. coli O157:H7 was applied to detecting the
pathogen on beef carcass surfaces (Berry, 2000). The method used a selectively
inducible green fluorescent protein (GFP) reporter gene in a plasmid-transformed
strain of E. coli O157:H7 inoculated onto beef carcass surfaces. Expression of stress-
related genes could be evaluated by quantitation of fluorescence in cells on the food
surface. Although this procedure does not reflect naturally contaminated beef, it did
show the probable locations of the bacteria on the meat. This approach using reporter
gene technology should be useful in studying the genetic responses of the bacteria
when exposed to stresses and antimicrobial interventions in various food systems.
SHIGELLA SPP.
Since Shigella spp. are closely related to E. coli, they would be expected to show
analogous stress responses. Indeed, the acid resistance response of Shigella has been
considered to be analogous to that of E. coli (Gorden and Small, 1993), and a
homologous alternative sigma factor, encoded by an rpoS allele, has been demon-
strated in E. coli and S. dysenteriae (Small, 1994).
An important stress response contributing to virulence in S. dysenteriae is acid
tolerance (Small, 1994). Since the infectious dose of Shigella is low (10-200 CFU)
in humans and is considerably less than that of E. coli and most other Enterobacte-
riaceae (Small, 1994), it is likely that other factors for Shigella virulence are involved
in addition to acid resistance, but these determinants have not yet been identified.
In reviewing the literature, published studies were not found on the impact of stress
responses on growth and survival of Shigella in foods.
YERSINIA ENTEROCOLITICA
Stationary phase cells of Y. enterocolitica showed increased resistance to acid, but the
resistance was dependent on the presence of urea in the medium and an active urease
enzyme (Koning-Ward and Robins-Browne, 1995). The catabolism of urea to form
ammonia and carbon dioxide through urease activity probably provided a buffering
effect that promoted gastric passage. Thus, Y. enterocolitica appears to have a unique
mechanism of induced acid tolerance compared to most other Enterobacteriaceae.

CAMPYLOBACTER JEJUNI
Members of the genus Campylobacter are among the major causes of food-related
bacterial gastroenteritis, and their incidence is increasing in several countries (Mead
et al., 1999). Due to the success of Campylobacter spp. in eliciting gastrointestinal
symptoms, it would be expected that Campylobacter spp. would elicit stress
responses that promoted survival on food vectors and during passage in animal and
human gastrointestinal tracts. However, available evidence indicates that Campylo-
bacter spp. lack a stationary phase stress response analogous to most other Gram-
negative foodborne pathogens (Kelly et al., 2001). Unexpectedly, resistance of
Campylobacter jejuni to thermal stress (50°C) or aeration was greatest in the expo-
nential phase of growth and declined in early stationary phase.
Analysis of the recently available genomic sequence supports that C. jejuni
NCTC 11351(Parkhill et al., 2000) lacks rpoS homologues that have been clearly
demonstrated to be involved in various stress responses in Salmonella and E. coli
(Hengge-Aronis, 2000). It is surprising that more research has not been published
on C. jejuni, considering the importance of the organism in foodborne disease.
VIBRIO PARAHAEMOLYTICUS AND VIBRIO CHOLERAE
Acid adaptation promoted resistance to various stresses in V. parahaemolyticus

(Koga et al., 1999). Induction in vitro of an acid tolerance response (ATR) was
demonstrated to increase infectivity of V. cholerae in a mouse model, but the ATR
apparently was not induced during passage in humans (Merrell et al., 2002). The
impact of stress responses on the survival of V. parahaemolyticus, V. cholerae, or
other vibrios in foods appears to not have been studied. Again, such studies would
be valuable in food and water microbiology since many vibrios are extremely
important causes of food and waterborne disease.
PSEUDOMONAS AERUGINOSA
An rpoS-dependent stress response has been demonstrated in the important spoilage

organisms, P. aeruginosa, P. putida, and P. fluorescens (Ramos-González and Molin,
1998; Sarniguet et al., 1995; Suh et al., 1999). Mutations in rpoS affected heat
resistance and virulence of P. aeruginosa in an animal model (Suh et al., 1999). The
importance of stress responses for infection of plant substrates or in growth and
survival in foods has not been reported in the pseudomonads.
GRAM-POSITIVE BACTERIA
Much less is known about the molecular biology and practical food aspects of stress
responses in Gram-positive bacteria than in their Gram-negative counterparts
(Hecker and Völker, 2001; Price, 2000; Storz and Hengge-Aronis, 2000). The alter-
native sigma factor σB is mainly responsible for the induction of genes encoding
stress proteins, although anti-sigma factors and other molecular mechanisms are
operative in certain Gram-positive species.

In general, Gram-positive bacteria are more resistant than Gram-negatives to
environmental stresses including heat, irradiation, osmotic stress, and acid (Roberts
et al., 1996) and thus the possibility that they can induce stress responses and further
increase their resistance is a concern for food microbiologists. Several genera of
Gram-positive bacteria including Bacillus and Clostridium are able to form
endospores, which can impart extraordinary physical and chemical resistance prop-
erties to the organisms (Setlow and Johnson, 2001). Although sporulation can be
considered a specialized and complex form of stress response, its coverage is beyond
the scope of this chapter. The mechanisms of resistance and importance of spores
in food microbiology has recently been reviewed (Setlow, 2000; Setlow and Johnson,
2001). The following discussion focuses on stress responses in important food-
related Gram-positive vegetative organisms.
STAPHYLOCOCCUS AUREUS
S. aureus has long been recognized as among the most osmotolerant foodborne
bacterial pathogens, surviving or even growing in foods in the aw range of 0.88 to
0.91. Most strains of S. aureus only produce their characteristic enterotoxins at
slightly elevated water activities (aw ~0.9) compared to the lower minimal aw that
supports growth.
Several investigators have shown that environmental and growth parameters
influence the resistance properties of S. aureus. Shebuski et al. (2000) demonstrated
that the growth medium had a marked effect on heat resistance of S. aureus. Growth
of S. aureus at an aw value of 0.94 increased its thermal tolerance at 60°C. The
authors also provided evidence for stress-induced heat-shock proteins as well as the
accumulation of compatible solutes that contributed to enhanced thermal tolerance
of S. aureus. The σB regulon has been identified and contributes to survival of
S. aureus during harsh conditions including food processing and during food storage
(Chan et al., 1998; Hecker and Völker, 2001). Staphylococcal thermonuclease, lipase,
and α-hemolysin are hyperproduced in certain σB mutants (Hecker and Völker,
2001). Interestingly, overexpression of the σB operon led to thickening of the cell
wall and resistance to beta-lactams (Morikawa et al., 2001), which implies that this
response — characterized by thickening of the cell wall — could result in enhanced
resistance to food processing procedures and to antimicrobials in foods. The hyper
mutant acquired resistance to the lytic activity of lysostaphin, and had increased
yields of carotenoids, which can protect microorganisms against ROS (Johnson and
Schroeder, 1996). A chromosomal locus encoding several ORFs in S. aureus also
contributed to oxidative defense and is induced by the cell-wall antibiotic oxacillin
(Singh et al., 2001).
Biofilm formation in S. aureus and S. epidermidis appears to be induced by
stress responses. In S. epidermidis, formation of biofilms was influenced by ethanol
and salt stress (Knobloch et al., 2001; Rachid et al., 2000). Salt stress resulted in
biofilm formation on food packaging material (Le Magrex-Bebar et al., 2000). These
studies clearly show that stress responses in food-related staphylococci can influence
food safety and spoilage by increasing resistance properties and by inducing the
formation of biofilms.

LISTERIA MONOCYTOGENES
Due to the importance of L. monocytogenes in foods, several studies have addressed

its stress responses and associated resistance properties, though few studies have iden-
tified the roles that stress responses exhibit for survival in actual food systems. Induced
acid tolerance has been demonstrated for L. monocytogenes (Davis et al., 1996; Hill
et al., 1995; Kroll and Patchett, 1992; Phan-Thanh et al., 2000). As with other foodborne
pathogens and spoilage organisms, expression of acid tolerance was growth phase
dependent and varied according to type of organic acid exposure and the strain of the
pathogen. Molecular studies showed that L. monocytogenes contains a σB controlled
stress response (Becker et al., 1998) that is analogous to B. subtilis, and that induction
of gene expression by σB contributes to enhanced osmotolerance, acid resistance and
virulence (Becker et al., 1998; Hecker and Völker, 2001; Wiedmann et al., 1998).
Adaptation of L. monocytogenes to sublethal stresses has been demonstrated to
protect the pathogen on exposure to a variety of normally lethal conditions present
in certain foods (Lou and Yousef, 1997). Sublethal exposure to ethanol (5% v/v),
acid (HCl, pH 4.5 to 5.0) H2O2 (500 ppm) or NaCl (7% w/v) added to cultures during
the exponential phase for 1 h protected against subsequent exposure to ethanol (17.5%
v/v), NaCl (25% w/v), H2O2 (0.1%), or acid (pH 3.5). The authors emphasized that
“stress hardening” should be considered in design and evaluation of food processing
technologies for control of L. monocytogenes and other foodborne pathogens.
In contrast to certain other bacterial species, acid adaptation of L. monocytogenes
was initially reported not to provide cross-protection against an activated lactoper-
oxidase system (activated-LPS) (Ravishankar and Harrison, 1999). Later, it was
reported that acid adaptation of L. monocytogenes with lactic acid did result in cross-
protection against an activated-LPS in tryptic soy broth (Ravishankar et al., 2000).
The enhanced survival of acid-adapted cells was greater at the lower pHs tested
(3.5 and 4.0 compared to 4.5). These authors also provided evidence of the altered
expression of several proteins in the acid-adapted cells. This latter study supports
that stress responses of L. monocytogenes would enhance its survival in various dairy
products and probably other foods in which ROS are formed.
Although the role of acid-adaptation and other stress responses has been inves-
tigated in vitro, relatively few studies have addressed the impact of stress responses
on survival of L. monocytogenes in foods. Acid adaptation increased survival of
L. monocytogenes in acidic foods and during milk fermentation (Gahan et al., 1996;
Hill et al., 1995). Heat shock of L. monocytogenes in log or stationary growth phases
and in broth or minced meat affected thermotolerance of the pathogen (Farber and
Brown, 1990; Jorgensen et al., 1999). The heat resistance following heat shock was
dependent on growth phase, pH and lactic acid concentration. In particular, the
concentration of lactic acid appeared to most strongly influence the heat resistance.
The D-values at 60°C were two to six-fold higher in minced beef than in TPB,
emphasizing the importance of food constituents on heat resistance. Heat resistance
was found to be dependent on various conditions of culture including pH, acidulant,
and growth temperature (Juneja et al., 1998). Our laboratory found evidence that
pre-growth of L. monocytogenes in salt-enriched media increased its salt resistance
in buffer systems and in commercial cheese brines (Larson et al., 1993, 1999).

Since L. monocytogenes is more resistant than many foodborne pathogens to
organic acids and other inhibitors, it can be difficult to control or eradicate in foods
and in food processing facilities. Methods to sensitize the organism to inhibitors and
sanitizers could be valuable for its control. In an interesting study, exposure of
L. monocytogenes to ethanol sensitized the pathogen to low pH, organic acids, and
osmotic stress (Barker and Park, 2001). The combination of organic acids, low pH
and ethanol was effective as a listericidal treatment (Barker and Park, 2001). Our
laboratory has shown that certain lipophilic terpenoids sensitized L. monocytogenes
to various inhibitors compatible with foods (B. Brehm-Stecher and E. A. Johnson,
2001, unpublished data). In both of these studies, damage to the cytoplasmic mem-
brane and increased leakiness appeared to initiate the killing. Other membrane active
agents including glycerol monolaurate and hop beta acids are also strongly inhibitory
to L. monocytogenes (Larson et al., 1996; Wang et al., 1992, 1993; Oh and Marshall,
1993). The cytoplasmic membrane has been suggested as an important target for
cell inactivation in stressed cells of various food-related bacteria (Russell et al.,
1995). Food-compatible agents that damage the cytoplasmic membrane of L. mono-
cytogenes could lead to novel technologies for inactivation of this recalcitrant and
persistent pathogen. On the other hand, alteration of the cell envelope could also
promote resistance to various antimicrobial agents such as nisin (Davies et al., 1996).
A simple method for evaluating the adaptive response of L. monocytogenes in
foods has been proposed (Bolton and Frank, 1999) that, if verified for different
strains of L. monocytogenes, could be quite valuable for assessing the physiological
state of cell populations. A fertile research area should be the development of
methods to quantitatively detect individual cells of various species of foodborne
pathogens and spoilage organisms exhibiting in vivo stress responses in foods.
BACILLUS SPP.
The σB regulon in Bacillus subtilis has served as a paradigm for the expression and
control of stress-related genes in many Gram-positive organisms (Hecker and Völker,
2001; Price, 2000). Although such a regulon also would be expected to occur in
food-related Bacillus species such as Bacillus cereus, very little information is
available on the presence of these genes in food-related bacilli, and the role of such
a stress system in food processing and during food storage has not been investigated.
As mentioned above, the importance of endospores formed by bacilli, clostridia, and
other endospore-formers on food safety and spoilage has recently been reviewed
(Setlow and Johnson, 2001).
CLOSTRIDIUM SPP.
Clostridia produce resistant endospores and are able to survive many food processing
procedures. Several species can grow under low pH conditions and either spoil foods
or produce harmful protein toxins such as C. perfringens enterotoxin and C. botu-
linum neurotoxins (Bahl and Dürre, 2001; Johnson, 2000; Rood et al., 1997). Despite
the importance of clostridia to food spoilage and human disease, our understanding
of stress responses is very rudimentary. Stress responses have mainly been studied

at the physiological level and in nonpathogenic species (Woods and Jones, 1986).
Very little published information is available related to the contribution of stress
responses to clostridia in food systems. Acid shock markedly increased the thermal
tolerance of C. perfringens (Villarreal et al., 2000). Heat adaptation at 46°C increased
the heat resistance of C. perfringens, and Western blots provided evidence for the
expression of at least four major putative heat shock proteins (Novak et al., 2001).
Prior heat shock also increased the thermal resistance of ten strains of C. perfringens
in a beef gravy system (Juneja et al., 2001). Heat-shock genes and chaperones have
been shown to be expressed in the nonpathogen Clostridium acetobutylicum (Bahl
et al., 1995). The involvement of protein phosphorylation has been detected in
response to stress (Balidimos et al., 1990), possibly indicating a two-component
kinase sensing and signalling system involved in the stress response of C. acetobu-
tylicum. Two-component signalling systems have been implicated in stress responses
and sensing of the environment in a large number of organisms. Considering the
importance of toxigenic clostridia to food safety, it would be valuable for researchers
to investigate stress responses in relation to food processing and survival during
storage in this group of organisms.
The genomic sequences that are becoming available for various clostridia includ-
ing C. perfringens and C. botulinum should facilitate a genomic approach to iden-
tifying stress-related genes in these organisms. Our laboratory has found that a σB
homolog is present in the C. botulinum type A genome based on the sequence posted
on the Sanger web site (www.sanger.ac.uk). Further analysis should provide insight
into the role of σB in protection against environmental stresses and in toxin produc-
tion in C. botulinum.
IMPACT OF STRESS ADAPTATION

ON THE PERFORMANCE OF BENEFICIAL
MICROORGANISMS IN FOOD FERMENTATIONS
The benefit of certain groups of microorganisms in food fermentations and preser-
vation has long been appreciated (Adams and Nout, 2001; Pasteur, 1866; Pederson,
1976; Steinkraus, 1996). The major beneficial groups of microorganisms important
in food fermentations are yeasts, particularly Saccharomyces cerevisiae and related
fermentative ascomycetous yeasts, and lactic acid bacteria (LAB). Stress adaptation
and tolerance responses have been observed in a variety of fungi (Jennings, 1993),
but have been most extensively studied in laboratory and industrial strains of
S. cerevisiae (Brul and Coote, 1999; Hohmann and Mager, 1997).
The ability of S. cerevisiae to tolerate environmental stresses has been deemed
a key to optimal performance in certain industrial fermentations such as baking,
brewing, winemaking, and distiller’s fermentations (Attfield, 1997). During these
fermentations, yeasts are exposed to a variety of environmental stresses including
elevated temperatures, oxygen radicals and other oxidants, hyperosmolarity, dessi-
cation/rehydration, and freezing/thawing. S. cerevisiae has evolved to rapidly
respond to environmental stresses (Attfield, 1997).
Compared to bacterial responses, yeasts and fungi have different molecular mech-
anisms leading to stress adaptations, particularly the maintenance of intracellular

levels of ATP for repair of cellular damage and for expulsion of H+ by the activity
of plasma membrane ATPase. S. cerevisiae also rapidly activates various signalling
pathways to the cytoplasm and nucleus in stressful situations including those involv-
ing RAS-adenylate cyclase, protein kinase A, and MAP-kinase (Brul and Coote,
1999; Hohmann and Mager, 1997; Mager and De Kruijff, 1995; Ruiz and Schüller,
1995). Exposure to heat and oxidative stresses also leads to molecular damage in
fungi, and ROS appear to play a central role in stress-induced injury in yeast,
particularly in conditions of low water activity and RH, dehydration, freezing, heat,
and presence of organic acids and alcohols (Attfield, 1997; Costa et al., 1997;
Davidson et al., 1996; Moradas-Ferreira et al., 1996).
Industrial strains of baker’s yeast are much more resistant to ROS compared to
most laboratory strains (Attfield, 1997). The molecular mechanisms of high levels of
resistance to ROS have provided experimental strategies for rational development of
improved yeast strains for industrial fermentations (Attfield, 1997). The response of
fungi to osmotic stress is one of the better understood adaptive responses (Attfield,
1997; Hohmann and Mager, 1997). Exposure of yeast to hyperosmotic stress involves
expression of genes governing glycerol/polyol biosyntheses, efflux of cations, as well
as a general stress response (Attfield, 1997). Accumulation of glycerol and certain
other polyols is essential for tolerance of hyperosmotic stress. Lastly, the accumulation
of high levels of trehalose (10 to 15%) leads to robustness and enhanced ability of
yeast to withstand dehydration and freezing in industrial preparation of starter strains,
and for rapid availability of energy during inoculation of new fermentations.
Lactic acid bacteria (LAB) are used for a variety of food fermentations, such as
in the production of fermented dairy products, where they may encounter a variety
of stresses such as heat, acid, salt, low oxygen tension, and antimicrobial agents.
The molecular aspects of stress responses in LAB are covered elsewhere (Duwat
et al., 2000; Klaenhammer et al. in Chapter 6 of this book; Sanders et al., 1999).
The study of stress responses in Lactococcus lactis is increasing in order to optimize
its fermentation performance (Kim et al., 1999; Sanders et al., 1999). Various ben-
eficial phenotypic traits of LAB in food fermentations such as rapid acidification,
selective proteolysis, tolerance of osmotic and stresses, resistance to ROS, and ability
to thrive in nutrient poor conditions and at low temperatures are influenced by stress
responses in various species of LABs (O’Sullivan and Condon, 1997; Sanders et al.,
1999). Furthermore, adaptation to a particular stress can lead to tolerance of other
stresses (Sanders et al., 1999). In Lactococcus lactis, the ability to withstand freezing
and lyophilization is an important attribute of lactic acid bacteria developed as starter
cultures. The ability of commercial L. lactis ssp. lactis and L. lactis ssp. cremoris
to withstand freezing at –60°C for 24 h was significantly improved by a prior 25
min heat shock at ~40°C or by a 2 h cold shock at 10°C (Broadbent and Lin, 1999).
Thermal stress treatments also enhanced the resistance of several LAB strains to
lyophilization. Analysis of membrane fatty acid composition suggested that
enhanced resistance to freezing and lyophilization may be related to cell membrane
lipid composition, but that other factors were involved as well in the enhanced
resistance properties.
Other bacterial fermentation or spoilage LAB including Leuconostoc mesenteroi-
des (McDonald et al., 1990), Lactobacillus delbrueckii subsp. bulgaricus (Lim et al.,

2000) and Lactobacillus alimentarius (Lemay et al., 2000) elicit stress responses. It
will be valuable to determine which stress responses contribute most importantly to
desired fermentation parameters in actual food systems, as this will facilitate the
development of strains with optimal fermentation characteristics.
CROSS-PROTECTION AMONG MICROBIAL

STRESS RESPONSES
Cells adapted to a particular stress such as acid often show enhanced survival on
subsequent exposure to a different stress such as heat or salt. This toleration of distinct
stresses after adaptation to an individual stress has been termed cross-protection. A
variety of phenotypic properties have been shown to be afforded by cross-protection
including virulence, cell morphology, antibiotic and food antimicrobial resistance,
resistances to heat, acid, salt, surface active agents, and nutritional requirements
(Archer, 1996; Leyer and Johnson, 1993; Rowan, 1999). Acid-adaptation induced
cross-protection against heat, salt, an activated lactoperoxidase system (generating
ROS), and surface active agents in Salmonella enterica serovar Typhimurium (Leyer
and Johnson, 1993). Acid adaptation also increased cell-surface hydrophobicity,
which could affect the ability of Salmonella to bind to surfaces and to form biofilms.
Similarly, in Vibrio parahaemolyticus, acid adaptation induced cross-protection
against heat, crystal violet, bile and deoxycholic acid (Koga et al., 1999). Acid-
adapted salmonellae showed increased resistance to organic acid rinses on beef
surfaces (Dickson and Kunduru, 1995). Since general stress responses are actively
being elucidated in many species of Gram-negative and Gram-positive bacteria, it is
expected that many more species will be found to induce cross-protection against
chemical and environmental stresses. It is anticipated that cross-protection will mark-
edly affect the survival of pathogens in foods containing multiple barriers. Rowe and
Kirk (1999) emphasized that understanding the food conditions leading to stress and
the kinetics of stress responses would enable more accurate estimates of risk assess-
ment and development of adequate safety systems in food processing operations.
A long-standing and debatable phenomenon is whether certain classes of muta-
tions in microorganisms occur randomly as initially proposed by Luria and Delbrück
(1943), or whether mutations are induced by selections encountered in stressful
environments (see Cairns et al., 1998; Rosche and Foster, 2000). Cairns et al. (1998)
showed that Lac+ mutations accumulated with time when Lac- cells were grown in
selective conditions for lactose utilization. Archer (1996) and Rowan (1999) have
emphasized that adaptive mutations induced by stress could have considerable
importance in food safety.
CONCLUSIONS AND PERSPECTIVES

Study of stress responses in foodborne pathogens and in organisms involved in
beneficial fermentation organisms is a relatively new field. There is little doubt that
stress responses in these organisms have a marked impact on food safety and quality.
Unfortunately, there is a paucity of studies on the practical importance of stress

responses of microorganisms in actual food systems, and much more research is
needed. For these studies, standard strains and growth conditions should be used
among laboratories for concise comparison of the results.
Stress responses probably have a much greater impact on food microbiology
than is currently appreciated. For example, many food processes and formulations
are tested for safety by inoculation with cells harvested from exponential phase.
However, cells grown to the stationary phase or adapted to various stresses will have
greater resistance than exponential cells. Our studies support the hypothesis that it
is important in laboratory food challenge studies and in testing of food processing
procedures to use stressed or adapted cells, since the use of healthy exponentially
growing cultures may inaccurately represent their survival state in the actual food
environment (Leyer and Johnson, 1992, 1993). The survival of foodborne pathogens
in certain acidic food products such as mayonnaise, salad dressings and sauces
(Smittle, 2000) may warrant re-evaluation using acid-adapted populations of cells.
Similar considerations should be given to foods stabilized by brining or by many
other food preservation processes that could trigger stress responses. The physio-
logical stress state of the pathogens may also affect current trends in risk assessment,
predictive modeling, HACCP programs, and in reaching a food safety objective.
The practical research of stress responses in foods could be complemented by
the elegant and currently more extensive studies of the molecular biology of these
responses. For example, it is becoming possible with the development of genetic
tools in many foodborne pathogens to create isogenic mutants, and strains can be
constructed that possess mutations in single genes involved in specific and general
stress responses. These mutants could then be evaluated for their survival of food
processing operations and for survival during storage of foods.
Newer applications of stress response analysis include comparative genomic and
proteomic views of stress responses in microbial species (Koonin et al., 2000;
Neidhardt and VanBogelen, 2000) and the utilization of stress responses for envi-
ronmental monitoring and molecular toxicology (LaRossa and van Dyck, 2000). The
rapidly increasing availability of complete genome nucleotide sequences (Koonin
et al., 2000; Wodicka et al., 1997) and amino acid sequences of expressed open
reading frames (ORFs) (Neidhardt and VanBogelen, 2000) in food-related organisms
will enable the identification of genes and proteins involved in stress responses and
evaluation of their importance in the physiology of food-related organisms. For
example, the complete genome of a strain of S. cerevisiae has been sequenced with
the identification of over 6000 ORFs and 5800 predicted protein coding regions
(Dujon, 1996). About one-third of these ORFs have completely unknown function
(Attfield, 1997; Dujon, 1996; Goffeau, 2000). As emphasized by Attfield (1997),
much more research is needed in physiological research for industrial strains of yeast
within the context of industrially relevant conditions. An understanding of physio-
logical and genetic determinants of stress responses and industrial performance will
provide rational methods using classical breeding approaches or recombinant DNA
technology for improvement of microbial strains. The use of genome-wide expres-
sion monitoring (Wodicka et al., 1997) will also be useful for following the temporal
expression of food microorganisms as they encounter stresses and induce adaptive
responses during industrial food fermentations.

The food industry has long applied combinations of sublethal intrinsic, extrinsic,
and implicit inhibitory factors for the control of undesirable organisms in foods and
to promote the growth of desirable microbes. As the public demands foods with
enhanced freshness and appeal, the industry is using milder processes and reducing
levels of antimicrobials in many food products. In turn, this results in more frequent
exposure of pathogens and spoilage organisms to sublethal stresses, which could induce
resistance responses and compromise the safety and shelf-life of these food products.
This trend further supports the need for practical studies of stress in food microbiology,
risk assessment, and preventive programs. Although the study of stress responses and
their impact on food safety and quality is in its gestation phase for most food-related
organisms, increased study should result in our understanding of the microbiology of
food systems and enhancement of the safety and quality of our food supply.
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Adaptation or Resistance
5 Responses of
Microorganisms to Stresses
in the Food Processing
Environment
Sadhana Ravishankar and Vijay K. Juneja
CONTENTS
Introduction
Biofilms
Bacterial Attachment
Stages in Biofilm Formation
Methods of Studying Biofilms
Control of Biofilms
Chemical Control
Biological Control
Chemical Stress
Acidic and Alkaline Treatments
Phosphates and Other Chemicals
Ozone
Sanitizer Stress
Chlorine and Chlorinated Compounds
Nonchlorinated Compounds
Sanitizer Stress Adaptation and Cross-Protection
Metal Ion Stress
Links to Antibiotic Resistance
Adaptation to Heavy Metal Ions and Cross-Protection
Antibiotic Stress
Cross-Resistance
The authors wish to acknowledge Dr. Ravishankar Palanivelu for his assistance with the figures. The
authors are also grateful to Drs. Sizer, Slade, and Palumbo for critical review of the manuscript, and to
Ms. Vasuhi Rasanayagam for her assistance with the citations.

Other Stresses
Adaptation to Starvation Stress
Adaptation to Osmotic Stress
Cross-Protection
Adaptation to Oxidative Stress
Cross-Protection
Conclusions
References
INTRODUCTION
Foodborne bacteria are exposed to a variety of stresses in the environment. Often-
times, they are able to tolerate such stresses, survive and/or grow in food and cause
spoilage as well as illness. If the stress is mild, it causes injury to the bacteria and
if it is severe, it causes inactivation. Injured bacteria in food are of concern, since
they can revive when favorable conditions are encountered, as well as multiply and
grow in food. Such mild stresses are very often encountered by the bacteria in food
as well as in the food processing environment. For instance, with the present day
consumer demand for fresh-like, preservative-free food products with good nutri-
tional quality, minimal processing is done in which mild treatments are given to the
food product. The bacteria once exposed to a mild stress are able to tolerate further
severe stresses. This ability of the bacteria is called stress adaptive response (SAR)
or stress hardening, which enables the bacterium to resist further homologous as
well as heterologous stresses (Yousef, 2000).
In the food processing environment, several treatments are given to the food to
preserve its quality as well as shelf-life. The environment and the equipment used
for processing in a plant handling wet processes are regularly or periodically cleaned
to keep them, as well as the processed food, contamination free. Under such con-
ditions bacteria are exposed to a variety of chemicals, sanitizers, heavy metal ions,
antibiotics, etc. If these treatments are not severe enough, the bacteria survive and
are able to adapt to even harsher treatments. These bacteria can form microcolonies
on the equipment surfaces or other areas of the plant which, in course of time, form
biofilms. Also, there are certain areas either in the equipment or other places in a
plant that are inaccessible or hard to reach for cleaning, and the bacteria escape
treatment. These areas are also most vulnerable for biofilm formation.
Once biofilms have been formed it becomes very difficult to eradicate them. For
instance, Listeria monocytogenes is a foodborne pathogen well known for its pres-
ence in processing plants (Smoot and Pierson, 1998; Cox et al., 1989) and for the
formation of biofilms, due to which the food industry has incurred heavy losses
especially in dairy (Mafu et al., 1990) and processed ready-to-eat meat products.
This bacterium is easily disseminated by aerosols and contaminated food products
in the processing plants (Cox, 1996) and can survive in aerosols (Spurlock and
Zottola, 1991). When this bacterium forms biofilms it has an enhanced resistance
to sanitizers (Frank and Koffi, 1990). Listeria monocyotgenes was isolated from
domestic, retail and industrial refrigerators in Greece (Sergelidis et al., 1997). Pathogens

such as Listeria, Staphylococcus aureus and Salmonella were isolated from a poultry
abattoir in South Africa (Geornaras et al., 1997) and Salmonella from healthy swine
and from abattoirs in Brazil (Lázaro et al., 1997). In a poultry slaughtering facility
the main airborne contaminants (bioaerosols) were bacteria and the highest count
was found in the shackling area and decreased toward the packaging area (Lutgring
et al., 1997). The microbial load of the floor after cleaning in different types of food
processing facilities was assessed and it was determined that a milk site had the
lowest load followed by the pastry site where the load decreased initially after
cleaning and then increased, while a meat site had the highest load with a rapid
increase (Mettler and Carpentier, 1998). Salmonella serovars were isolated from a
citrus processing plant both inside the premise (in the juice as well as surface of the
fruits) and outside the premise, from amphibians captured outside the plant (Parish,
1998). In plants handling wet processes, enough moisture and other favorable con-
ditions to promote microbial growth are found.
In plants where processing takes place under dry conditions, bacteria are able
to enter the plant through air, raw ingredients, worker traffic and other means. They
are exposed to dehydration and are able to adapt to such conditions, survive and
contaminate food products. For instance, Salmonella has been a problem due to its
contamination from the plant environment in dry dairy products, grain products,
chocolate products and others, causing several recalls (Gabis and Faust, 1988).
Aerobic spore-formers such as Bacillus species are the predominant microflora in
the food packaging material such as paper and board and these bacteria were found
to produce enzymes which degrade papermaking chemicals and were resistant to
industrial biocides (Väisänen et al., 1989). In dry processing plants, bacteria may
encounter areas where there is lack of nutrients. In such conditions bacteria are
exposed to starvation stress (Figure 5.1). Some other stresses that a bacterium may
encounter in a processing environment during exposure to chemicals, sanitizers, or
otherwise, include oxidative stress, osmotic stress, acidic/alkaline stress, etc.
(Figure 5.1). According to Bower and Daeschel (1999), the resistance responses of
bacteria in food environments are conferred by various factors including innate
structures such as impermeable outer membrane of bacterial cell, mechanisms for
antibiotic inactivation, and biofilm formation on food processing surfaces as an
adaptive response to prevent the hazardous effects of cleaners and sanitizers. Anti-
biotic resistant bacteria present in food animals, exhibiting cross resistance to bio-
cides, entering a food processing plant may pose a contamination risk to the pro-
cessed product. Antibiotic residues present in meat and milk have contributed to the
development of resistant bacteria (Brady and Katz, 1992; Brady et al., 1993). In this
chapter, various aspects dealing with the adaptation as well as resistance responses
of bacteria to various stresses in the food processing environment such as chemicals,
sanitizers, metal ions, starvation and antibiotics will be discussed.
BIOFILMS
In food processing plants, microorganisms are able to attach to solid surfaces and
form microcolonies. These, along with various nutrients, minerals and organic matter,
deposit together forming biofilms. Biofilms are defined as bacterial populations

FIGURE 5.1 Various stresses encountered by bacteria in a food processing environment.
adhering to a surface or to each other in aggregates enclosed by a matrix of polysac-

charides (Poulsen, 1999). The organisms become sessile in a biofilm and the matrix
forms a protective barrier against the effect of antimicrobial agents. Biofilms serve
two purposes for the forming microorganisms: protection from hostile environment
as well as acting as a trap for nutrient acquisition (Mattila-Sandholm and Wirtanen,
1992). Bacteria in a biofilm are exposed to starvation, dehydration and oxidative
stresses. As a result of these exposures bacteria adapt to these stresses. In addition
this exposure may cross-protect the bacteria against other stresses. Stress signaling
and cell to cell communication occur in a biofilm community. These could be possible
reasons for the enhanced resistance of biofilm bacteria compared to those that are in
a planktonic state. Hence biofilms and their resistances are discussed in this chapter.
Biofilms have been described as self-regulating in that they give rise to other
biofilms (Hood and Zottola, 1995). Once the biofilm grows in size, it breaks into
pieces allowing for more cells to attach to each piece of the matrix, which then
grows in size. This breaking up process could be attributable to possible alteration
in the properties of cell surface or attached substratum (Marshall, 1992). Food
processing plants have abundant exposed surfaces available and accessible for bio-
film formation and a typical biofilm can contain in excess of 107 cells/cm2 (Holah
and Kearney, 1992). Persistent L. monocytogenes isolated from poultry and ice cream
plants were able to show enhanced adherence capabilities to stainless steel even with
a short contact time (1 h) (Lunden et al., 2000). Thus they can become a real threat
to the processing industry. Biofilms have created problems in the industry in every-
thing from corroding water pipes to computer-chip malfunctions (Potera, 1996).

BACTERIAL ATTACHMENT
All spoilage and pathogenic microorganisms can form biofilms under suitable con-
ditions. However, bacteria belonging to the genera Pseudomonas, Enterobacter,
Flavobacterium, Alcaligenes, Staphylococcus and Bacillus have a tendency to form
biofilms more than others (Mattila-Sandholm and Wirtanen, 1992). It may take
several hours to days for the microorganisms to form a biofilm on a surface. Bacteria
adhere at different rates to different substrates as described by Marshall (1992).
Some bacteria possess the needed structures such as fimbriae or pili as well as other
extracellular polymeric substances and are able to attach quickly to surfaces. This
is called passive adhesion involving a physico-chemical reaction. Others require
prolonged exposure to attach to a surface, whereby a physiological response is
needed and this is called active adhesion. Initially at this stage the bacteria are not
very firmly attached, still showing some Brownian movement, and can be removed
by moderate shear. A transition from loose to firm attachment occurs causing irre-
versible adhesion.
Some bacteria attach more efficiently when starved, while others attach effi-
ciently when nutrients are available. Listeria monocytogenes was able to compete
and form biofilms with 8 other species at low nutrient levels at 10°C on stainless
steel coupons, and a Flavobacterium species stimulated the biofilm formation of
L. monocytogenes (Jeong and Frank, 1994). Listeria monocytogenes was also able
to form biofilms under nutrient limiting conditions involving a chemically defined
minimal medium with certain nutrients such as mannose, trehalose and tryptone
enhancing biofilm development, and alteration of phosphate to levels other than those
present in minimal medium reduced biofilm development (Kim and Frank, 1995).
Pseudomonas putida cells that grew very slowly in a flow chamber biofilm were
able to grow faster when provided with a readily metabolizable carbon source
(Sternberg et al., 1999). A particular strain of Pseudomonas attaches immediately
on high energy surfaces but slowly on low energy surfaces (Marshall, 1992). Bacteria
have their own approaches of attachment initiation to surfaces and involve specific
microbial surface structures such as flagella. Flagella may be directly required for
attachment and initiation of biofilm formation or indirectly required through enabling
mobile organisms to reach a surface as well as move along a surface in a developing
biofilm (Pratt and Kolter, 1998). Escherichia coli cells lacking flagella or having
paralyzed flagella were unable to initiate the initial biofilm formation (Pratt and
Kolter, 1998).
It has been observed that biofilms have capillary water channels through which
water and nutrients are transported and distributed to various areas (Costerton et al.,
1995). These capillary water channels also serve to transport oxygen to the inner
areas of the biofilm (Poulsen, 1999). The inner areas of the biofilm, however, may
receive low oxygen due to the diffusion limitations and exhaustion of oxygen by
the outer areas and thus it is possible that both aerobic and anaerobic species of
microorganisms can thrive in a biofilm (Poulsen, 1999). Also, the properties of the
substratum surface such as surface free energy, charge, roughness or toxicity towards
a specific microbial species influence the types or species of microorganisms colo-
nizing a surface (Marshall, 1992). In case of microorganisms with different mor-

phological structures, variants with rough morphology were able to attach better and
form biofilms than variants with smooth morphology (Pringle et al., 1983; Herald
and Zottola, 1988; Sasahara and Zottola, 1993).
There is either competition or cooperation among certain bacterial species as
well as coaggregation with certain species in mixed biofilm populations (Geesey
et al., 1992). Allison et al. (1998) found that cell to cell signaling mechanisms
involving homoserine lactones promote surface attachment and biofilm formation
and that enzymatic degradation of extracellular polymeric substance is involved in
detachment and dispersal of biofilms under conditions of starvation. Chemical
signals that are freely diffusible and employ N-acyl homoserine lactones involved
in biofilm development and maintenance also have been found in the case of other
Gram-negative bacteria (Davies et al., 1998; Heys et al., 1997).
Listeria monocytogenes grown in a medium with lactic acid showed enhanced
adhesion to stainless steel and cells appeared more hydrophobic when attached to
stainless steel at 4°C (Briandet et al., 1999). Sasahara and Zottola (1993) found that
L. monocytogenes alone was not able to attach to glass while P. fragi could. When
mixed with P. fragi culture, L. monocytogenes was able to attach to glass and form
biofilms, and the authors suggested that L. monocytogenes, a non-exopolymer pro-
ducer, needs an exopolymer producing organism such as P. fragi for attachment and
this is more important than other requirements such as hydrophobicity, surface charge
or flagellar mobility, particularly for attachment to glass. Stainless steel surfaces
(reactive) were found to be colonized and attached by 10-fold more coliforms and
heterotrophic bacterial cells when compared to an inert surface such as polycarbonate
(Camper et al., 1996). However, if stainless steel slides were replaced with polycar-
bonate slides, these could harbor the same amount of bacteria as did stainless steel.
Growth media and conditioning of the attaching surface were found to be
contributing factors in the attachment of P. fragi, S. typhimurium and L. monocyto-
genes to stainless steel (Hood and Zottola, 1997a). Environmental factors such as
temperature and pH affected the attachment of L. monocytogenes to stainless steel
and Buna-N rubber, with rate of adhesion being slow at alkaline conditions (Smoot
and Pierson, 1998a). The addition of trypsin to attachment medium decreased the
adhesion by 99.9%, implying that proteins are involved in the initial attachment of
this organism to surfaces (Smoot and Pierson, 1998b).
Aeromonas hydrophila was able to attach to stainless steel and form biofilms at
28°C, but not at 42 or 4°C and this could be a problem in seafood and aquaculture
processing plants if effective sanitation measures are not applied (Farid et al., 1998).
In a poultry processing plant, bacteria from the rinse of whole broiler carcasses were
found to attach to stainless steel and form biofilms (Arnold, 1998). In such a biofilm,
mixed populations of E. coli and S. aureus were found. When grown as a pure culture
separately, E. coli cells formed extracellular fibrils while S. aureus did not
(Figure 5.3). However, in mixed culture, the formation of extracelluar fibrils was
different from what was seen in pure culture. Thus the physiological behavior of
the bacterial species changes in a mixed population biofilm and each species is able
to adapt and grow in a competitive environment.
Gram-negative bacteria were better able to adhere to glass and form biofilms
with a higher population than Gram positives (Sommer et al., 1999). E. coli cells

FIGURE 5.2 Initial steps in biofilm formation.
attached and formed biofilms on stainless steel and the formation was affected by
the nutrient conditions of the medium used (Dewanti and Wong, 1995). These authors
found that in a nutrient limiting medium, the cells were shorter and more hydropho-
bic due to starvation stress, but an extensive and thicker extracellular matrix was
produced when compared to those grown in nutrient rich medium. On chlorinated
polyvinyl chloride pipe and glass surfaces, Klebsiella pneumoniae, Salmonella enter-
itidis and E. coli attached and formed biofilms, with K. pneumoniae forming the
most populated and metabolically active biofilm followed by S. enteritidis and then
E. coli, respectively (Jones and Bradshaw, 1996). L. monocytogenes produced copi-
ous amounts of attachment fibrils, while E. coli did not, on stainless steel surfaces
after one week incubation (Mustapha and Liewen, 1989). Biofilm formation in meat
processing plants was studied by fixing stainless steel chips adjacent to food contact
surfaces and cast iron chips in floor drains, and Pseudomonas, Klebsiella, Aeromonas
and Hafnia were found to produce biofilms (Hood and Zottola, 1997b).
Microorganisms attached to vegetable surfaces are not completely removed by
washing and minimal processing and thus can grow and form biofilms during storage
(Carmichael et al., 1999). Bacteria were able to attach and form biofilm on the
surface of lettuce with pseudomonads being the dominating microflora (Carmichael
et al., 1999). On apples, E. coli O157:H7 was observed at depths up to 70 µm below
the skin, with greater numbers on puncture wounds and greater attachment levels
on the intact skin, lenticels, russet areas and floral tubes (Burnett et al., 2000).
STAGES IN BIOFILM FORMATION

The process of biofilm formation occurs in several stages. The initial steps in biofilm
formation are depicted in Figure 5.2. The various stages involved in biofilm forma-
tion explained in the proposed theories are shown in Table 5.1. Marshall et al. (1971)
explained it to be a two stage process. The first stage is called the reversible stage,
in which the bacterial cells are in close proximity, but not yet in actual contact with
the substrate or surface, and they are held close together by electrostatic and hydro-
phobic interactions and van der Waals forces. The second stage is called the irre-
versible stage, in which the cells attach to the surface by producing complex polysac-

FIGURE 5.3 Escherichia coli and Staphylococcus aureus in pure (A and B) and mixed culture
(C) biofilm attached to stainless steel. The products of extracellular fibrils in single culture
biofilm are different from that of mixed culture biofilm. (From Arnold, J.W. 1998. Poultry
Avian Biol. Rev., 9(1):1–9. With permission.)
TABLE 5.1
Various Stages in Proposed Biofilm Formation Theories
Busscher & Characklis &
Weerkamp, Cooksey, 1983;
Marshall 1987; Characklis, 1984; Gilbert Bos
et al., Notermans Characklis, Ganesh Kumar & et al., et al.,
Stages 1971 et al., 1991 1981 Anand, 1998 1993 1999
Reversible adhesion √ √
Irreversible adhesion √ √ √
Transport of material √ √ √ √
to surface
Adsorption √ √ √ √
Consolidation √
Adhesion √ √ √
Co-adhesion √
Adaptation √
Growth and biofilm √ √
formation
Colonization and √ √ √
biofilm formation
Detachment and √ √
dispersal
charides and the attachment involves hydrophobic, dipole–dipole, ion–ion, covalent

bonds and hydrogen interactions. The cells multiply forming microcolonies and
eventually form biofilm with cells embedded within the polysaccharide matrix. Many
of the chemical and physical characteristics of the matrix, such as binding to metal
ions and viscoelastic behavior, depend on the type of polysaccharide (Christensen,
1989).
A three stage process has been proposed by Busscher and Weerkamp (1987)
and Notermans et al. (1991), which involves 1) adsorption of cells to the surface,
2) consolidation involving electrostatic interactions, van der Waals forces and other
interactions, and, finally, 3) colonization.

Characklis (1981) described a four stage process of biofilm formation, involving
1) transport of the molecules to the surface by diffusion or turbulent flow,
2) adsorption of the molecules at the surface, 3) adhesion of microorganisms to the
surface using van der Waals, electrostatic and hydrophobic interactions, and
4) colonization resulting in biofilm formation.
A five stage process of biofilm formation has also been described (Characklis
and Cooksey, 1983; Characklis, 1984; Ganesh Kumar and Anand, 1998). The five
stages include 1) transport of organic and inorganic material to the surface and
conditioning of the surface, 2) adsorption of conditioning film and adhesion of
microorganisms, 3) attachment of cells, growth and formation of microcolonies
4) formation of biofilm and bacterial metabolism within the film and 5) detachment
and dispersal of biofilms.
Gilbert et al. (1993) describe the process of surface colonization in six steps as
follows: 1) arrival of bacteria in close proximity to the surface, 2) reversible adhesion
to the surface, 3) irreversible adhesion to the surface, 4) adaptation to an attached
phenotype, 5) growth and division, microcolony and biofilm formation, and
6) dispersion.
Bos et al. (1999) provide an excellent review of the physico-chemistry of micro-
bial adhesive interactions in which they describe the initial steps in biofilm formation
in six stages: 1) adsorption of conditioning film components, 2) microbial transport
and aggregation, 3) adhesion of single organisms and microbial coaggregation, 4) co-
adhesion between microbial pairs, 5) exopolymer production and establishment of
firm irreversible adhesion, and 6) growth. All these models explain a similar phe-
nomenon but in different ways.
METHODS OF STUDYING BIOFILMS

Bacteria in the biofilms are quantitated by different methods such as swabbing,
rinsing, agar flooding, agar contact (Ganesh Kumar and Anand, 1998), microscopic
beads (Oh and Marshall, 1995), scraping (Frank and Koffi, 1990; Costerton and
Lappin-Scott, 1989) and vortexing (Mustapha and Liewen, 1989; Oh and Marshall,
1995). Another method utilizing Robbins device has been described and used in
which biofilms are formed on small coupons (metal or plastic studs) simulating a
surface, by exposing them to large amounts of liquid for about 3 to 4 weeks, after
which the studs are removed and the amount of biofilm formed is estimated (Cos-
terton and Lappin-Scott, 1989; Jass et al., 1995). The Robbins sampler also could
be used to determine the concentrations of antimicrobial agents needed to inactivate
biofilm bacteria and hence can be useful in designing such treatments (Costerton
and Lappin-Scott, 1989).
Other techniques of monitoring biofilms include epifluorescence microscopy
(Wirtanen and Mattila-Sandholm, 1993; Blackman and Frank, 1996), scanning elec-
tron microscopy (Blackman and Frank, 1996; Farid et al., 1998), environmental
scanning electron microscopy (Little et al., 1991), scanning confocal laser micros-
copy (Caldwell et al., 1992; Debeer et al., 1997; Carmichael et al., 1999; Sternberg
et al., 1999), light section microscopy (Marshall et al., 1989) atomic force microscopy

(Beech, 1996), interference reflection microscopy (Marshall et al., 1989), nuclear
magnetic resonance (Blenkinsopp and Costerton, 1991), Fourier transformation
infrared spectrometry (Nichols et al., 1985; Bremer and Geesey, 1991; Cheung et al.,
2000), quartz crystal microbalance (Nivens et al., 1993) and cellular automation
(Wimpenny and Colasanti, 1997).
Some researchers have used the direct viable count methods for enumeration of
biofilms and assessing the efficacy of disinfection (Lytle et al., 1989; Feipeng et al.,
1993; Leriche and Carpentier, 1995). The impedance method was the most effective
enumeration method for biofilm bacteria due to its ability to quantitate both reversibly
and irreversibly attached cells while some other methods require additional steps to
remove reversibly attached cells (Mosteller and Bishop, 1993). Control of the growth
rate of biofilms has been achieved using the Perfused biofilm fermenter (Gilbert et al.,
1989) and Sorbarod filters (Hodgson et al., 1995). Methods for studying biofilms
have been reviewed by Ladd and Costerton (1990) and Nivens et al. (1995).
CONTROL OF BIOFILMS
In the food processing industry biofilms are a source of pre- or post-processing
contamination and hence care should be taken to avoid the formation of biofilms.
Proper cleaning and sanitation are needed to avoid formation or to eradicate the
formed biofilm. The areas of the processing equipment most prone to biofilms
include gaskets made of Buna-n rubber or Teflon, pipe elbows, caps in dead-end
areas, the vacuum breaker near the pasteurizer, backplates of pumps, conveyer belts,
as well as drains, floors and other stainless steel surfaces (Czechowski, 1991). Food
processing surfaces such as stainless steel, nylon, Teflon and polyester floor sealant
support the growth of L. monocytogenes biofilms, with polyester floor sealant and
stainless steel allowing the most, Teflon allowing an intermediate and nylon allowing
the least formation when incubated at 21°C in tryptic soy broth (Blackman and
Frank, 1996).
Chemical Control
If the process conditions are different from those required for optimum growth of
microorganisms, biofilm formation can be prevented (Poulsen, 1999). However, in
many processing conditions, this is not the case and so other methods such as
maintaining proper hygiene or frequent cleaning and sanitizing are required. Frequent
cleaning with a gap of about 8 h was found to remove the attached organisms easily
and prevented biofilm formation (Zottola, 1994). The best way to control biofilms
is to effectively clean and then sanitize the surfaces to which they are attached.
Initial cleaning with a detergent should be done in such a way that it dissolves the
biofilm and removes it from its attachment site. A sanitizing step following this will
inactivate the surface attached microorganisms. Factors affecting the efficacy of a
sanitizing agent include the type of the sanitizer, concentration used, cleaning tem-
perature and time, flow rate of the sanitizing solution, hardness of water for diluting
the sanitizer, age of the biofilm, and the type of surface to be cleaned (Czechowski,
1991). Disinfection of K. pneumoniae attached to glass surfaces by chlorine
depended on the surface, biofilm age, encapsulation and nutrients, while disinfection

by monochloramine was only affected by surfaces (LeChevallier, 1988a). It is a
generally accepted fact that inactivation of biofilms is difficult since the sanitizer or
the toxic compound has to penetrate the exopolysaccharide barrier. However, Nichols
(1989) proposed that the cells deeply embedded in a biofilm may have a different
cellular physiology compared to the cells at the surface and hence their sensitivity
to a particular sanitizer or toxic substance may be different from those at the surface.
A number of researchers have investigated the efficacy of various disinfectants
and sanitizers in controlling biofilms. Chlorine and monochloramine were tested for
their efficacy against planktonic and attached organisms (Yu et al., 1993). During
formation of biofilms there was no change in the susceptibility of the planktonic cells
to these disinfectants. There was no difference between the resistance of attached and
planktonic cells. Monochloramine proved to be more effective in eliminating the
attached organisms than free chlorine. Monochloramine was more effective in pen-
etrating the biofilm and inactivating bacteria while free chlorine reacted with a number
of compounds before it penetrated into the biofilms (LeChevallier et al., 1988b).
In another study with Enterobacter cloacae, about 50% of the cells survived
exposure to 0.5 mg chlorine per liter and multiple exposures to chlorine did not
increase resistance of the cells (Lytle et al., 1989). In the same study it was found
that 25 mg chlorine was better in inactivation than 0.5 or 2 mg. Maintaining 1 mg/L
free chlorine residual was not sufficient to control coliform biofilms in water distri-
bution pipelines (LeChevallier et al., 1987). Chlorine (0.5 mg/L) was ineffective in
removing the total bacterial population in the biofilm formed in a water distribution
system consisting of coliforms, K. pneumoniae and other bacteria (Morin et al.,
1996). Monochloramine at 2 mg/L was ineffective in controlling K. pneumoniae and
P. aeruginosa biofilms (Huang et al., 1995). These authors also studied the respira-
tory activity of these biofilms during disinfection with monochloramine and they
found that there was spatially nonuniform loss of respiratory activity with less
activity near the biofilm–bulk fluid interface and more activity near the substratum
or in the center microcolonies. The reason was attributed to transport limitation and
the depletion of the sanitizer near the substratum or center of microcolonies as
compared to the surface.
Wirtanen and Mattila-Sandholm (1992) studied biofilm formation of P. fluore-
scens, L. monocytogenes and B. subtilis and found that at least 2 h are needed for
biofilm formation and that the resistance of the formed biofilms to chlorine was
higher when formed in milk when compared to meat medium. Older biofilms of
Pseudomonas were more resistant to chlorine and this resistance was attributed to
changes in the metabolic activity of bacteria in the biofilms or production of extra-
cellular compounds that can react with chlorine, preventing its diffusion (Sommer
et al., 1999). In the same study the authors also found that both large and small
biofilms had similar resistance to chlorine, and they attributed the reason to the
formation of microcolonies having similar resistances.
Mechanically scrubbing and cleaning the surface along with disinfectants is one
effective way of eliminating biofilms. In a study by Exner et al. (1987) various
chemical disinfectants were tested for their efficacy in controlling biofilms against
mechanical cleaning. Aldehydes and chlorine were effective at 10% concentration,
peracetic acid at 0.5% and hydrogen peroxide at 1.5% with 30 min to 1 h exposure

times. Mechanical cleaning was the most effective with scanning electron micro-
graphs of treated biofilms showing clean surfaces and no microorganisms being
isolated after the treatment. In a food factory environment, biofilms formed by
Staphylococcus aureus and Pseudomonas aeruginosa were most effectively elimi-
nated by mechanical cleaning using a high pressure spray and mechanical floor
scrubber than by chemical methods involving detergents (Gibson et al., 1999). In
the same study applying an alkaline, acidic or neutral detergent prior to water spray
did not prove effective in removing biofilms. The acidic detergent was most bacte-
ricidal towards S. aureus causing about a six-log reduction, while alkaline detergent
was bactericidal towards P. aeruginosa, causing four- to five-log reductions. The
authors concluded that since mechanical spray and scrub have potential to dissem-
inate bacteria through aerosols, it might be more effective to utilize a mechanical
treatment followed by chemical methods such as using bactericidal detergents to
eliminate the contamination.
Bacteria in a biofilm are more resistant to the effect of sanitizers than the free
living cells and, the older the biofilm, the greater the resistance to sanitizer treatments.
Oh and Marshall (1995) found that a combination of monolaurin at 50 µg/ml and
heating at 65°C for 5 min effectively controlled L. monocytogenes adhered on to
stainless steel. They also found that planktonic cells were more sensitive to the
treatment than attached cells; 1-day-old biofilms were more sensitive than 7-day-old
biofilms; and resistance in a nutrient rich environment was better than that in a nutrient
lacking environment. Attached cells of L. monocytogenes and S. typhimurium were
found to be more resistant to trisodium phosphate than the planktonic cells (Somers
et al., 1994). Attached cells of B. subtilis and P. aeruginosa on stainless steel and
polyurethane surfaces were more resistant to iodophor, peracetic acid and hydrogen
peroxide mixture, and chlorhexidine gluconate than the planktonic cells (Lindsay and
von Holy, 1999). Cells of S. aureus, E. coli, S. enteritidis and L. monocytogenes
attached to PVC, Teflon, Plexiglas, wood, rubber and stainless steel were more
resistant to QAC (quaternary ammonium compound) sanitizer than non-attached ones
(Dhaliwal et al., 1992). A non-foaming acidic sanitizer and a hypochlorite sanitizer
were not effective on P. putida biofilms formed on rubber surfaces in that they caused
merely injury; resuscitation of the samples had growth equal to those of untreated
controls (Chumkhunthod et al., 1998). E. coli O157:H7 was able to attach to meat
grinder surface from contaminated meat and the surfaces that came in contact with
the processed meat having the lowest fat level were the hardest to clean; chlorine and
peroxyacetic acid sanitizer effectively reduced the bacterial count but following
enrichment, injured cells were recovered (Farrell et al., 1998). Rinsing with super-
heated water was not effective in removing Bacillus biofilms from Teflon and stainless
steel; however increasing the alkaline phase wash of alkaline-acid treatment and
addition of EDTA to the alkaline detergent proved effective (Wirtanen et al., 1996).
The effects of chemical treatments including electrolytes, dimethyl sulfoxide
and Tween 20 on microbial adhesives and biofilm matrix polymer using P. aerugi-
nosa were investigated using interferon reflection microscopy and light section
microscopy (Marshall et al., 1989). There was contraction of the microbial polymers
and adhesives during treatment with electrolytes and expansion during treatment
with Tween 20. The dimethyl sulfoxide treatment caused contraction on the adhesive

but had no effect on biofilm polymer. It was concluded that both adhesive and
polymer have acidic groups and undergo hydrophobic reactions. However, it was
unclear as to the difference in the behavior of the adhesive and polymer to dimethyl
sulfoxide reaction, whether it was due to differences in their structure or due to their
proximity to glass surface.
Both pure and mixed culture biofilms of P. flourescens and B. cereus were
equally affected by sanitizers such as chlorine, iodophor, peracetic acid, acid anionic
and fatty acid sanitizers; however, pure culture biofilms of Y. enterocolitica were
more resistant to these sanitizers than when they were in a mixed culture (Mosteller,
1993). Sodium periodate, cetyltrimethylammonium bromide, and sodium hydroxide
inhibited attachment of P. fragi to stainless steel, and sodium dodecylsulfate, sodium
periodate and sodium hydroxide effectively removed attached cells (Herald and
Zottola, 1989). Aeromonas hydrophila biofilms on stainless steel surfaces were
inactivated by chlorine with exposure to 25 ppm for 1 min for 8-h biofilms, and
75 ppm for 1 min for 8-day-old biofilms (Farid et al., 1999). Peroctanoic acid (Cords,
1993) was more effective on binary species biofilms of P. aeruginosa and L. mono-
cytogenes than peracetic acid and chlorine on stainless steel coupons (Fatemi and
Frank, 1999). Ozone was suggested as a potential disinfectant against P. flourescens
biofilms, where the ozone treated cells appeared non-intact and shriveled (Bott, 1991).
The effect of 58 chemical compounds including antibiotics, detergents, surfac-
tants, nutrients and inorganic and organic acid salts on swarming (movement of
elongated swarm cells across a surface) of Bacillus species was studied and it was
found that most of the compounds tested inhibited swarming through their action
on flagellar mechanisms and motility (Thampuran and Surendran, 1996). Since
motility is an important feature facilitating bacterial biofilm formation, these chem-
ical compounds could play a role in impeding motility and thus biofilms.
A buffered organic acid anionic acid surfactant (containing citric acid, EDTA,
sodium lauryl sulfate and an anionic detergent) exhibited sanitizing efficiency by
inactivating S. aureus, S. typhimurium, L. monocytogenes and P. aeruginosa on
formica countertop surfaces, both in the presence and absence of protein (Restaino
et al., 1994). QACs and chlorine sanitizer effectively reduced S. aureus populations
on abraded and smooth stainless steel and polycarbonate surfaces but not on mineral
resin (Frank and Chmielewski, 1997). A number of sanitizers (iodophors, amphot-
erics, QAC, chlorine, Biguanide) were assessed for efficacy using a surface disin-
fectant test against attached cells of P. aeruginosa, S. aureus and Proteus mirabilis
and attached cells were found to be 10 to 100 times more resistant than free cells
(Holah et al., 1990). Chlorine (25 ppm), QAC (20 ppm) and iodine(12.5 ppm) were
effective against P. fluorescens, Staphylococcus haemolyticus and Bacillus in a
simulated water coolant system at 25 and 4°C (Overdahl and Zottola, 1991). Attach-
ment of P. aeruginosa and Staphylococcus epidermidis to glass and stainless steel
coupons was loosened by treatment with disinfectants such as sodium hypochlorite,
Dodigen™, and sodium dodecylsulfate, while Tween-80 strengthened the attachment
of S. epidermidis to stainless steel (Eginton et al., 1998).
Some lactic acid bacteria are capable of producing ropy slime on the surface of
cooked meat products and vacuum packaged meats. The efficacy of sanitizers was
tested against the ropy slime forming lactobacilli, and QACs, peracetic acid sanitizers

and hydrogen peroxide were found effective, while chlorine sanitizers and polyhex-
amethylene biguanide chloride were ineffective (Mäkelä et al., 1991).
Biological Control
Apart from detergents and sanitizers, the use of enzymes for biofilm control has
been investigated. Johansen et al. (1997) tried a combination of enzymes to break
up Staphylococcus and Pseudomonas biofilms on steel and polypropylene. A com-
bination of oxidoreductases and polysaccharide hydrolyzing enzymes was found to
be the most effective in removing biofilm as well as being bactericidal. A combi-
nation of glucose oxidase and lactoperoxidase was bactericidal, but did not remove
biofilms, and a mixture of polysaccharide hydrolyzing enzymes removed the biofilm
but did not prove to be bactericidal. Other enzymes that have been used for control-
ling biofilms include proteases (Aldridge et al., 1994), cellulase (Wiatr, 1990),
polysaccharide lyases (Sutherland, 1995) and lactoperoxidase (Thomas et al., 1983).
The antimicrobial peptides magainins and defensins were found to be bacteri-
cidal toward rough strains of S. typhimurium compared to smooth ones (Rana and
Blazyk, 1989). Bacterial cells respond to an antimicrobial peptide by altering their
membrane composition (Brul and Coote, 1999). L. monocytogenes cells showing
resistance to nisin demonstrated enhanced levels of zwitterionic phosphatidyl-
ethonalamine and lowered levels of anionic phosphatidylglycerol and cradiolipin
(Crandall and Montville, 1998). The phospholipid membrane composition of nisin
resistant L. monocytogenes was different from that of nisin susceptible strains
(Verheul et al., 1997). Treatment of stainless steel coupons with skim milk before
inoculation of microorganisms reduced the attachment of the organisms, and indi-
vidual milk proteins such as α-casein, β-casein, κ-casein and α-lactalbumin inhibited
the adhesion of S. aureus and L. monocytogenes (Barnes et al., 1999). Concanavalin
A inhibited the attachment of P. fragi to stainless steel, and trypsin was effective in
removal of attached cells (Herald and Zottola, 1989).
Nisin films adsorbed onto silica surfaces prevented the growth of L. monocyto-
genes while the organism was able to grow on a non-nisin silica surface (Bower
et al., 1995). Nisin spray was effective in reducing populations of Gram-positive
bacteria such as Brochothrix thermosphacta, Carnobacterium divergens and Listeria
innocua on the surface of beef carcass tissue (Cutter and Siragusa, 1994a). Pratt and
Kolter (1998) found that α-methyl-d-mannoside inhibited biofilm development on
polycarbonate, polystyrene and borosillicate glass, and hence mannose could be used
in antimicrobial treatments to treat and prevent biofilms in the food processing plants.
Bdellovibrios, predatory microorganisms that can grow within the periplasm of
Gram-negative bacteria and prey upon them, have been found to be capable of
removing E. coli O157:H7 and Salmonella attached on the surfaces of food process-
ing equipment (Fratamico and Cook, 1996).
CHEMICAL STRESS
Bacteria are exposed to a variety of chemicals in the food processing plant environ-
ment. They may adapt to the chemical stresses during this exposure and exhibit

enhanced resistance to the chemicals they are further exposed to. In E. coli and other
enterobacteria extracellular induction components called alarmones present in the
environment warn the organisms of any chemical stresses in the environment by
cross talk leading to induction of tolerance responses or adaptation in the organisms
(Rowbury, 2001). The response of the bacteria to chemicals depends on various
factors: the type of the chemical and the bacteria, the concentration of the chemical,
exposure type, and other physical, chemical and biological environmental factors
(Levine and Case, 1997). The efficacy of acetic acid spray wash against E. coli
O157:H7 on beef surface depended on the initial inoculum level and the inoculation
menstrum but not on the spray temperature or tissue type (Cutter et al., 1997).
Any chemical compound causing microbial inactivation is also referred to as
biocide (Russell and Russell, 1995). Biocides have enhanced activity at elevated
temperatures, and other factors influencing biocidal activity include: presence of
non-ionic surfactants, materials such as polyvinyl chloride or nylon to which a
preservative agent may bind, or rubber in which the agent may partition, thereby
resulting in reduced effect (Russell, 1992). The Gram-positive cocci are generally
more susceptible to biocidal action than Gram-negative cocci. Excellent literature
reviews on effects of chemicals and biocides on microorganisms have been published
(Levine and Case, 1997; Levine and Black, 1996; Levine and Rachakornkij, 1994;
Russell and Rusell, 1995). Some of those specific to foodborne microorganisms and
their actions are summarized in Table 5.2.
Nirmalakhandan et al. (1994) studied the toxic effects of a variety of chemicals
alone and in combinations and found that the effect was additive. In their study they
also developed an approach to analyze and predict chemical toxicity using the
molecular structural features of the constituent chemicals. The toxicities of selected
industrial chemicals to microorganisms and aquatic organisms were compared and
it was found that aquatic organisms were more sensitive to these chemicals than
microorganisms (Vaishnav and Korthalis, 1990). Some possible reasons for the low
sensitivity of microorganisms were 1) the presence of cell wall and capsule barriers
through which the chemical has to travel to reach the cell membrane or the target
component, 2) endospore formation under adverse conditions, 3) spontaneous muta-
tions, and 4) ability of microbial cells to detoxify certain chemicals. These are also
some possible reasons for the adaptation and resistance of bacteria to chemical
stresses in the food processing plants. When Leuconostoc mesenteroides was exposed
to ethanol and sodium arsenite, there was an increase in the expression of 70 and
60 kDa proteins while the total protein synthesis was reduced (Salotra et al., 1995).
ACIDIC AND ALKALINE TREATMENTS

Some of the bacterial control methods used in the food industry include acidic and
alkaline treatments. Organic acid sprays and rinses are used to reduce or eliminate
surface contamination. Oftentimes, bacteria develop resistance to these treatments
which could arise from adaptation. Hot acid sprays of 1.5% acetic, citric and lactic
acids were ineffective in reducing E. coli O157:H7 populations on beef surface
(Brackett et al., 1994). Lactic acid was found to be ineffective in reducing E. coli
and Salmonella typhimurium populations on beef (Anderson and Marshall, 1990).

TABLE 5.2
Mechanism/Action of Various Chemical/Biocidal Compounds
on Microorganisms
Compound Mechanism/Action
Alcohols Leakage of cytoplasmic constituents, disruption of

membrane, inhibition of membrane ATPase and transport
proteins
Antimicrobial peptides, proteins and Cell wall hydrolysis, inhibition of protein synthesis, cell
enzymes membrane disruption
Carbanyl cyanide m-chlorophenyl hydrazone Inhibition of electron transport and oxidative
phosphorylation
Chlorhexidine diacetate Cell wall disruption, protoplast/spheroplast lysis, leakage
of cytoplasmic constituents, inhibition of membrane
ATPase, coagulation of DNA, RNA, protein
Chlorine Inhibition of mRNA, protein synthesis and oxidative
phosphorylation, release of dipicolinic acid from spores,
spore coat and cortex degradation
Cycloserine D Inhibition of cell wall protein synthesis
Dinitrophenol Dissipation of membrane proton motive force (pmf),
inhibition of transport and enzymes in the cytoplasm and
membranes
Ethidium bromide Inhibition of transcription
Ethylenediamine-tetraaceticacid Cell wall disruption
Ethylene oxide Alkylation of proteins and nucleic acids
Glutaraldehyde Binding to cell wall components and inhibition of essential
functions, cross linking of proteins, RNA and DNA,
reaction with spore outer layer
Hexachlorophane Protoplast/spheroplast lysis, leakage of cytoplasmic
constituents, cytoplasmic coagulation, interference in
electron transport
Hydrogen peroxide Oxidation of DNA, RNA, proteins and lipids, removal of
coat protein, lysis of spore protoplast
Iodine compounds Oxidation of sulfhydryl groups of proteins and disruption
of protein structure
Long chain polyphosphates Cell wall binding and chelation of metal ions
Mercury compounds Reaction with enzyme thiol groups
Naturally occurring compounds from plants Membrane perturbation, membrane rupture
A. Spice essential oils Oxidative cleavage of disulfide bonds and inactivation of
B. Isothiocyanates extracellular enzymes, formation of reactive thiocyanate
C. Thymol radicals aiding in oxidation of biomolecules
Plasma membrane disruption
P-aminobenzoic acid Inhibition or interference with peptidoglycan layer
synthesis
Parabens Dissipation of proton motive force, inhibition of transport
and enzymes in cytoplasm and membrane
Peracetic acid Disruption of thiol groups
Phenols Leakage of cytoplasmic constituents

Mechanism/Action of Various Chemical/Biocidal Compounds
on Microorganisms
Compound Mechanism/Action
Quaternary ammonium compounds Disruption of cell wall and leakage of cytoplasmic

constituents, coagulation of DNA, RNA, protein
Silver salts Reaction with sulfhydryl groups of proteins to form
mercaptides
Weak organic acids Membrane disruption, inhibition of metabolic reactions,
accumulation of anions and protons inside the cell,
thereby affecting the intracellular pH homeostasis,
dissipation of pmf
(Adapted from Brul, S. and Coote, P., Int. J. Food Microbiol., 50, 1, 1999; Lee, R.M. et al., 1994; Russell,
A.D. and Russell, N.J., Symp. Soc. General Microbiol., 53, 327, 1995.)
E. coli was found to be resistant to 3% acetic acid on beef (Greer and Dilts, 1992).
E. coli O157:H7 was more resistant to acetic acid compared to S. typhimurium and
L. monocytogenes on beef (Dickson, 1991). Acetic and lactic acids were ineffective
while fumaric acid was effective in reducing populations of E. coli O157:H7,
S. typhimurium and L. monocytogenes on beef (Podolak et al., 1995). However, acid
adapted Salmonella strains were sensitive to lactic acid rinse on beef (Dickson and
Kunduru, 1995). Lactic acid (2 to 5%) when used to decontaminate meat increased
the generation times of Yersinia enterocolitica and L. monocytogenes by up to two-
fold while at 1% there was no effect (van Netten et al., 1997). Acetic, lactic and
citric acids at 1, 3 and 5% concentrations brought about one- to two-log reductions
in E. coli O157:H7 populations but did not inactivate the pathogen completely on
beef tissue, while greater reductions were achieved for P. fluorescens with the same
treatments (Cutter and Siragusa, 1994b).
Acetic and lactic acids and trisodium phosphate reduced the counts of E. coli
O157:H7, L. innocua and C. sporogenes on beef surface to less than 1.3 logs (Dorsa
et al., 1997). A combination of sodium hydroxide and acetic acid was effective in
eliminating L. monocytogenes biofilms attached to glass, while a combination of
sodium chloride with acids was not effective (Arizcun et al., 1998). Catfish fillets
inoculated with L. monocytogenes were dip treated with various acids and such
treatments resulted in 16-, 7.5-, 4.3-, 3.7- and 3.4-fold reductions in the population
of the organism with tartaric, succinic, acetic, malic and propionic acids, respectively,
while no effect from tannic acid treatment was seen (Marshall and Bal’a, 1995).
Fumigation of mung bean seeds with gaseous acetic acid (242 µl acid/l of air for
12 h at 45°C) inactivated E. coli O157:H7 and S. typhimurium, but not L. monocy-
togenes (Delaquis et al., 1999).
Microorganisms grown in mild acid or alkalinity in broth were able to resist
stronger acidic or alkaline conditions and habituation to acid in broth can occur in
as small a duration as 15 min and to alkalinity in 30 to 60 min (Rowbury et al., 1989).
This shows that the time required for bacteria to adapt to stresses could be very

short. Tolerance to alkaline (pH 10 to 11.5) conditions was induced in E. coli by
adapting at pH 8.5 to 9.5 and this induction required protein synthesis, which was
inhibited by glucose and amiloride but not by L-leucine, FeCl3 or FeSO4 (Rowbury
et al., 1996).
PHOSPHATES AND OTHER CHEMICALS

A variety of chemical treatments were used to inactivate pathogens and spoilage
organisms on chicken skin (Hwang and Beuchat, 1995). Trisodium phosphate (1%)
and 1% lactic acid were effective in reducing populations of Salmonella species,
L. monocytogenes and psychrotrophs from chicken skin. Addition of 5% Tween 80
and 0.05% sodium hydroxide to trisodium phosphate effectively reduced Salmonella
species and psychrotrophs but not L. monocytogenes, due to its resistance to alka-
linity of sodium hydroxide. Ten percent solutions of sodium tripolyphosphate, mono-
sodium phosphate, sodium acid pyrophosphate or sodium hexametaphosphate were
not effective against Salmonella species or L. monocytogenes. Trisodium phosphate
and cetylpyridinium chloride with a high pressure spraying action reduced
S. typhimurium on chicken skin by 1.5 to 2.5 logs (Wang et al., 1997). Trisodium
phosphate, cetylpyridinium chloride, acetic acid and grapefruit seed extract brought
about 1.5- to 2.0-log reduction in S. typhimurium populations on chicken skin (Xiong
et al., 1998). Cetylpyridinium chloride was effective against E. coli O157:H7,
S. typhimurium and mesophilic bacteria on beef lean and adipose tissue surfaces
after cleaning and during storage (Cutter et al., 2000) and against S. typhimurium
on chicken skin (Breen et al., 1997). Trisodium orthophosphate alone and in com-
bination with KCl at 3% level and 60 sec contact time caused 99.6% reduction in
the surviving populations of P. aeruginosa, B. cereus and Moraxella osloensis in
fish and shell fish (Bender and Brotsky, 1993). Trisodium phosphate was more
effective than sodium tripolyphosphate and sodium metaphosphate in reducing total
coliform and aerobic plate counts on catfish frames (Marshall and Jindal, 1997).
Trisodium orthophosphate at 4 to 12% concentrations and alkaline pH (>11.5)
was also effective in reducing or retarding growth of Salmonella and Campylobacter
on poultry (Bender and Brotsky, 1994). Trisodium phosphate was effective in reduc-
ing planktonic as well as biofilm populations of E. coli O157:H7, Campylobacter
jejuni and S. typhmurium but not L. monocytogenes on stainless steel and Buna-N
rubber (Somers et al., 1994). In the same study it was found that biofilms of
S. typhimurium and L. monocytogenes formed on stainless steel were more resistant
to trisodium phosphate treatment than the ones formed on Buna-N rubber. Trisodium
phosphate was better than acetic acid and phosphate buffered saline rinses in reduc-
ing levels of E. coli O157:H7 and K12 on beef tissue (Fratamico et al., 1996).
Trisodium phosphate was effective on 48-h-old S. enteritidis biofilms on glass flow
cells, but not on 72-h-old biofilms; bacteria within small crevices that were artifi-
cially created on the substrate escaped treatment (Korber et al., 1997).
Long chain polyphosphates (0.5 to 1%) were found to inhibit Clostridium tyrobu-
tyricum growth in processed cheese spreads (Loessner et al., 1997). The antibacterial

mechanism of long chain polyphosphates was studied using S. aureus as the test
organism and it was found that these compounds act by binding to the cell wall and
chelate metals and remain in a bound state (Lee et al., 1994b). Sodium chlorate was
found to be bactericidal towards E. coli O157:H7 and S. typhimurium DT104 in rumen
contents in vitro (Anderson et al., 2000). These organisms possess respiratory nitrate
reductase, which can reduce chlorate to toxic chlorite and hence, this compound may
have antimicrobial action against other bacteria possessing these enzymes. A water
wash followed by acidified sodium chlorite effectively reduced the populations of E. coli
O157:H7 and S. typhimurium on beef carcasses; however, bacteria were still detected
after treatment in 30 to 50% of the carcasses (Castillo et al., 1999). A combination of
hot water wash and lactic acid spray was effective in removing E. coli O157:H7,
S. typhimurium and other Enterobacteriaceae on beef carcasses (Castillo et al., 1998).
Triclosan (an antimicrobial agent used in hand soaps and other toiletries) incorporated
plastic showed antimicrobial activity against a number of bacteria including E. coli,
Bacillus species, S. typhimurium, S. aureus and Shigella flexneri, but not on meat
surfaces, probably due to the presence of fatty acids (Cutter, 1999). Sodium hexameta-
phosphate combined with hop resins inhibited the growth of E. coli in laboratory
medium and mashed potatoes, and the mechanism might be due to damage to the cell
membrane; however, complete inactivation was not achieved (Fukao et al., 2000)
OZONE
Recently, ozone has gained popularity as a disinfectant due to its several advantages
over chlorine: 1) the reaction of ozone with organic compounds does not produce
toxic or carcinogenic compounds, unlike chlorine; 2) ozone is unstable and so it
does not persist in the environment after use; 3) the cost of ozone generation unit
and its maintenance is comparable to or less than cost of chlorine compounds; and
4) ozone does not require heat thereby saving the cost of power consumption (Greene
et al., 1993). Compared to chlorine, lower concentrations and shorter contact times
are required for inactivating microorganisms by ozone (Kim et al., 1999).
The potential applications of ozone as a disinfectant in a processing plant
include: controlling microbial growth in water recirculating systems such as cooling,
washing, and product fluming operations; its role in clean in place systems to control
microbial growth; and controlling microbial growth on surfaces exposed to the
environment (Bott, 1991). Ozone acts on the bacterial cell membrane causing cell
lysis and on sulphydryl groups of bacterial enzymes as well as on bacterial nuclear
region. Gram-negative bacteria were more sensitive to ozone than Gram-positive
ones; however effectiveness of ozone was reduced in food based systems such as
milk or meat broths (Moore et al., 2000). Ozonated water was compared to chlorine
for its effectiveness towards inactivating organisms in milk biofilms and ozone was
found to be as effective as chlorine (Greene et al., 1993). Ozone caused a 5.6 and
4.4 log reduction, while chlorine brought about 4.6 and 4.2 log reductions in pop-
ulations of P. fluorescens and Alcaligenes faecalis, respectively. The various appli-
cations of ozone in food processing are discussed by Kim et al. (1999).

SANITIZER STRESS
In a processing plant, sanitizers are used daily to reduce microbial load, minimize
access to microbes and prevent contamination. However, we still hear about the
source of contamination causing outbreaks arising from the processing plant. One
possible reason is bacteria escaping or surviving the sanitizer treatment and contam-
inating the food. Surviving the sanitizer treatment may arise from adaptation to
sanitizer stress and becoming more resistant to the treatment. Non-psychrotrophic
pathogens present in food industry premises are probably the most resistant to
disinfectants though they might be present in few numbers, and this resistance could
be due to their exposure to adverse conditions in such an environment (Leriche and
Carpentier, 1995).
CHLORINE AND CHLORINATED COMPOUNDS

There are several sanitizers available for use in the food industry of which chlorine
and chlorinated compounds form the active ingredient. The inactivation by chlorine
has been described to involve inhibition of mRNA and protein synthesis, and oxi-
dative phosphorylation in bacterial cells (Bunduki et al., 1995). Dychdala (1991)
provides more information on the mechanisms of action of chlorine on bacteria.
Chlorinated water is often used in the food industry for washing fresh produce
and other commodities. Dipping of fresh cut watercress, onions and potatoes in
chlorine solution increased the microbial counts during storage and this could have
been due to chlorine damaging the produce cut tissues, which could cause cellular
fluid release that can promote microbial growth (Park and Lee, 1995; Gunes et al.,
1997). Lettuce inoculated with E. coli O157:H7 from bovine feces was washed with
chlorinated water and it was found that washing with 200 ppm chlorinated water
was not more effective than a water wash with 1 and 5 min hold time (Beuchat,
1999). Neither tap water nor hypochlorite was effective in removing Pseudomonas
and other Enterobacteriaceae from lettuce leaf surface (Adams et al., 1989). It is
possible that the organisms lodge in inaccessible areas such as stomata and cut edges
and escape treatment.
The efficacy of chlorinated water in inactivating E. coli O157:H7, Salmonella
or L. monocytogenes on whole apples, tomatoes and lettuce was studied (Beuchat
et al., 1998). Chlorine at 2000 ppm was effective in inactivating the pathogens on
these vegetables. There was a difference in the inactivation of Salmonella and
L. monocytogenes using 200 and 2000 ppm chlorine on apples while the two treat-
ments were similar in inactivating E. coli O157:H7 on both apples and tomatoes.
On lettuce leaves chlorine treatment was not as effective as on other produce. There
was about a log difference between water wash and chlorine wash (200 ppm chlo-
rine). Chlorine at 60 and 110 ppm was effective in removing Salmonella montevideo
on raw tomatoes (Zhuang et al., 1995).
Chlorine at 200 ppm reduced the population of E. coli O157:H7 on fresh and
cut edges of lettuce by about one log, but total inactivation was not achieved (Takeuchi
and Frank, 2000). When fresh cut lettuce and cabbage were washed with 200 ppm
chlorine, there were about 1.7- and 1.2-log reductions, respectively, in the population

of L. monocytogenes (Zhang and Farber, 1996). In this study no more than a one-
log reduction was achieved with the other sanitizers and disinfectants (chlorine
dioxide, trisodium phosphate, lactic and acetic acids). Vinegar (5.2% acetic acid)
and chlorine (200 ppm) were effective against Shigella sonnei on whole and chopped
parsley (Wu et al., 2000). Chlorine at 200 mg/L was not very effective on L. mono-
cyotgenes in that it reduced the population of the organism by 2 logs on Brussels
sprouts compared to a water wash which brought about a 1.5-log reduction (Brackett,
1987). Chlorine at 1040 ppm did not effectively eliminate 102 to 103 Salmonella
stanley cells on alfalfa seed, but when alfalfa seed with 101 to 102 CFU/g of the
organism was treated with 2040 ppm of chlorine the organism was reduced to
undetectable levels (Jaquette et al., 1996). Chlorine in the form of Ca(OCl)2 (Š2000
ppm), acidified ClO2 (Š100 ppm), acidified NaClO2 (Š500 ppm) and also hydrogen
peroxide (Š0.2%) and trisodium phosphate (Š1%) was effective in reducing E. coli
O157:H7 from alfalfa seeds; however, the few surviving cells could grow during
sprouting and germination (Taormina and Beuchat, 1999) and hence alternative
measures may be needed to prevent growth. This survival and growth could be due
to enhanced resistance arising out of stress adaptation. Hypochlorous acid, the active
form of chlorine, loses its activity when encountering organic material and hence,
the efficacy of chlorine could be limited on fresh produce which has organic matter
on the surface (Beuchat et al., 1998).
Alfalfa sprouts have been vehicles of salmonellosis in recent years (Ponka et al.,
1995). Copious amounts of biofilms of naturally occurring microflora were present
on alfalfa, broccoli, clover and sunflower sprouts, which were more abundant on
the cotyledon region, and these can afford protection for the colonizing pathogenic
organisms (Fett, 2000). Effectiveness of chemicals in reducing Salmonella popula-
tions on the surfaces of alfalfa sprouts has been investigated. Ethanol (80%), hydrogen
peroxide (6%) and sodium hypochlorite or calcium hypochlorite at chlorine concen-
trations of 1800 to 2000 µg/ml effectively reduced Salmonella populations on alfalfa
seeds by more than 1000-fold (Beuchat, 1997). However, in this study viable Sal-
monella cells were still detected on seeds soaked in these chemical solutions for
10 min. The reason was that Salmonella in hard to reach areas such as crevices and
area between the cotyledon and testa of the seed could have escaped the treatment
or the severity of the treatment. When the bacterial cells escape the severity of the
treatment, there is a possibility of becoming adapted to that chemical treatment and
hence, surviving the treatment.
The effect of chlorine on sanitizing poultry products and fish has been the subject
of investigation by many researchers. Chlorination after sonication (aids in detaching
bacterial cells) was effective on inactivating S. typhimurium cells attached to broiler
skin, while either treatment alone was not effective (Lillard, 1993). Peroxidase
catalyzed sanitizer and chlorinated water were equally effective on inactivating
S. typhimurium and S. enteritidis on chicken egg shells (Kuo et al., 1997) and
Enterococcus faecalis and P. aeruginosa on inert carriers and egg shell surfaces
(Kwon et al., 1997); peroxidase catalyzed sanitizer was also effective against
S. typhimurium on chicken breast skin and broiler carcasses (Bianchi et al., 1994),
and on egg shell surfaces of hatchery eggs (Kuo et al., 1996). Chlorine at 100 and
200 mg/L was more effective in inactivating Yersinia enterocolitica and natural

microflora on the shell of eggs than 1 and 3% acetic and lactic acids (Favier et al.,
2000). Washing eggs with QAC and sodium hypochlorite prevented S. enteritidis
penetration and no damage to the egg shell surface occurred, while sodium carbonate
wash facilitated the penetration of organisms into the shell and resulted in recon-
tamination (Wang and Slavik, 1998). S. aureus isolated from defeathering machinery
in poultry processing plants were eight times more resistant to chlorine than the
natural chicken skin microflora, and this may be due to their growth in clumps as
well as to the production of extracellular slime (Bolton et al., 1988). Chlorinated
water was effective in removing L. monocytogenes from fish (King Salmon) surface;
however, a Listeria free product could not be achieved (Bremer and Osborne, 1998).
Chlorine dioxide is an oxidizing agent and a sanitizer which has a good potential
for sanitizing surfaces in the food industry. It can be used either as a gas or liquid,
with the liquid form being more powerful than the gaseous form. Chlorine dioxide
has advantages over chlorine because it does not react with ammonia or other
nitrogenous compounds, is not affected by pH and is less reactive with organic
matter than chlorine. The efficacy of chlorine dioxide in sanitizing epoxy surfaces
in aseptic juice storage tanks was studied and it was found that 10 mg/L chlorine
dioxide gas with a 30 min exposure time effectively inactivated the inoculated juice
spoilage organisms, Lactobacillus buchneri, Leuconostoc mesenteroides, Saccharo-
myces cerevisiae, Candida sp., Eurotium sp. and Penicillium sp., leaving a clean
epoxy surface (Han et al., 1999). Chlorine dioxide (1.3 ppm) effectively reduced the
bacterial load of cucumber hydrocooling water (Reina et al., 1995) and poultry
chiller water (Tsai et al., 1995). Chlorine dioxide was more effective than aqueous
chlorine on inactivating streptomycin resistant L. monocytogenes on fish cubes and
in the rinse solutions from these cubes (Lin et al., 1996). However chlorine dioxide
spray (20 ppm) was not effective for removing fecal contamination on beef carcass
tissue (Cutter and Dorsa, 1995).
NONCHLORINATED COMPOUNDS
Apart from chlorine, quaternary ammonium compounds (QAC), iodine compounds,
biguanides and acid anionic sanitizers are also commonly used in the food industry.
QACs are hydrophilic cations, which can adsorb easily to negatively charged bac-
terial surface, and into the cell wall, thereby causing disruption of the cytoplasmic
membrane causing leakage of cytoplasmic contents (Merianos, 1991). Gram-negative
bacteria are more resistant to the action of QACs than Gram-positive bacteria. The
resistance of P. aeruginosa to benzalkonium chloride was attributed to an increase
in the content of cellular fatty acids (phospholipids and fatty and neutral lipids),
thus resulting in a reduction in the permeation of the sanitizer through the cell wall
(Sakagami et al., 1989).
QAC at 50 ppm for 1 min was effective in inactivating L. monocytogenes (more
than a four-log reduction) on smooth as well as porous stainless steel surfaces, while
200 ppm of sodium hypochlorite for 2 min and 400 ppm of the sanitizer for 2 min
were required to inactivate the organism on smooth and porous surfaces, respectively,
and QACs were found to be more effective between the two (Mustapha and Liewen,
1989). In the same study cells incubated for 1 h on stainless steel surface were more

resistant to sodium hypochlorite treatment than those incubated for 24 h, and the
reason was attributable to the presence of more moisture after 1 h than after 24 h.
QACs (25 ppm), acid anionic sanitizer (25 ppm) and chlorine containing sanitizers
(50 ppm) were effective in inactivating Listeria species, with QACs being the most
effective sanitizing agent, although L. monocytogenes was the most resistant to QACs
and acid anionic sanitizers and L. innocua the most resistant to chlorine sanitizers
(Sallam and Donnelly, 1992). Adherent cells of L. monocytogenes on glass slides
when treated with Benzalkonium chloride and acid anionic sanitizer with or without
heat (55°C) were reduced by two to three logs in the first 30 sec, followed by a
resistant population still remaining viable for at least 20 min, while planktonic cells
grown in broth were rapidly inactivated (Frank and Koffi, 1990). L. monocyotgenes
cells attached to stainless steel for 8 days were more resistant to hypochlorite and
heat treatment (65°C for 3 min) than those attached for 4 h (Lee and Frank, 1991).
These studies show that L. monocytogenes survives treatments with sanitizers espe-
cially when adhered to surface and, hence, proper cleaning and sanitizing measures
to inactivate adherent cells are needed.
The efficacy of a variety of chemicals and sanitizers on inactivating L. mono-
cytogenes attached to a variety of equipment contact surfaces was studied (Krysinski
et al., 1992). The most resistance was found on polyester/polyurethane surfaces,
followed by solid polyester and etched stainless steel. Chlorine and iodophor were
ineffective on stainless steel, while none of the biocides were effective on polyes-
ter/polyurethane. The most effective sanitizers were acidic quaternary ammonia,
peracetic acid and chlorine dioxide, moderately effective were mixed halogens, acid
anionics and fatty acid sanitizers and least effective were chlorine, iodophor and
neutral quaternary ammonia. Some of these least effective ones are commonly used
in the food industry. QACs were found more effective on reducing L. monocytogenes
attached to chitin (contains organic matter) than chlorine and iodine sanitizers
(McCarthy, 1992). QAC (100 and 200 ppm), chlorine and acid anionic sanitizers
were more effective than iodine and quaternary ammonium detergent sanitizers in
removing extracellular material from L. monocytogenes and S. typhimurium biofilms
on various surfaces (Ronner and Wong, 1993). Chlorine, Zepamine A (QAC), and
Ultra-Kleen (peroxide-based powder) were effective in removing L. monocytogenes
(suspended in phosphate buffered saline) attached to gloves, while Zep-i-dine (iodine
based sanitizer) and Zep Instant Hand Sanitizer (60% ethanol) were ineffective.
However, in the presence of crab cook water that had organic matter, only Ultra-
Kleen was effective and all other sanitizers failed (McCarthy, 1996). The efficacy
of hand sanitizers was tested using real soil involved in the food processing, and
hand washing with a mild soap and water for 20 sec was better than 70% alcohol
hand sanitizer (Charbonneau et al., 2000).
Hydrogen peroxide is a commonly used disinfectant for sterilizing packaging
material for aseptic filling of foods. The ability of hydrogen peroxide to sanitize
eggs has been studied (Sheldon and Brake, 1991; Padron, 1995). Hydrogen peroxide
vapor was effective for surface decontamination and prevention of decay in canta-
loupes (Sapers and Simmons, 1998). The antibacterial action of hydrogen peroxide
is believed to be due to its breakdown mediated by transition metal ions into cytotoxic
radicals such as hydroxyl radicals, which can then initiate oxidation of biomolecules

such as DNA, RNA, proteins and lipids; this breakdown can be initiated by reducing
agents and peroxidases (Juven and Pierson, 1996; Halliwell and Gutteridge, 1992).
The effect of hydrogen peroxide on spores is stronger at high temperatures due
to radical formation. Spores of Bacillus species were inactivated by hydrogen per-
oxide at elevated (sublethal) temperatures and neutral pH, and extended exposures
or higher concentrations of the disinfectant caused damage to the spore coat, cortex
and protoplast (Shin et al., 1994). In this study it was found that some transition
metal ions protected spores against the action of hydrogen peroxide while no pro-
tective effect was seen in the case of vegetative cells and, hence, the authors con-
cluded that antibacterial mechanism of hydrogen peroxide towards spores is different
from that of vegetative cells. Hydrogen peroxide was found to have a synergistic
effect with peracetic acid towards inactivating bacteria isolated from water (P. aerug-
inosa, E. coli and S. aureus) (Alasri et al., 1992) and for disinfecting ultrafiltration
membranes by inactivating spores of Bacillus species (Alasri et al., 1993). This
synergy was maintained or increased with increasing contact time and it allowed for
usage of less concentration of each biocide.
Fresh fruits are surface washed with sanitizing agents and the efficacy of various
agents in inactivating microorganisms on the surface has been studied. E. coli
O157:H7, Salmonella and other natural microflora on surfaces of cantaloupes, honey-
dew melons and asparagus were effectively inactivated by chlorine (200 to 2000 ppm),
acidified sodium hypochlorite, Tsunami™ (contains peracetic acid) and hydrogen
peroxide, with the first three chemicals being more effective than hydrogen peroxide
and water on the pathogens (Park and Beuchat, 1999). Hydrogen peroxide was less
effective than chlorine in inactivating E. coli on cantaloupe surfaces at 4°C, while
both sanitizers were ineffective at 20°C after 24 to 72 h storage (Ukuku et al., 2000).
Acetic acid (5%) and 80 ppm peroxyacetic acid were more effective than
hypochlorite or phosphoric acid treatments for sanitizing cider apples bringing about
three-log reductions in E. coli O157:H7 populations and causing no injury (Wright
et al., 2000). Salmonella chester on apple disks was found to be resistant to hydrogen
peroxide, trisodium phosphate, calcium hypochlorite and sodium hypochlorite, and
the reasons could be attributed to the firm attachment of the organism to the stem
and calyx region as well as partial resistance of the bacteria to these sanitizers (Liao
and Sapers, 2000). Peroxyacetic acid, chlorine dioxide or a chlorine-phosphate buffer
solution were studied for their efficacy on removing non-pathogenic E. coli O157:H7
on apple surface and none of these sanitizers could bring a five-log reduction of the
organism at the manufacturer’s recommended concentration (Wisniewsky et al.,
2000). Immersing fresh Valencia oranges in hot water at 80°C for 1 min or 70°C
for 2 min was more effective in causing five-log reductions than chemical sanitizers
such as chlorine, chlorine dioxide, mixture of peroxyacetic acid and hydrogen
peroxide, acid anionic sanitizer and trisodium phosphate treatments for 8 min, which
caused about two- to three-log reductions in the populations of E. coli and other
natural microflora on the surfaces of oranges (Pao and Davis, 1999). When orange
fruits were washed with various cleaners followed by potable water rinse, an alkaline
sodium orthophenylphenate was more effective than others in reducing populations
of E. coli; however, adding sanitizers to the washing solutions did not improve the

effect (Pao et al., 2000). A number of sanitizers were tested for their efficacy in
inhibiting citrus spoilage organisms and organism on citrus fruit surface, and QACs,
chlorine dioxide and iodophor were very effective, requiring low concentrations for
inhibition, while peracetic acid, hypochlorite, dimethyldicarbonate and a phosphoric
acid anionic sanitizer required higher concentrations to inhibit (Winniczuk and
Parish, 1997). Chlorine, acidified sodium chlorite, trisodium phosphate, Tsunami,
and hydrogen peroxide were ineffective in inactivating Alicyclobacillus acidoterres-
tris spores on apple surfaces (Orr and Beuchat, 2000). A sanitizer named Oxonia
active (a mixture of hydrogen peroxide, peracetic acid, acetic acid and 1-hydroxy-
ethylydene-1,1-diphosphonic acid) was more effective on Clostridium spores than
on Bacillus spores, with B. cereus being the least sensitive to the sanitizer (Black-
istone et al., 1999).
SANITIZER STRESS ADAPTATION AND CROSS-PROTECTION

Previous exposure to one stress is known to offer cross-protection against certain
other stresses in bacteria. Starved planktonic cells of L. monocytogenes exhibited
greater resistance to benzalkonium chloride than non-starved cells (Ren and Frank,
1993). Cold temperature reduced the efficacy of iodophor and QACs against
L. monocytogenes strains especially at lower concentrations and shorter exposure
times, while low temperature did not affect the efficacy of chlorine (Tuncan, 1993).
Using higher concentrations and longer exposure times may overcome these limi-
tations. L. monocytogenes cells, when exposed to sublethal levels of various disin-
fectants and sanitizers, chlorine, iodophor, QACs, citric, lactic and propionic acids
(chemical shock), were not resistant to lethal levels of these compounds (Pickett
and Murano, 1996). However, exposure to sublethal levels of acid anionic sanitizer,
increased the resistance of L. monocytogenes cells to further exposure at minimum
inhibitory concentrations or lethal levels of the same sanitizer. When the acids were
pH adjusted to result in dissociation of the acid and the organism was exposed,
increased resistance was observed. When E. coli O157:H7 was exposed to sublethal
levels of peroxyacetic acid, it exhibited enhanced tolerance to peroxidative stress in
hydrogen peroxide (Zook et al., 2001). However, this exposure did not cross-protect
the bacterium against thermal stress as well as acetic acid stress. An increase in cell
density enhanced the resistance of P. aeruginosa biofilm to iodine (Brown and
Gautier, 1993). Nutrient starvation, increased cell density and increased production
of exopolymeric substance increased the resistance of S. typhimurium in binary
species biofilm with P. aeruginosa to chlorine sanitizer (Leriche and Carpentier,
1995).
Exposure of E. coli cells to free chlorine for less than 1 sec activated the heat
shock and soxRS regulons, but not the oxyR or SOS regulons or universal stress
protein (uspA) (Dukan et al., 1996). Exposure of E. coli O157:H7 to chlorine prior
to heat treatment resulted in an increase in the heat shock proteins (Dukan et al.,
1996). However, pre-exposure to chlorine did not result in an increase in the D-value
of the organism at 58°C in apple juice (Folsom and Frank, 2000). Acid adaptation
of S. typhimurium increased the resistance of the organism towards an acid anionic
sanitizer, but sensitized the cells to hypochlorous acid (due to oxidation of essential

cell components, energy depletion, and changes in cell membrane permeability) and
iodine (Leyer and Johnson, 1997). Exposure to sublethal levels of trisodium phos-
phate increased the sensitivity of C. jejuni, E. coli, P. flourescens and S. enteritidis
to lysozyme and nisin (De Melo et al., 1998). Antibiotic resistant as well as sensitive
strains of L. monocytogenes and S. typhimurium were equally sensitive to acid
anionic sanitizers (Lopes, 1998). Pre-exposure of L. monocytogenes cells to starva-
tion, ethanol, acid and hydrogen peroxide enhanced its thermal resistance (Lou and
Yousef, 1996).
METAL ION STRESS

Another stress that a bacterium can be exposed to in a food processing plant is metal
ions. Heavy metals are a group of 65 metallic elements with varying physical,
chemical and biological properties and are able to exert toxic effects on microor-
ganisms (Gadd, 1992). However, some bacteria are able to tolerate these effects due
to intrinsic properties, metabolic changes during interaction with a metal or envi-
ronmental factors, while some others can resist the effects by certain detoxification
mechanisms. Some detoxification mechanisms include: metal chelation by iron
chelating compounds such as siderophores, and other chelators such as ethylenedi-
aminetetriaceticacid (EDTA) or nitrilotriaceticacid (Schets and Medema, 1993);
presence of metal binding extracellular polymers or metal binding proteins such as
metallothioneins and phytochelatins; methylation of certain compounds such as
mercury, lead, or tin, which become volatile and are lost in the environment or by
removal of alkyl or aryl groups (Gadd, 1992). A particular protein (PsMTA) was
expressed in E. coli as a carboxyterminal extension of glutathione-s-transferase and
this protein exhibited metal binding properties (Tommey et al., 1991). Transcription
of the fliC gene encoding flagellin, the protein of flagellae in E. coli was found to
be regulated by heavy metal ions such as aluminum, copper, iron and nickel (Guzzo
et al., 1991). Gram-negative bacteria are generally more resistant to the action of
heavy metals than Gram-positive bacteria.
Bacteria are able to adapt to the stress from metal ions and are able to survive.
The molecular mechanisms of genetic adaptation to xenobiotic compounds including
heavy metals are discussed by van der Meer et al. (1992). According to the authors,
the various molecular and biochemical processes leading to such an adaptive
response in bacteria include 1) induction of specific enzymes to degrade the heavy
metal ions or other pollutants, 2) ability to grow and metabolize the substrate, and
3) selection of mutants possessing altered enzymatic capabilities or unique/novel
metabolic activities.
The inhibitory effect of various electroplated coatings of cobalt, zinc, copper,
silver, chromium and cobalt-containing alloys of nickel, zinc and chromium etc. on
Enterococcus faecalis, methicillin-resistant S. aureus, E. coli, P. aeruginosa and
K. pneumoniae was tested and depended on the amount of hydrogen peroxide pro-
duced from the surface of the coating, with the greatest inhibition occurring from
the highest amounts of hydrogen peroxide (10–6 mmol/cm2) producing coating surfaces

(Zhao et al., 1998). Bacteria when exposed to heavy metal ions respond by reducing
their physiological activity (Scrader and Cardamone, 1985).
The ability of Aeromonas hydrophila to attach to various surfaces such as
stainless steel, copper and polybutylene was studied and the fewest number of
attached cells were found on copper; these cells had altered surfaces compared to
the cells attached on other surfaces (Assanta et al., 1998). This was attributed to the
antimicrobial effect of copper on the bacterium affecting its ability to attach, and
this may increase the susceptibility of the attached cells to sanitizers. Cuprous oxide
can react with adsorbed cells on the surface and the resulting product involving free
cupric ions could inhibit or resist adhesion (Jonas, 1991). A similar finding was
reported with Legionella pneumophila in which fewer cells of the organism were
found attached on copper surfaces compared to polybutylene (Lee and West, 1991).
When copper resistant E. coli was grown in a copper supplemented medium, an
increased production of exopolymer was seen (Chao and Chen, 1991). The copper
binding abilities from exopolymers of copper resistant as well as copper sensitive
Pseudomonas strains were studied and both bound the same amount of copper ion;
however, the sensitive strain could not grow in media with copper while the others
did and it was found that the low pH of the medium inhibited the growth (Chao and
Chen, 1991). Thus the exopolymer production and acid tolerance of the organism
may play a role in survival of the microorganism in environments with high con-
centrations of heavy metals.
Microorganisms require certain metal ions in low amounts for their growth; for
instance, L. monocytogenes requires magnesium and iron for growth in a minimal
medium (Premaratne et al., 1991). Supplementing the growth medium with metal
ions such as zinc, magnesium, manganese, cobalt and calcium pantothenate increased
the propionibacteria biomass yield, while there was no influence on the production
of volatile fatty acids (Kujawski et al., 1992). Growth inhibition of L. monocytogenes
(in brain–heart infusion broth at 19°C, pH 6.0) by sodium polyphosphate was
reversed by low concentrations of polyvalent metal ions such as manganese, calcium,
magnesium and zinc, but not by copper, cobalt, nickel and aluminum; however,
growth inhibition of the organism did not occur in mineral rich foods such as pureed
beef, green beans and sweet potatoes (Zaika et al., 1997).
The antibacterial effects of certain food grade phosphates such as sodium ultra-
phosphate, sodium polyphosphate glassy and tetrasodium pyrophosphate on S. aureus
were reversed by polyvalent metal ions such as calcium, magnesium and iron, when
these metal ions were added to the media containing polyphosphates either before or
after inoculation of the organism (Lee et al., 1994a). The lost enzymatic activity by
EDTA of a 52-kDa metalloproteinase was restored by metal ions such as zinc,
calcium, manganese, magnesium and iron (Kim and Kim, 1993). These studies show
the protective effects of low concentrations of metal ions towards microorganisms.
LINKS TO ANTIBIOTIC RESISTANCE

Often resistance to metal ions is related to antibiotic resistance and common plasmids
seem to be involved. An E. coli strain carrying the robA plasmid from a cyclohexane

tolerant mutant strain exhibited increased tolerance to solvents and increased resis-
tance to antibiotics and heavy metal ions such as silver, mercury and cadmium
(Nakajima et al., 1995).
Staphylococcus aureus strains that were resistant to mercury also carried peni-
cillinase plasmids (Silver and Misra, 1988). Marine Vibrio species isolated from
yellowtail fishes in Japan were found to be resistant to lead acetate, cobalt chloride,
sodium arsenate and nickel sulfate as well as to the antibiotic aminobenzylpenicillin
(Hayashi et al., 1993). Some Gram-negative bacteria carry plasmids exhibiting resis-
tances to antibiotics as well as to heavy metal ions such as arsenic, cadmium, cobalt
and nickel (Lyon and Skurray, 1987).
ADAPTATION TO HEAVY METAL IONS AND CROSS-PROTECTION

Inducible resistance to heavy metal ions and its cross-protection against other stresses
have been observed in bacteria. Bacillus subtilis exhibited growth at lethal levels of
zinc when previously exposed to mild concentrations of the metal ion and this was
a chromosomally determined resistance (Podlesek et al., 1993) as opposed to a
plasmid determined resistance (Nies, 1992). This inducible response involved a
change in protein expression of the organism with the induction of a 150 kDa protein
and suppression of a 127 kDa protein. No inducible cadmium resistance was
observed in the organism and adaptation to zinc did not offer any cross-protection
against cadmium and vice versa (Podlesek et al., 1993). Cadmium tolerance to lethal
concentrations was induced in E. coli pretreated with mild concentrations of the
metal ion as well as with mild heat; however, adaptation to mild concentrations of
cadmium did not induce thermotolerance in the organism (Inbar and Ron, 1993).
In another study, pre-exposure of E. coli cells to a mild concentration of cadmium
offered cross protection against lethal temperatures (VanBogelen et al., 1987). Cad-
mium tolerance was found to induce 40 stress proteins (Blom et al., 1992). Listeria
monocytogenes strains from various sources were tested for the presence of plasmids
and 40% of food isolates did possess plasmids; more than 95% of plasmid positive
strains were cadmium resistant, implying that cadmium resistance in L. monocytogenes
is plasmid determined (Lebrun et al., 1992). Cadmium resistance in S. aureus was
found to be chromosomally determined (Lyon and Skurray, 1987; Witte et al., 1986).
ANTIBIOTIC STRESS
In recent years several antibiotic resistant strains of bacteria have emerged, a source
of concern. One such example of a foodborne pathogen that has emerged due to its
resistance to multiple antibiotics is Salmonella typhimurium DT104. In 1985, pas-
teurized milk from a contaminated dairy plant was implicated in an outbreak of S.
typhimurium (involving 180,000 cases) which was resistant to five antibiotics (Ryan
et al., 1987). New antibiotics are manufactured to combat resistant organisms by
altering their chemical structure either to escape bacterial defenses or weaken them.
Bacteria are able to resist those new antibiotics as well. Some resistant bacteria are
able to transfer this resistance to sensitive cells (Russell and Chopra, 1996). A
number of mechanisms play a role in the development of these resistances. The

resistance could be either intrinsic or acquired. The intrinsic resistance is due to
some inherent property of the organism such as virulence. Acquired resistance
usually involves a mutation and sometimes mutation can occur in genes that regulate
multiple functions within a cell (Shlaes, 1993); and this single mutation could confer
resistance to multiple antibiotics (Murray, 1991).
According to Russell and Chopra (1996), some mechanisms of antibiotic resistance
include antibiotic alteration, insensitive or modified target site, impaired antibiotic
uptake, enhanced efflux of antibiotic, and absence of an enzyme and metabolic pathway.
In addition, access to the target site may be prevented (Neu, 1992) or there is overpro-
duction of the target site (Miller and Sulavik, 1996). A mutation in a target enzyme
could occur, resulting in decreased binding to the site, or an alternative reaction bypass-
ing the antibiotic-sensitive step could occur (Volk et al., 1996). Efflux of the antibiotic
is usually by cell membrane proteins that are plasmid or transposon encoded and they
bind the antibiotic and push it out of the cell (Service, 1995; Russell and Day, 1996).
Resistance in some Gram-negative bacteria occurs through alteration in the permeabil-
ity of the outer membrane proteins called porins, with these proteins having restrictive
channels than the normally expressed protein (Volk et al., 1996). It has been shown in
studies with E. coli that the antibiotic resistant systems show similarities at the amino
acid sequences and among their genetic regulators (Miller and Sulavik, 1996). Some
antibiotics such as aminoglycosides, β lactams and chloramphenicols are inactivated
by hydrolysis or by formation of inactive derivatives by the bacterial cell (Davies,
1994). Starvation or nutrient deprivation and growth rate contribute towards the resis-
tance of biofilm bacteria to the action of antibiotics (Brown et al., 1988).
The major groups of antibiotics that have been used against microorganisms
include β lactams, fluoroquinolones, aminoglycosides, chloramphenicols, tetra-
cyclines, MLS group (macrolides, lincosamides, streptogramins) and antibiotics that
inhibit cell wall precursor biosynthesis. The mechanism of action of some antibiotics
and bacterial resistance mechanisms against these are listed in Table 5.3. Russell
and Day (1996) and Volk et al. (1996) have given detailed descriptions of actions
of various classes of antibiotics.
Several researchers have investigated the effect of antibiotics on foodborne
bacteria. Older biofilm cells of S. aureus were resistant to tobramycin and cephalexin
under iron limited conditions, while the planktonic cells were susceptible, and the
younger biofilm cells which were more resistant than planktonic cells could be
eradicated by the combinations of the two antibiotics (Anwar et al., 1992). A similar
phenomenon was seen with P. aeruginosa, where the older biofilms grown under
iron limited conditions were resistant to tobramycin and piperacillin (Anwar and
Costerton, 1990). The exopolysaccharides in the biofilms may bind the antibiotics
and prevent penetration into the cell (Anwar et al., 1992) or embedded biofilm cells
may be able to produce enzymes that can degrade the antibiotic (Giwarcman et al.,
1991). Moreover, the old biofilm cells are under starvation stress which may confer
protection against the action of antibiotics.
The resistance of S. aureus isolated from fish processing plant workers to various
antibiotics was studied and the highest percent resistance was seen for ampicillin
followed by penicillin and then tetracycline, polymyxin-B, erythromycin, kanamy-
cin, neomycin, chloramphenicol and streptomycin in decreasing order (Sanjeev and

TABLE 5.3
Antibiotic Action on Microorganisms and Mechanisms of Resistance
Antibiotic Mode of Action Resistance Mechanism
Aminoglycosides Inhibition of protein synthesis Aminoglycoside modifying enzymes, reduced

ribosomal binding
Bacitracin, Blocks cell wall peptidoglycan Unknown
Ristocetin synthesis
Chloramphenicol Inhibition of protein synthesis Modification of the target (ribosomal 50S subunit)
Cycloserine Inhibition of cell wall peptidoglycan Altered transport, production of elevated enzyme
synthesis levels
Erythromycin Inhibition of protein synthesis Modification of target by producing methylating
enzymes
Fosfomycin Interference with bacteria cell wall Altered transport due to chromosomal mutation
synthesis, enzyme inactivation
Fusidic acid Inhibition of protein synthesis Target site, factor G involved in translocation is
modified
Mupirocin Interference with protein synthesis Altered target (second resistant enzyme Isoleucyl
tRNA synthetase)
Penicillin, Blockage of cell wall synthesis Altered penicillin binding proteins or absence of
Cephalosporin target enzyme
Polymyxin Distortion of outer membrane Unknown
Quinolones Inhibition of synthesis or Modified target (DNA Gyrase)
metabolism of nucleic acids
Rifampin Inhibits DNA-directed RNA Reduced DNA polymerase binding
polymerase activity
Rifampicin Inhibition of mRNA synthesis Modification of RNA polymerase
Streptomycin Inhibition of protein synthesis Modification of target (S30 ribosomal unit)
Sulfonamides Modification of energy metabolism Modified target (dihydroperoate synthetase)
Tetracyclines Inhibition of protein synthesis Active efflux from the cell
Trimethoprim Modified energy metabolism Excessive production of dihydrofolate reductase
Vancomycin, Inhibition of cell wall peptidoglycan Modified target
Teicoplanin synthesis
(Adapted from Davies, J., Science, 264, 375, 1994; Neu, H.C., Science, 257, 1064, 1992; Russell, A.D. and
Day, M.J., Microbios, 85, 45, 1996; Service, R.F., Science, 270, 724, 1995; Volk, W.A. et al., in Essentials
of Medical Microbiology, 5th ed., Lippincott-Raven, Philadelphia, 1996, 253.)
Mahadeva Iyer, 1988). S. aureus and E. cloacae were exposed to single or combi-
nations of antibiotics for a period of time and minimum inhibitory concentrations
(MIC) determined, and an increase in the MIC for S. aureus was seen, indicating
that a “safe” level of antibiotics appearing in foods can select for antibiotic resistant
population of the organism (Brady and Katz, 1992; Brady et al., 1993).
The antibiotic resistance of Salmonella is believed to come from animal sources
where animals are fed antibiotics to increase feeding efficiency and thereby weight
gain, and bacteria gains resistance to the drug. Outbreaks of salmonellosis are linked
to the consumption of foods from animal origin (Epling and Carpenter, 1990).

S. typhimurium DT 104 isolated from poultry sources were found resistant to ampi-
cillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, penicillin and spec-
tinomycin (Rajashekara et al., 2000) and the resistance was found to be chromosoma-
lly integrated (Threlfall et al., 1994). Salmonella serotypes isolated from different
parts of slaughtered pigs were found resistant to tetracycline (Bhattacharyya et al.,
1991). In another study Salmonella isolated from pork carcasses were found to be
resistant to penicillin, trimethoprim, ampicillin and tetracycline, and moderately
resistant to streptomycin, kanamycin and chloramphenicol (Epling and Carpenter,
1990). Use of antibiotics in the feed for pigs undergoing asymptomatic S. typhimu-
rium infection could cause an increase in antibiotic resistant organisms (Ebner and
Mathew, 2000). Salmonella isolated from fish and crustaceans were found resistant
to single or multiple antibiotics such as bacitracin, penicillin and novobiocin, and
the reason could be attributed to human wastewater mixing in aquaculture ponds in
Third World countries as well as use of antibiotics in aquaculture ponds (Hatha and
Lakshmanaperumalsamy, 1995). In Salmonella wein, a 74 kDa iron repressible outer
membrane protein and a plasmid were related to multiple antibiotic resistance,
bacteriocin resistance and aerobactin production (Visca et al., 1991).
Salmonella, Staphylococcus, and some other members of the Enterobacteriaceae
family isolated from slaughterhouse and retail chicken samples exhibited multiple
antibiotic resistance, with slaughterhouse samples showing higher resistance than
retail samples, since the animals are fed subtherapeutic doses in chicken farms
(Manie et al., 1998). Use of tetracycline and penicillin in feed should be avoided
since the resistance from these is easily transferred causing multiple resistance.
Coliforms (E. coli, Enterobacter, Citrobacter, Klebsiella and Serratia) isolated from
slaughtered chickens showed multiple antibiotic resistance; the highest resistance is
seen for tetracycline, followed by cephalotine, cotrimoxazole and nalidixic acid
(Turtura et al., 1990). Aeromonas hydrophila isolated from chicken during various
processing stages were found to be multiple antibiotic resistant with the greatest
resistance observed for ampicillin and cephalothin and most resistant isolates were
recovered from carcasses immediately after evisceration and from chill water sam-
ples (Barnhart and Pancorbo, 1992). Campylobacter strains isolated from poultry
abattoir effluent and two sewage purification plants (one receiving mixed sewage
including that of poultry abattoir and the other one not receiving any sewage from
meat processing) were resistant to quinolones, with the resistance of isolates from
the sewage purification plant receiving mixed sewage being higher than that of those
from plant receiving no meat processing sewage (Koenraad et al., 1995). Campylo-
bacter strains isolated from poultry and humans were found resistant to fluoroqui-
nolones, and cross resistance to nalidixic acid was also seen, and the transmission
route was suggested to be from chicken to man and not vice versa (Endtz et al., 1991).
Antibiotic resistance of Enterococcus isolated from turkeys fed virginiamycin
was studied and E. faecium was found resistant to ampicillin, gentamicin and
quinupristin-dalfopristin antibiotics but not to virginiamycin, with strains from older
turkeys showing greater ampicillin and quinupristine-dalfopristine resistance (Welton
et al., 1998). Enterobacteriaceae isolated from minced meat were resistant to ceph-
alothin followed by sulfisoxazole, ampicillin and tetracycline with 48% of the isolates
showing multiple resistance; however, no resistance to a disinfectant was seen

(Stecchini et al., 1992). Antibiotic resistance of enterococcal isolates from pork,
water and clinical sources was compared and pork isolates showed lower antibiotic
resistance in general than water or clinical isolates except in case of tetracycline,
and with cefazolin and imipenem the resistance was higher than clinical isolates,
while water isolates showed greater resistance in case of cephalosporins, amikacin,
gentamicin, imipenem and rifampin (Knudtson and Hartman, 1993). Enterococci
isolated from raw meat from Italy were found resistant to vancomycin and this
resistance was associated with resistance to methicillin, teicoplanin, erythromycin,
tetracycline and chloramphenicol; however, susceptibility to imipenem, rifampin and
ampicillin in some isolates was observed (Pavia et al., 2000).
Bacteria in noncarbonated mineral water were resistant to nalidixic acid and other
antibiotics with 51% of the isolates showing multiple resistance; this is of public
health concern, since mineral water is widely consumed (Massa et al., 1995). Plesi-
omonas shigelloides strains isolated from blue crab were susceptible to gentamicin,
nalidixic acid and tetracycline and resistant to ampicillin, carbenicillin, kanamycin
and streptomycin, with streptomycin resistance being linked to small size plasmids
(Marshall et al., 1996). Gram-negative psychrotrophs isolated from vegetables showed
multiple antibiotic resistance patterns, with high resistance to chloramphenicol; how-
ever, less than 10% of the isolates showed resistance to mezlocillin-ticarcillin-
gentamicin or ceftizoxime-norfoxacin and although 50% of the strains were QACs
tolerant, sensitive strains were inactivated by QACs (Fernandez-Astorga et al., 1995).
Listeria monocytogenes strains isolated from Italian meat products were resistant
to antibiotics such as tetracycline, co-trimoxazole and erythromycin and the resis-
tance was not plasmid mediated (Barbuti et al., 1992). However, antibiotic resistance
(to chloramphenicol, erythromycin, streptomycin and tetracycline) in L. monocyto-
genes was found to be mediated by a 37 kb plasmid, which was also self transferable
to other organisms such as Enterococcus faecalis, Streptococcus agalactiae and
S. aureus (Poyart-Salmeron et al., 1990). Erythromycin resistance in Listeria species
was associated with the presence of ermC genes encoding rRNA methylases and
this was found transferrable to L. monocytogenes, L. innocua and E. faecalis (Roberts
et al., 1996). These studies showed the transfer potential of antibiotic resistance
genes from one organism to the other. Listeria species isolated from raw milk were
tested for their antibiotic resistance and most isolates were resistant to sulfisoxazole;
only two L. innocua and none of L. monocytogenes were resistant to tetracycline
and the resistance in L. innocua was not plasmid mediated (Slade, 1991). Salmonella
and Listeria species were isolated from sausage samples in Greece and some Sal-
monella strains were resistant to ampicillin, chloramphenicol and tetracycline, while
all Listeria isolates were sensitive to penicillins and aminoglycosides, but resistant
to cephalosporins (Abrahim et al., 1998).
CROSS-RESISTANCE
The aquisition of a plasmid in a bacteria that confers antibiotic resistance could alter
the cell envelope which in turn could render the bacteria susceptible to a subsequent
biocidal treatment (Russell, 1991). This might suggest a cross-resistance existing
between antibiotics and biocides (Russell and Day, 1996). There is evidence in the

literature that antibiotic resistance, especially if plasmid mediated, could offer pro-
tection against biocides. For instance, antibiotic resistant strains of S. aureus and
S. epidermidis exhibited resistance towards chlorhexidine and quaternary ammonium
compounds (Russell, 1997). Antibiotic resistance in E. coli offered cross-protection
against some household disinfectants such as pine oil (Moken et al., 1997).
OTHER STRESSES
Some other stresses that a bacterium may undergo in a food processing environment
include starvation, osmotic and oxidative stresses. The oxidative and osmotic stresses
may also be encountered when the organism is exposed to a chemical, sanitizer or
other compounds. Bacteria in a biofilm formed on any equipment or other surfaces
in a food processing environment may undergo starvation and oxidative stresses.
ADAPTATION TO STARVATION STRESS

Starvation in general makes a bacterium more resistant in the environment and
protects against several subsequent stresses. Stationary phase cells in general are
able to resist harsh conditions better than log phase cells. The mechanism by which
a cell regulates its metabolism under starvation conditions is described as the strin-
gent response, which involves induction of stationary phase specific genes as well
as stimulation of certain biosynthetic pathways (Nystrom, 1994). In E. coli the sigma
factor, σS, regulating general stress response is induced during the stationary phase
(Loewen and Hengge-Aronis, 1994).
The response to starvation in Vibrio species has been characterized by Kjelleberg
et al. (1993) in which the starved cells of the organism undergo morphological
differentiation into ultra-micro sized (smaller) cells, which undergo cell division
with no major increase in cell biomass; these cells can regain their normal size under
conditions of sufficient nutrient availability. This ability is seen in carbon starved
cells, but not in nitrogen or phosphorus starved cells and only carbon starvation can
offer cross-protection against other stresses. The authors describe three adaptive
phases of the nutrient starved Vibrio cells; the first phase involves some physiological
changes causing a decrease in the rate of macromolecular synthesis, an increase in
the rate of protein degradation and temporary accumulation of ppGpp. The second
phase causes changes in membrane fatty acid composition, degradation of reserve
material and resistance development to other stresses. The third phase involves
changes in the metabolic activities causing reduced rate of respiration and synthesis
of RNA, protein and peptidoglycan; however, the cell does not cease to develop,
recover and grow. Also there are induction and repression of a number of stress
proteins. The chaperones DnaK and GroEL have been found to be induced in Vibrio
under conditions of starvation (Holmquist et al., 1993).
Under conditions of starvation, cells are able to retain ribosomes in excess of
that required for protein translation (Flärdh et al., 1992; Kjelleberg et al., 1993) and
degradation of ribosomes occurs very slowly with the half life of ribosomes estimated
to be 80 h during the first 72 h of starvation (Flärdh et al., 1992). Bacterial α-glucan
phosphorylases are involved in the regulation of endogenous glycogen metabolism

during starvation, sporulation and other stresses (Schinzel and Nidetzky, 1999).
Phosphorylases provide energy to the bacterial cells and help them to adapt to the
quickly changing environment (Böck and Schinzel, 1998). In E. coli, the glycogen
phosphorylase is found most active during the stationary phase and under substrate
deprived conditions (Yang et al., 1996). Nutrient starvation in S. aureus cells caused
accumulation of higher levels of ppGpp and ppGp nucleotide (Crosse et al., 2000).
Listeria monocytogenes undergoes stress in the absence of energy yielding
substrates. Growth of L. monocytogenes in different solutions lacking all the required
nutrients for growth, for instance, tap water, deionized water, normal saline and
different dilutions of phosphate buffered saline, was studied (Lee, 1995). None of
these promoted the growth of the organism with complete death occurring in deion-
ized water in less than 5 days. The population decline in 0.3 mM phosphate buffered
saline was more rapid compared to normal saline, and the number of survivors were
more in tap water after 25 days. Starved cells were prevented from lysis in 200 mM
phosphate buffered saline. When starved in salts solution the population increased
initially and then declined more than 3 logs after 30 days and then remained constant
for 120 days. Escherichia coli O157:H7 was able to survive in water at cold tem-
peratures and there were changes in the outer membrane proteins (Wang and Doyle,
1998). Escherichia coli, Shigella and S. typhimurium cells incubated in seawater,
distilled water, phosphate buffer and phosphate buffered saline exhibited increased
acid resistance, the greatest resistance being induced with exposure to seawater and
in stationary phase cells and this resistance partly depended upon rpoS sigma factor
and de novo protein synthesis (Gauthier and Clément, 1994).
ADAPTATION TO OSMOTIC STRESS

Microorganisms respond to osmotic stress in three phases: the cytoplasm shrinks or
swells due to efflux or influx of water from hyper- or hypo-osmolarity, the cell then
undergoes biochemical changes to restore volumes compatible with growth and then
growth occurs (Brown and Edgley, 1980). Bacteria are able to accumulate proline
and glycinebetaine either by increased synthesis or uptake under conditions of
osmotic stress (Csonka, 1989). Escherichia coli K12 has the ability to convert choline
to glycinebetaine under conditions of osmotic stress which can act as an osmopro-
tectant (Landfald and Strøm, 1986). Accumulation or increase in some other solutes
such as potassium (Meury et al., 1985), glutamate (Botsford, 1984), trehalose (Dupray
et al., 1995; Larson et al., 1987) and γ-aminobutyrate (Measures, 1975) has been
observed in bacteria during osmotic stress. Two-dimensional gel electrophoretic
analysis of E. coli exposed to osmotic stress showed three proteins to be synthesized
(Clark and Parker, 1984). The transcription of the genes kdp, proU proP, ompF and
ompC is osmotically regulated (Csonka, 1989).
Cross-Protection
Salmonella manhattan exhibited enhanced survival in seawater with prior exposure

to wastewater, which caused synthesis of trehalose that can act as an osmoprotectant,

and there was accumulation of trehalose and repression of two degradative cytoplas-
mic enzymes during exposure to seawater (Dupray et al., 1995). Pre-exposure to
heat and ethanol enhanced the survival of L. monocytogenes cells in 25% sodium
chloride (Lou and Yousef, 1997). Acid adapted cells of S. typhimurium had enhanced
tolerance towards salt (Leyer and Johnson, 1993). In B. subtilis pre-exposure to mild
salt or mild heat offered protection against lethal salt levels, but pre-exposure to
mild salt did not induce thermotolerance as did mild heat shock (Volker et al., 1992).
Compared to other stresses, thermal induction seems to offer global protection
against several other lethal stresses in bacteria.
ADAPTATION TO OXIDATIVE STRESS

When bacteria are exposed to reactive oxygen species such as superoxide and
hydrogen peroxide, they undergo oxidative stress. The oxidant radicals can react
with amino acids and the resulting derivatives can inactivate enzymes (Wolff et al.,
1986). The oxidative stress in bacteria is regulated by the oxyR system for hydrogen
peroxide and SoxRS system for superoxide (Farr and Kogoma, 1991). Hydrogen
peroxide treatment in E. coli induced the expression of katG, dps, ahpCF and gorA
genes, but not oxyR and this expression was oxyR dependent and σS independent,
and exposure to sodium chloride induced the expression of the same genes including
oxyR, which implies that the two stresses involve overlapping genetic control
(Michan et al., 1999).
E. coli, when grown anaerobically, required superoxide dismutase for a smooth
transition to growth in an aerobic environment, and a sudden transfer to aeration
(superoxide presence) in the absence of superoxide dismutase causing oxidative
stress, blocked amino acid biosynthesis and affected growth (Kargalioglu and Imlay,
1994). Exposure of E. coli cells to ozone induced catalase and superoxide dismutase
(Whiteside and Hassan, 1987). Some of the proteins induced during oxidative stress
and regulated by oxyR have been identified as antioxidant enzymes such as catalase,
alkyl hydroperoxide reductase and glutathione reductase (Christman et al., 1985).
Cross-Protection
Oxidation reduction potentials of chemically modified water (with added chlorine,

bromine and acids) and electrolyzed oxidizing water were found to enhance inacti-
vation of E. coli O157:H7 (Kim et al., 2000). Aeromonas hydrophila exhibited sen-
sitivity to hydrogen peroxide at 1 mmol/L; however, tolerance to the sanitizer was
developed when the cells pre-exposed to a sublethal level of hydrogen peroxide
exhibited tolerance to lethal levels (Landre et al., 2000). This tolerance involved the
synthesis of a set of protective polypeptides and also was dependent on the growth
phase and stock culture age, with stationary phase and older cells exhibiting higher
resistance to hydrogen peroxide (Landre et al., 2000). Starvation of E. coli cells
induced tolerance to heat or hydrogen peroxide (Jenkins et al., 1988). In this study,
the cells starved for 4 h afforded the maximum and better protection compared to
cells exposed to sublethal stresses of heat, hydrogen peroxide or ethanol, and this

response involved synthesis of protective proteins, with three proteins found common
to heat, hydrogen peroxide and ethanol stresses In this study adaptation of E. coli
to hydrogen peroxide or ethanol did not result in enhanced heat resistance. However,
S. typhimurium cells when exposed to sublethal levels of hydrogen peroxide pro-
duced some heat shock proteins and exhibited enhanced thermal tolerance (Christ-
man et al., 1985). It is possible that different sets of stress proteins are produced in
the two organisms with different stresses and hence there is difference in the cross-
protection responses.
Pre-exposure of Enterococcus faecalis cells to mild concentrations of hydrogen
peroxide enhanced their survival under lethal concentrations of the peroxide. An
acid pretreatment at large and, to a limited extent sodium chloride and heat but not
ethanol and alkalinity, afforded protection against hydrogen peroxide (Flahaut et al.,
1998). Some of these responses involved de novo protein synthesis, especially those
induced by hydrogen peroxide and sodium chloride. However, in E. coli, alkalinity
was found to enhance oxidative stress (Rowbury, 1997) and, in B. subtilis, ethanol
but not heat enhanced protection against hydrogen peroxide (Dowds, 1994).
Adaptation of L. monocytogenes to various stresses such as acid, ethanol, hydro-
gen peroxide, heat or sodium chloride enhanced its survival in higher concentration
of hydrogen peroxide (Lou and Yousef, 1997). Acid adaptation of L. monocytogenes
strains offered protection against an activated lactoperoxidase system involving
hydrogen peroxide in tryptic soy broth (Ravishankar et al., 2000) but not in skim
milk (Ravishankar and Harrison, 1999). E. coli O157:H7 cells exposed to heat were
sensitive to oxygen in that they did not recover as well on aerobic media as they
did on anaerobic media (Bromberg et al., 1998). Hence, recovering thermally
stressed E. coli O157:H7 on aerobic media may give misleading results. Moreover,
a hurdle approach involving thermal treatment followed by oxidative stress may
prove useful in inactivating this pathogen. When cells of E. coli O157:H7, S. enter-
itidis and L. monocytogenes were grown, heated and recovered anaerobically, the
heat resistance of these organisms was higher than when they were grown, heated
and recovered aerobically (George et al., 1998). These results may have serious
implications on the safety of sous vide food products as well as foods with a low
redox potential.
CONCLUSIONS
The stress response of bacteria and the physiological and molecular mechanisms
behind these responses are emerging areas of research. We have gained some under-
standing of these responses and further investigation is still needed. The availability
of modern biotechnological tools has made it feasible to understand the responses
of the bacterium to an external environmental stress; we need to identify the impor-
tant ones under given conditions (Brul and Coote, 1999) so that proper control
strategies can be devised and implemented. Control of resistant microorganisms can
be done through changing current practices of antibiotic use, developing newer
antibiotics, utilizing alternative practices such as competitive exclusion, preventing
bacterial adhesion and biofilm formation, applying a multiple hurdle approach, etc.
(Bower and Daeschel, 1999).

In food processing plants, maintaining a clean environment is very important,
and this can be achieved through an effective sanitation program. Chemical cleaning
with detergents, accompanied by manual scrubbing of food contact surfaces and
followed by sanitizer application, is an effective way to control biofilms. When
designing food contact equipment, care should be taken to select the appropriate
type of material to prevent microbial adhesion and importance should be given to
the cleanability of the equipment. Employees working in a food processing plant
should be properly educated about the importance of maintaining a hygienic envi-
ronment. A better understanding of the physiology of microbial adaptive responses
and thereby devising appropriate novel approaches to combat them can help us
achieve the goal of increasing the safety of minimally processed foods and a safer
food supply in the world.
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Stress Adaptations of
6 Lactic Acid Bacteria
Hany S. Girgis, James Smith, John B. Luchansky,
and Todd R. Klaenhammer
CONTENTS
Introduction
Heat Shock Response and Thermotolerance
Thermotolerance
Heat Shock Genes
Classification
groE Operon: groES and groEL
dnaK Operon: hrcA, grpE, dnaK, and dnaJ
clp Family of Genes: clpB, clpC, clpE, clpP, and clpX
Cold Stress Response and Cryoprotection
Physiological Response and Adaptation
Cold-Stress Genes and Gene Products
Acid Adaptation
Tolerance and Adaptation to Low pH
Proton Movement: H+-ATPase
Arginine Deaminase (ADI) Pathway
Degradative Amino Acid Decarboxylases
Citrate Transport System
Alkaline Stress Response
Osmotic Stress
Compatible Solute
Protein Synthesis during Osmotic Shock
Oxidative Stress
Tolerance and Adaptation to Oxidative Stress
Regulation and Function of Oxidative Stress Response Proteins
NADH Oxidase/NADH Peroxidase
Glutaredoxin and Thioredoxin
Superoxide Dismutase
recA, fpg, and DNA Damage
Starvation
Overlapping Regulatory Networks and Cross-Protection

The Future
Conclusions
References
INTRODUCTION
Lactic acid bacteria (LAB) are widely used in dairy and other food fermentations
for their ability to impart desirable flavor and rheological attributes to food, and for
their ability to inhibit unwanted bacteria (Gilliland, 1985). During a successful
fermentation, LAB must be able to tolerate the accumulation of toxic byproducts of
their growth such as lactic acid and hydrogen peroxide, withstand antimicrobial
agents produced by neighboring microorganisms, and endure deleterious environ-
mental conditions required for the proper fermentation of a raw food item. Further-
more, LAB used as probiotics must be able to adapt to harsh conditions such as the
acidic environment of the stomach during ingestion and the low temperatures asso-
ciated with freezing prior to their storage and distribution. The LAB are equipped
with complex stress response mechanisms that provide a selective advantage in
compromising environments. The last few years have seen a tremendous increased
interest in these stress response systems in LAB. The impetus for this heightened
awareness is sparked by the industrial goal of minimizing losses associated with
reduced cell viability upon inoculation in food and during passage through the
gastrointestinal tract. Also, with the increased incidence of food poisoning, research-
ers are looking to enhance tolerance to environmental stress in LAB as a way to
improve food safety. This chapter discusses the literature investigating adaptation to
many of the harmful factors that LAB encounter in food systems and in the envi-
ronment. Recently, a review article was published discussing the stress response
mechanisms in Lactococcus lactis (Sanders et al., 1999).
This chapter presents an extensive review of the existing knowledge on many
of the species of LAB comprising six genera — Streptococcus, Lactococcus, Entero-
coccus, Leuconostoc, Oenococcus, and Lactobacillus. Occasionally, references will
also be made to systems that have been thoroughly investigated in model organisms
such as Escherichia coli and Bacillus subtilis.
HEAT SHOCK RESPONSE AND THERMOTOLERANCE

THERMOTOLERANCE
The heat shock response refers to an abrupt increase in temperature causing the
induction of a small group of proteins called heat shock proteins. Heat shock proteins
play essential physiological roles as molecular chaperones in protecting cells against
damage due to thermal stress by binding to cellular proteins in a manner that maintains
their native conformation and minimizes denaturation (Martin et al., 1992; Craig
et al., 1993). The heat shock response is involved with a variety of other challenges
and conditions such as ethanol (Strauss et al., 1987), osmotic (Vachova et al., 1994),
acid (Heyde and Portalier, 1990), alkaline (Taglicht et al., 1987), oxidative (Morgan
et al., 1986; Ericsson et al., 1994) stresses, and DNA damage (Lage and Menezes,

1994). See “overlapping regulatory networks and cross-protection section” below.
The regulation of the heat shock response has been studied extensively in E. coli
(Bukau, 1993; Yura et al., 1993; Gross, 1996) and B. subtilis (Hecker and Völker, 1990;
Völker et al., 1994; Hecker et al., 1996). The heat shock response in E. coli is regulated
by the rpoH-encoded alternative sigma factor σ32. The heat shock response is mediated
through a variety of mechanisms in B. subtilis and other Gram-positive microorgan-
isms. See “classification” below for a description of the regulatory mechanisms.
Microorganisms exposed to a sublethal heat treatment acquire the transient
ability to withstand subsequent lethal heat challenges and this phenomenon is called
acquired thermotolerance (Lindquist, 1986; Hahn and Li, 1990; Mackey and Derrick,
1990; Boutibonnes et al., 1991). Heat-induced thermotolerance has been achieved
in numerous LAB such as Lactococcus lactis (Boutibonnes et al., 1991; Whitaker
and Batt, 1991), Lactobacillus bulgaricus (Teixeira et al., 1994), Enterococcus faeca-
lis (Boutibonnes et al., 1993; Flahaut et al., 1996), Streptococcus thermophilus (Auf-
fray et al., 1995), Lactobacillus acidophilus (Broadbent et al., 1997), Lactobacillus
casei (Broadbent et al., 1997), Lactobacillus helveticus (Broadbent et al., 1997), and
Lactobacillus collinoides (Laplace et al., 1999). The induction of heat shock proteins
in response to thermal stress has been analyzed by SDS-PAGE in E. faecalis
(Boutibonnes et al., 1993; Flahaut et al., 1996), L. lactis (Auffray et al., 1992; Kil-
strup et al., 1997; Broadbent and Lin, 1999), S. thermophilus (Auffray et al., 1995),
Oenococcus oeni (Guzzo et al., 1994, 1997), Lb. acidophilus, Lb. casei (Broadbent
et al., 1997), Lb. helveticus (Broadbent et al., 1997), and Lb. collinoides (Laplace
et al., 1999). The induction of heat shock proteins and the acquisition of thermotol-
erance has also been observed in organisms exposed to a variety of growth-limiting
hazards such as chemicals, UV irradiation, viral infection, and pH shifts. Although
heat-induced thermotolerance is accompanied by the induction of heat shock pro-
teins, the contribution of heat shock protein synthesis to the development of acquired
thermotolerance remains controversial (Yamamori and Yura, 1982; VanBogelen
et al., 1987; Hahn and Li, 1990; Sanchez and Lindquist, 1990; Smith and Yaffe,
1991; Boutibonnes et al., 1992; Weber, 1992).
HEAT SHOCK GENES

Classification
Heat shock genes are classified according to the mode of regulation and fall within
four general classes as described for the Gram-positive model microorganism,
B. subtilis. Class I genes are organized in two operons, the groE operon and the
dnaK operon. The CIRCE (controlling inverted repeat of chaperone expression)
operator sequence serves as a cis-acting regulatory element and a binding site for a
repressor protein named HrcA (for heat regulation at CIRCE [Schulz and Schumann,
1996]), the first gene product of the dnaK operon and a negative regulator of class I
heat shock genes (Yuan and Wong, 1995; Schulz and Schumann, 1996). In B. subtilis,
expression of class II genes is dependent on an alternative sigma factor named sigma
B; the synthesis and activity of sigma B increase under stress conditions. No such
regulator of class II heat shock genes has been identified in LAB.

Class III genes are defined as those lacking a CIRCE element that are sigma B
independent. Members of this class include clpC, clpE, and clpP. Class III genes
are negatively regulated by a repressor protein called CtsR (for class three stress
gene repressor [Derre et al., 1999]), the product of the first gene in the clpC operon
(Krüger and Hecker, 1998; Derre et al., 1999; Nair et al., 2000). Class IV stress
response genes are expressed independent of HrcA, sigma B, and CtsR and the
regulatory mechanisms remain to be identified (Hecker et al., 1996). Examples of
class IV stress response genes are ftsH, lonA, and htpG.
groE Operon: groES and groEL
The gene products of the groE (or groESL) operon are the widespread and highly
conserved classical heat shock chaperone proteins, GroES and GroEL. As chaperone
proteins, GroES and GroEL function to protect the cells against heat shock by
binding to cellular proteins in a manner that maintains their native conformation and
minimizes denaturation (Craig et al., 1993). Understanding the manner in which
these proteins function is facilitated through the description of the three-dimensional
molecular structure of GroES and GroEL. The GroEL protein is tetradecameric,
consisting of two stacked rings with seven subunits in each ring forming a barrel-
shaped structure. The GroES protein is heptameric, resembling a dome-shaped
structure (Hartl, 1996). After partially denatured proteins enter the GroEL hydro-
phobic chamber, GroES forms a dome enclosing the chamber, and thereby creating
a protected environment wherein proteins can fold into native structures (Houry
et al., 1999).
Amino acid alignment data suggest that the bicistronic groE operon is highly
conserved, containing only two genes and always in the same order: groES followed
by groEL (Segal and Ron, 1996). A defining characteristic is the presence of a highly
conserved CIRCE operator sequence (TTAGCACTC-N9-GAGTGCTAA), which
preceeds the first structural gene in both the groE and dnaK operons (Zuber and
Schumann, 1994; Yuan and Wong, 1995; Mogk et al., 1997) and the dnaJ gene in
L. lactis (van Asseldonk et al., 1993). The CIRCE operator sequence serves as a
binding site for HrcA, the first gene product of the dnaK operon and a negative
regulator of class I heat shock genes (Yuan and Wong, 1995; Schulz and Schumann,
1996). In most bacteria, the CIRCE is transcribed with the corresponding genes and
participates in the regulation of expression at both the DNA and mRNA levels (Zuber
and Schumann, 1994; Yuan and Wong, 1995).
Among the LAB in which the groE operon has been cloned and characterized
are L. lactis (Kim and Batt, 1993), Lb. helveticus (Broadbent et al., 1998), and
Lactobacillus johnsonii (Walker et al., 1999). Two sets of CIRCE elements were
found flanking the promoter region of the groE operon in Lb. helveticus and Lb.
johnsonii, whereas one CIRCE element was found downstream of the promoter in
L. lactis (see Table 6.1). The presence of two copies of CIRCE elements rather than
one could provide a stronger level of negative regulation. This stronger level of
negative regulation may be manifested in a higher temperature required for heat
shock induction. Although the results of northern hybridization showed increased
expression of the groE operon in these three organisms after heat shock, the induction

TABLE 6.1
Structure and Location of CIRCE Elements in LAB
Organism
Gene/Operon (References) Upstreama Inverted Repeatb Downstreama Inverted Repeatb
groE Bacillus subtilis None TTAGCACTC-N9-GAGTGCTAA

(Li and Wong, 1992)
Lactococcus lactis None TTAGCACTC-N9-GAGTGCTAA
(Kim and Batt, 1993)
Lactobacillus helveticus TTAGCACTA-N9-AAGTGCTAA TTAGCACTT-N9-GAGTGCTAA
(Broadbent et al., 1998)
Lactobacillus johnsonii TTAGCACTC-N9-AAGTGCTAA TTAGCACTT-N9-GAGTGCTAA
(Walker et al., 1999)
dnaK Bacillus subtilis None TTAGCACTC-N9-GAGTGCTAA
(Wetzstein et al., 1992)
Lactococcus lactis AAATTAGCACTC-N9-GAGTGCTAATTT TTAGCACTT-N9-GAGTGCTAA
(Eaton et al., 1993)
Lactobacillus sakei None TTAGCACTC-N9-AAGTGCTAA
(Schmidt et al., 1999)
Streptococcus mutans None TTAGCAGTC-N9-GAGTGCTAA
(Jayaraman et al., 1997)
dnaJ Lactococcus lactis AATTAGCACTCTT-N5-AAGAGTGCTAATT None
(van Asseldonk et al., 1993)
a Orientation of “upstream” and “downstream” is in reference to the transcriptional start site.
b Underlined sequences are the conserved motifs in a number of Gram-positive heat shock genes.

temperature was higher, 52 and 55°C, for Lb. helveticus and Lb. johnsonii (which
contain two CIRCE elements), respectively, compared to 42°C for L. lactis (Kilstrup
et al., 1997).
Other forms of environmental stress can elicit a heat shock response (Lindquist,
1986; Georgopoulos, 1992), as shown by the induction of GroES in L. lactis by acid
and UV254nm irradiation (Hartke et al., 1997). Furthermore, induction of heat shock
proteins GroEL, GroES, and DnaK was also induced in response to salt stress in
L. lactis (Kilstrup et al. 1997), which was not the case when salt was added to the
growth medium of E. coli and B. subtilis (Clark and Parker, 1984; Hecker et al.,
1988; Hecker and Völker, 1990; Völker et al., 1994). The DnaK and GroEL homologs
in Leuconostoc mesenteroides were overexpressed in response to heat shock, cold
shock, and ethanol treatment (Salotra et al., 1995). The relationship between the
expression of heat shock proteins and enhanced tolerance to environmental stress
has been investigated in some LAB. In E. faecalis, the contribution of GroEL and
DnaK synthesis to thermotolerance was inconclusive, whereas de novo protein syn-
thesis was required (Flahaut et al., 1997). Heat shock induction of the groESL operon
in Lb. johnsonii provided some cross-protection against freeze injury (Walker et al.,
1999), demonstrating the potential to improve tolerance to environmental stress by
increasing chaperone concentration at opportune times.
dnaK Operon: hrcA, grpE, dnaK, and dnaJ
The DnaK or HSP70 (70 kDa) family of proteins are among the most well-known
heat shock proteins. These proteins are ubiquitous and have been found in all
prokaryotic and eukaryotic organisms examined to date. The DnaK heat shock
protein has been studied extensively in E. coli and is essential for viability at high
temperatures (Itikawa and Ryu, 1979; Paek and Walker, 1987). At optimum tem-
peratures for growth, DnaK is involved in the synthesis of RNA and DNA and in
cell division (Paek and Walker, 1987; Sakakibara, 1988). Whereas the groE operon
has maintained a highly conserved organization, the number and order of genes
within the dnaK operon have not been highly conserved (Segal and Ron, 1996). The
most common sequence of genes in the dnaK operon is hrcA-grpE-dnaK-dnaJ; this
organization has been found, for example, in Gram-positive bacteria such as B.
subtilis (Wetzstein et al., 1992), Staphylococcus aureus (Ohta et al., 1994), and
Clostridium acetobutylicum (Narberhaus et al., 1992; Behrens et al., 1993). In addi-
tion to these genes, the dnaK operon in S. aureus and C. acetobutylicum contains a
fifth gene located downstream of dnaJ. In B. subtilis, three additional genes are
transcribed (Homuth et al., 1997). In E. coli, grpE is not linked to dnaK (Lipinska
et al., 1988) and no hrcA homologue has been identified.
The dnaK operon has been identified in a number of LAB. In Lactobacillus
sakei and Streptococcus mutans, the dnaK operon consists of four heat shock genes
with the organization hrcA-grpE-dnaK-dnaJ (Jayaraman et al., 1997; Schmidt et al.,
1999). Results of northern hybridization showed induction of these genes by heat
shock, salt, and ethanol in Lb. sakei (Schmidt et al., 1999), whereas in S. mutans,
these genes were induced by heat, acid, and alkali shock (Jayaraman et al., 1997).
The induction of the dnaK operon by heat and salt in Lb. sakei is in agreement with

the results of similar experiments performed on L. lactis (Kilstrup et al., 1997).
Degenerate primers have been used to clone the dnaK operon from L. lactis (Eaton
et al., 1993). In L. lactis, the operon contains three heat shock genes with the
arrangement hrcA-grpE-dnaK followed by a fourth open reading frame (Eaton et al.,
1993). Unlike the genetic organization of the dnaK operon in Lb. sakei and S. mutans
and in most prokaryotic microorganisms, the L. lactis dnaK operon does not contain
a dnaJ gene; rather, the dnaJ gene is a separate transcriptional unit and is located
downstream of the dnaK operon (van Asseldonk et al., 1993).
Another unusual aspect of the L. lactis dnaJ gene is the presence of its own
CIRCE element and its location upstream of the transcriptional start site (van Assel-
donk et al., 1993). Normally, CIRCE elements are found downstream of the site of
transcription initiation. Also, the inverted repeat comprising the CIRCE element in
the L. lactis dnaJ gene is longer than the consensus sequence (van Asseldonk et al.,
1993), which may have an impact on the temperature required for heat shock
induction. Deletion of this inverted repeated resulted in a higher transcriptional level
at lower temperatures, in comparison to the wild-type gene, confirming its role in
the regulation of the heat shock response (van Asseldonk et al., 1993). The CIRCE
element preceding hrcA in the L. lactis dnaK operon was identical to the consensus
sequence (Eaton et al., 1993). In Lb. sakei and S. mutans, a CIRCE element which
differed by only one base from the consensus sequence (Hecker et al., 1996) was
found upstream of hrcA between the transcriptional and translational start sites (see
Table 6.1) (Jayaraman et al., 1997; Schmidt et al., 1999).
clp Family of Genes: clpB, clpC, clpE, clpP, and clpX
Prokaryotic and eukaryotic cells respond to harsh environmental conditions by

synthesizing a group of chaperone proteins and proteases, which together serve to
maintain quality control of intracellular proteins. As stated earlier, chaperone pro-
teins are responsible for promoting proper assembly of proteins and preventing
misfolding and aggregation (Craig et al., 1993). Proteases, on the other hand, degrade
permanently damaged proteins. A large family of proteins named Clp contains
members that exhibit both proteolytic and chaperone activities. Constituents of this
large family of proteins include ClpA, ClpB, ClpC, ClpD, ClpE, ClpP, ClpX, and
ClpY (Schirmer et al., 1996). The proteins comprising the Clp family are classified
according to structural features and sequence similarities. In E. coli, the ClpP protein
resembles the structure of the eukaryotic 26S proteasome (Kessel et al., 1995) and
contains a central barrel that can be flanked at both ends by associating with either
ClpA or ClpX ATPases (Kessel et al., 1995; Wang et al., 1997) to form ClpAP or
ClpXP, respectively. When not associated with one of these ATPases, ClpP functions
as a serine protease (Maurizi et al., 1990) and degrades peptides less than seven
amino acids long (Woo et al., 1989). In the absence of ClpP, the ClpA and ClpX
ATPases function as molecular chaperones (Wickner et al., 1994; Wawrzynow et al.,
1995). However, when associated, the ClpAP or ClpXP complex exhibits protease
activity against substrates with specificity determined by the associated ATPase
subunit. The first substrate degraded by the protease complex was casein, thus the
designation Clp for caseinolytic protease (Katayama et al., 1988).

In B. subtilis and other Gram-positive microorganisms, the gene encoding ClpP
(clpP) is among the class III heat shock genes (Msadek et al., 1998) which also
encode the ClpC (Krüger et al., 1994; Msadek et al., 1994), ClpX (Gerth et al., 1996),
and ClpE (Derre et al., 1999) ATPases. As stated above, class III genes are negatively
regulated by a repressor protein called CtsR (for class three stress gene repressor
(Derre et al., 1999)), the product of the first gene in the clpC operon (Krüger and
Hecker, 1998; Derre et al., 1999; Nair et al., 2000). Recently, the clpP gene was
identified in L. lactis (Frees and Ingmer, 1999) and was found to be induced by salt
(Kilstrup et al., 1997)) heat shock, low pH, and puromycin (Frees and Ingmer, 1999),
which is a tRNA analogue that prematurely terminates translation resulting in the
synthesis of truncated, misfolded peptides. In E. coli and L. lactis, puromycin induces
a heat shock response (VanBogelen et al., 1987; Frees and Ingmer, 1999). The L.
lactis clpP mutant degraded puromycyl-containing peptides at a reduced rate and
extent, relative to the wild type cells, suggesting that ClpP in L. lactis degrades
misfolded proteins caused by environmental stress (Frees and Ingmer, 1999). Fur-
thermore, L. lactis clpP mutants failed to grow at an elevated temperature (37°C)
and in the presence of puromycin. The CtsR binding sequence was found overlapping
the –35°C region of the L. lactis clpP promoter, suggesting that clpP expression
might be negatively regulated by a CtsR homologue in L. lactis.
The genes encoding ClpC, ClpE, and ClpB have also been identified in L. lactis,
with ClpE being part of a new Clp protein family. Northern blot analysis showed
that L. lactis clpB and clpE were strongly induced at the transcriptional level by
heat shock, whereas the clpC protein was only mildly induced (Ingmer et al., 1999).
Although clpC, clpE, and clpB are labeled as heat shock genes, mutants containing
disruptions in these genes responded as wild-type cells to heat and salt treatments.
However, clpE mutants showed increased sensitivity to puromycin, relative to the
wild type, suggesting that ClpE may play a role similar to ClpP in the degradation
of randomly folded proteins (Ingmer et al., 1999).
The clpX gene was cloned from O. oeni using degenerate primers based on
conserved regions in the amino acid sequence (Jobin et al., 1999). In the Gram-
negative microoganisms E. coli and Haemophilus influenzae, clpX is found in a gene
cluster with tig and clpP (Gottesman et al., 1993; Fleischmann et al., 1995). How-
ever, in O. oeni, as in B. subtilis, tig is located next to clpX but clpP is found at a
different location on the chromosome and is transcribed as a monocistronic gene
(Gerth et al., 1996). Although expression of the B. subtilis clpX gene is independent
of CtsR (Gerth et al., 1996), a region resembling the ctsR consensus recognition
sequence (Derre et al., 1999) was found overlapping the transcriptional start site of
the O. oeni clpX gene; however, it was not determined if this sequence had any
impact on the regulation of O. oeni clpX. The clpX mRNA transcript showed
increased expression after a temperature shift from 30 to 42°C. Expression was
relatively high during the exponential phase of growth and gradually declined to
undetectable levels as cells entered stationary phase. Although O. oeni mutants were
not constructed, B. subitilis clpX mutants exhibited impaired growth in response to
salt, ethanol, and heat (Gerth et al., 1998).

COLD STRESS RESPONSE AND CRYOPROTECTION
PHYSIOLOGICAL RESPONSE AND ADAPTATION
Whereas high temperatures diminish protein stability, low temperatures present a
cell with a wide array of challenges, such as decreased rate of enzymatic reactions,
lower affinity for substrate uptake (Nedwell and Rutter, 1994), decreased fluidity of
the cellular membrane (Wada et al., 1990), impaired activity of RNA polymerase
(Grau et al., 1994), and increased intracellular solute concentration which can invoke
osmotic injury on proteins (Franks, 1995). Death associated with freezing and
thawing is primarily attributed to membrane damage and DNA denaturation (Alur
and Grecz, 1975; Calcott and MacLeod, 1975; El-Kest and Marth, 1992). Bacterial
adaptation to low temperatures is an active process resulting in increased fatty acid
unsaturation (Murata and Wada, 1995) and polypeptide synthesis (Jones et al., 1987).
Many comprehensive reviews of the microbial cold shock response have been pub-
lished (Jones and Inouye, 1994; Wolska, 1994; Graumann and Marahiel, 1996;
Panoff et al., 1998).
The study of the cold shock response in LAB is particularly important because
these microorganisms are routinely exposed to a variety of stresses, including low
temperature conditions, during the production of fermented food products (Rallu
et al., 1996). For example, fermentations normally begin with the addition of a frozen
“starter” culture to “raw” food material. Therefore, understanding the cold shock
response in these organisms may contribute to the development of starter cultures
with a greater capacity for freeze tolerance. The physiological response to suboptimal
growth temperatures has been investigated in a number of LAB including L. lactis
(Panoff et al., 1994, 1995; Kim and Dunn, 1997), S. thermophilus CNRZ302 (Wout-
ers et al., 1999), S. thermophilus TS2 (Kim and Dunn, 1997), Lb. acidophilus CRL
639 (Lorca and de Valdez, 1999), E. faecalis JH2-2 (Thammavongs et al., 1996;
Panoff et al., 1997), Pediococcus pentosaceus PO2 (Kim and Dunn, 1997), and
Lb. helveticus LB1 (Kim and Dunn, 1997).
Increased capacity towards survival in extreme cold temperatures after precon-
ditioning at low positive temperatures, a phenomenon termed cryotolerance, was
achieved for S. thermophilus CNRZ 302 (Wouters et al., 1999), E. faecalis JH2-2
(Thammavongs et al., 1996), L. lactis subsp. lactis (Panoff et al., 1995; Kim and
Dunn, 1997), and P. pentosaceus, but not for L. lactis subsp. cremoris, Lb. helveticus
LB1, and S. thermophilus TS2 (Kim and Dunn, 1997). The addition of chloram-
phenicol to the growth medium, thereby inhibiting protein synthesis, during a cold
treatment abolished cryotolerance in L. lactis (Wouters et al., 1999). The precondi-
tioning treatment also imparted L. lactis subsp. lactis with an improved survival
capacity against freezing temperature (–20°C) and heat (52°C) challenge (Panoff
et al., 1995). Furthermore, exponential-phase L. acidophilus cells growing at 25°C
displayed a greater resistance to variety of environmental stresses including expo-
sures to ethanol, peroxide, lactic acid, and osmotic stress relative to cells growing
at the optimal temperature of 37°C (Lorca and de Valdez, 1999).

COLD-STRESS GENES AND GENE PRODUCTS
Similar to the heat shock response, bacteria respond to low temperatures by express-
ing a number of cold shock proteins (Jones et al., 1987) regulated at both transcrip-
tional (La Teana et al., 1991; Jones et al., 1992) and translational (Brandi et al.,
1996; Goldenberg et al., 1996; Jones et al., 1996; Panoff and Lucas, 1996) levels.
However, in contrast to heat shock proteins, which include chaperones and proteases
required for protein folding and degradation, respectively, cold shock proteins per-
form a variety of different functions in bacterial cells. One of the major outcomes
of low temperature exposures is the formation of stable DNA and RNA secondary
structures which interfere with efficient DNA replication and mRNA transcription
and translation. Cold shock proteins comprise a family of small (7 kDa) transiently
expressed proteins that function as RNA chaperones to facilitate translation of
mRNA by blocking the formation of secondary structures (Jiang et al., 1997).
Study of the cold shock response has defined a nomenclature for the specific
types of proteins expressed in response to low temperature. Low-temperature stress
proteins are generally labeled as cold shock proteins (CSPs), whereas cold acclima-
tion proteins (CAPs) or cold-induced proteins (CIPs) are labeled as such according
to the size of the protein and the method by which the organisms were transferred
to low temperatures. The CSPs are immediately and transiently induced upon an
abrupt shift to a low temperature (Jones et al., 1987; Lottering and Streips, 1995),
whereas CAPs are synthesized during continuous growth at low temperatures (Rob-
erts and Inniss, 1992; Whyte and Inniss, 1992; Berger et al., 1996). The CIPs are
defined as CSPs larger than 10 kDa (Graumann and Marahiel, 1996). Aside from a
single study that differentiates between CSPs and CAPs in E. faecalis (Panoff et al.,
1997), research on microbial response to low temperature at the protein level in
LAB has focused entirely on CSPs.
Much of the current understanding of the cold shock response has come from
investigations on E. coli and B. subtilis. E. coli contains a large family of CSPs,
consisting of nine proteins from CspA to CspI (Lee et al., 1994). Three proteins
(CspB, CspC, and CspD) have been identified in B. subtilis (Graumann et al., 1996).
All CSPs share high sequence similarity (over 40%) to eukaryotic Y-box proteins
(Wolffe et al., 1992) and both CspA of E. coli and CspB of B. subtilis recognize the
highly conserved Y-box sequence ATTGG (La Teana et al., 1991; Jones et al., 1992;
Graumann and Marahiel, 1994), which is present in regulatory regions of major
histocompatibility complex II genes (Sommerville and Ladomery, 1996). The Y-box
proteins serve regulatory functions at the transcriptional and translational level.
Accordingly, the CSP-homologous domain was shown to confer sequence-specific
binding to single-stranded DNA and RNA (Sommerville and Ladomery, 1996). Both
NMR and x-ray crystallography showed that the three-dimensional structures of
CspB (B. subtilis) and CspA (E. coli), the major cold shock proteins (Goldstein
et al., 1990; Graumann and Marahiel, 1994), displayed very similar five-stranded β-
barrel structures with outward-facing residues for ssDNA binding (Schindelin et al.,
1993; Schnuchel et al., 1993; Newkirk et al., 1994; Schindelin et al., 1994). Physi-
ological investigations have confirmed CspA and CspB as ssDNA-binding proteins
(Graumann and Marahiel, 1994; Newkirk et al., 1994). Furthermore, CspA and CspB

exhibit mRNA binding capacity because both have RNA-binding motifs, RNP-1
(ribonucleoprotein) and RNP-2 (Schindelin et al., 1993; Jones and Inouye, 1994;
Jiang et al., 1997).
Taken together, this information suggests that CSPs exhibit regulatory effects at
the transcriptional and translational level through protein–nucleic acid interactions.
Indeed, CspA enhanced the transcription of two cold-inducible genes, gyrA and hns
(La Teana et al., 1991; Jones et al., 1992), suggesting that CspA may function as a
transcriptional activator of other cold shock genes. Furthermore, CspA functions as
an RNA chaperone by binding to mRNA and preventing the formation of secondary
structures (Jiang et al., 1997). Although, the physiological contribution of CSPs to
low temperature adaptation is not well understood, cold shock causes a 200-fold
induction of CspA in E. coli (Jones et al., 1987; Goldstein et al., 1990). Additionally,
disruption of CspB in B. subtilis resulted in increased sensitivity to freezing (Will-
imsky et al., 1992) and deletion of the three known CSPs in B. subtilis was lethal
(Graumann et al., 1997).
Only recently has the study of cold shock genes and gene products been focused
on LAB, but it is now progressing quickly. The proteins expressed in response to a
shift from optimal to suboptimal temperatures in Lb. acidophilus (Lorca and
de Valdez, 1999), E. faecalis (Panoff et al., 1997), L. lactis (Panoff et al., 1994;
Broadbent and Lin, 1999; Wouters et al., 1999), and S. thermophilus (Wouters et al.,
1999) were extracted and viewed by two-dimensional SDS-PAGE gel electrophore-
sis. A cold shock gene showing high sequence similarity with the major cold shock
protein of E. coli (68%) and B. subtilis (70%) was cloned from L. lactis subsp. lactis
using a PCR-based approach (Kim and Dunn, 1997). This approach was taken a
step further to amplify the major cold shock protein from 11 other LAB strains (Kim
et al., 1998), suggesting that the major cold shock protein is highly conserved in
LAB. The first cold shock gene identified in LAB, cspB, was cloned in L. lactis
(Chapot-Chartier et al., 1997). Results of northern hybridization (Chapot-Chartier
et al., 1997) and cspB-directed B-galactosidase assays (Chapot-Chartier et al., 1997;
Wouters et al., 1998) indicate that the L. lactis cspB is cold-shock inducible (Wouters
et al., 1998).
Two additional cold-shock genes, cspL and cspP, were cloned from Lb. plan-
tarum (Mayo et al., 1997). The identification of five cold shock genes, cspA, cspB,
cspC, cspD, and cspE (Wouters et al., 1998), in L. lactis quickly followed. Four of
these genes were found to be clustered, for the first time, in two tandem groups
(cspA/cspB and cspC/cspD), whereas cspE was found as a single gene. These cold
shock genes can be divided, on the protein level, into two groups based on iso-
electric point (pI) and homology: CspA and CspC share 80% identical residues and
have a pI of 9, whereas CspB, CspD, and CspE share 85% identical residues and
have a pI of 5 (Wouters et al., 1998). Transcriptional analyses showed that cspA,
cspB, cspC, and cspD were cold-shock inducible, whereas cspE was not (Wouters
et al., 1998). The RNA-binding motifs (RNP-1 and RNP-2) (Schindelin et al., 1993;
Jones and Inouye, 1994; Schroder et al., 1995) were found within the L. lactis gene
products, including a putative β-barrel structure formed by five β-strands (Wouters,
2000). Expression levels of these CSPs, in response to low temperature treatment,
coincide with freeze survival (Wouters et al., 1999).

Perhaps the single greatest contribution to the understanding of the physiological
and regulatory roles of CSPs in LAB was made very recently by Wouters et al.
(2000), where attempts were made to overproduce these CSPs in L. lactis using the
nisin controlled expression (NICE) system (Kuipers et al., 1998). Using the NICE
system, CspB, CspD, and CspE were overproduced to high levels, whereas the
concentrations of CspA and CspC were limited due to low protein and mRNA
stability, respectively. The reduced stability of CspA is attributed to the presence of
an Arg residue at position 58 rather than a Pro residue, which are known to reduce
the entropy of unfolded proteins (Schindler et al., 1999). Replacing the Arg residue
at position 58 for a Pro residue increased the concentration of CspA* 20-fold upon
induction with nisin. Overproduction of CspA* resulted in the induction of CspE
and several CIPs. Likewise, overproducing CspC resulted in the induction of CspB
and the putative CspF and CspG proteins, in addition to several CIPs. This suggests
that CspA* and CspC may be transcriptional activators acting on Y-box motifs
(La Teana et al., 1991; Jones et al., 1992; Brandi et al., 1994) observed in the
upstream regions of the lactococcal csp genes (Wouters et al., 1998).
Overproduction of CspA, CspB, CspD, or CspE did not affect the level of any
of the other CSPs; however, overproduction of CspB and CspD increased the syn-
thesis of several CIPs, suggesting a regulatory role for these proteins. With respect
to adaptation to freeze-survival, overproduction of CspB and CspE resulted in
approximately a ten- and five-fold increased survival, respectively, compared to that
of non-induced cells after four repetitive freeze–thaw cycles. In a previous report,
overexpression of CspD in L. lactis cells enhanced survival after freezing approxi-
mately two- to ten-fold compared to the control cells (Wouters et al., 1999). Over-
production of CspA, CspC, or CspA* provided no additional freeze-protective effects
compared to control cells (Wouters et al., 2000). These results indicate that CspB,
CspE, and CspD are directly involved in the protection against freezing (Wouters
et al., 1999, 2000).
ACID ADAPTATION
TOLERANCE AND ADAPTATION TO LOW PH
The understanding of acid tolerance and adaptation in LAB is expected to contribute
to enhancement of probiotic survival through the gastrointestinal tract. Furthermore,
this understanding is important with regard to starter culture performance during
fermentation since cell growth is always accompanied by lactic acid accumulation.
Lactic acid poses a significant threat to the cell because, in a low pH environment,
organic acids remain protonated and uncharged and can thereby pass easily into the
cell through the cell membrane. At a similar extracellular pH, a strong inorganic acid,
such as HCl, is likely to be in a disassociated state and will not passively diffuse
through the cell membrane (Kashket, 1987). Accordingly, reducing the intracellular
pH of L. lactis and S. bovis was more effective when the extracellular pH was adjusted
with lactic acid than with HCl acid (Poolman et al., 1987; Cook and Russel, 1994).
Bacteria are equipped with a number of mechanisms that confer acid tolerance.
Among the mechanisms that will be reviewed in this chapter are proton translocation,

arginine deaminase (ADI) pathway, amino acid decarboxylation-antiporter reactions,
and the citrate transport system. The activation of these mechanisms is the result of
an altered pattern of gene expression when bacteria are confronted with a change
in the extracellular pH (reviewed by Olson, 1993). Gene expression-dependent
adaptation is evident upon analysis of whole-cell protein extracts separated by two-
dimensional gel electrophoresis. Changes in protein patterns have been observed
during acid adaptation in E. coli (Heyde and Portalier, 1990; Hickey and Hirschfeld,
1990), Salmonella typhimurium (Hickey and Hirschfeld, 1990; Foster, 1991, 1993),
Aeromonas hydrophila (Karem et al., 1994), and Listeria monocytogenes (Davis
et al., 1996). The adapted cells showed enhanced survival capacity against lethal
acid challenge relative to unadapted cells (Goodson and Rowbury, 1989; Foster and
Hall, 1990; Davis et al., 1996; O’Driscoll et al., 1996). This inducible adaptation to
acid is termed the acid tolerance response (ATR; Foster and Hall, 1990) and is
dependent on protein synthesis (Foster, 1991; Raja et al., 1991; Karem et al., 1994;
O’Hara and Glenn, 1994; Davis et al., 1996). The ATR has been observed in several
LAB such as Lc. mesenteroides (McDonald et al., 1990), Lb. plantarum (McDonald
et al., 1990), S. mutans (Belli and Marquis, 1991), Enterococcus hirae (Belli and
Marquis, 1991), and L. lactis (Hartke et al., 1996; Rallu et al., 1996; O’Sullivan and
Condon, 1997). The ATR was displayed in L. lactis in response to sublethal exposures
to lactic acid (Hartke et al., 1996), HCl (Rallu et al., 1996), and UV radiation (Hartke
et al., 1995). Furthermore, resting cells were much better adapted to low pH than actively
dividing cells (Hartke et al., 1994). Although the ATR was achievable in L. lactis, the
requirement for protein synthesis is controversial: L. lactis subsp. lactis IL1403 showed
acid adaptation in the presence of chloramphenicol (Hartke et al., 1996), whereas
L. lactis subsp. cremoris MG1363 (Rallu et al., 1996) and L. lactis subsp. cremoris 712
(O’Sullivan and Condon, 1997) did not. The ATR in L. lactis subsp. cremoris 712 also
conferred enhanced resistance to lethal doses of heat, ethanol, sodium chloride, and
hydrogen peroxide; however, with the exception of heat, mild treatments of other
environmental stresses did not induce tolerance to acid (O’Sullivan and Condon, 1997).
Cross-protection induced by acid has also been observed in S. typhimurium (Leyer and
Johnson, 1993; Lee et al., 1995) and L. monocytogenes (O’Driscoll et al., 1996).
To identify some of the genes associated with acid tolerance, insertional mutants
of L. lactis were prepared using the pG+host9:ISS1 plasmid (Maguin et al., 1996).
Twenty-one mutants were isolated based on their ability to grow under high temper-
ature and low pH, conditions under which wild-type L. lactis does not grow well (Rallu
et al., 2000). All of the mutants were acid tolerant and 11 were resistant to a variety
of stresses. Many of the insertions in acid-tolerant mutants took place in genes impli-
cated with glutamate/glutamine transporters, high-affinity phosphate transporters, and
purine metabolism. These results suggest an intimate relationship between stress
response mechanisms and cellular metabolic pathways in L. lactis (Rallu et al., 2000).
PROTON MOVEMENT: H+-ATPASE

The F0F1 ATPase functions to maintain a favorable intracellular pH and protect cells
during exposures to acidic environments by translocating protons to the environment
at the expense of ATP. The activity and number of proton-translocating ATPases

increases in several LAB as the extracellular pH is adjusted from neutral to pH 5.0
(Kobayashi et al., 1984; Belli and Marquis, 1991; Nannen and Hutkins, 1991).
Whereas the atp operon, encoding various subunits of the H+-ATPase, is minimally
affected by pH in E. coli (Kasimoglu et al., 1996) and constitutively expressed in
B. subtilis (Santana et al., 1994), acidification of the growth medium leads to elevated
gene expression in Lb. acidophilus (Kullen and Klaenhammer, 1999).
Proton-translocating ATPase is an important mechanism in maintaining cyto-
plasmic pH in L. lactis subsp. lactis and cremoris (Nannen and Hutkins, 1991),
Lb. casei (Bender and Marquis, 1987; Nannen and Hutkins, 1991), E. faecalis
(Kobayashi et al., 1984, 1986), E. hirae (Belli and Marquis, 1991), S. mutans (Bender
et al., 1986; Belli and Marquis, 1991), and Lb. acidophilus (Kullen and Klaenham-
mer, 1999). An acid-sensitive isolate of L. lactis contained a mutation in the ATPase
structural gene and was unable to maintain a neutral intracellular pH in an acidic
environment (Amachi et al., 1998). Furthermore, mutants of S. typhimurium lacking
the proton-translocating ATPase are extremely acid sensitive and do not display an
ATR (Foster and Hall, 1991).
ARGININE DEAMINASE (ADI) PATHWAY

Bacteria metabolize arginine by the arginine deaminase (ADI) pathway (Cunnin
et al., 1986). This pathway consists of three enzymes: arginine deaminase, ornithine
carbamoyltransferase, and carbamate kinase. A fourth component, identified in
L. lactis (Poolman et al., 1987), Lb. sake (Zuniga et al., 1998), and Pseudomonas
aeruginosa (Verhoogt et al., 1992), is a membrane-bound antiport protein that cat-
alyzes the exchange between arginine and ornithine. These enzymes catalyze the
conversion of arginine to ornithine, ammonia, and carbon dioxide and generate 1 mol
of ATP per mole of arginine consumed (Figure 6.1). By generating ammonia, the ADI
pathway is a mechanism for survival in acidic environments (Marquis et al., 1987).
HOOC-CH(-NH2)-(CH2)3-NH-C(=NH)-NH2 (arginine)
arginine deiminase
NH4+
HOOC-CH(-NH2)-(CH2)3-NH-CO-NH2 (citrulline)
ornithine carbamoyltransferase Pi
HOOC-CH(-NH2)-(CH2)3-NH2 (ornithine) H2N-CO- P (carbamoyl phosphate)
ADP
carbamate kinase
ATP
HCO3, NH4+
FIGURE 6.1 The ADI pathway. (From Cunin, R.N., Microbiol. Rev., 50: 314–352, 1986.
With permission.)

The development of acid tolerance depends on the rise in pH associated with
ammonia production (Marquis et al., 1987). The enzymatic properties of the ADI
pathway are well documented in a variety of bacteria (Stalon, 1972; Stalon et al.,
1972; Fenske and Kenny, 1976; Crow and Thomas, 1982; Cunnin et al., 1986).
The enzymes in the ADI pathway are inherently acid tolerant and are activated
in response to low pH (pH 2 to 3) in several species of Streptococcus. As such, these
enzymes allow bacteria to recover from acid stress severe enough to prevent the cell
membrane from functioning normally (Casiano-Colon and Marquis 1988). In most
LAB, the ADI pathway is repressed by glucose and induced by arginine (Simon
et al., 1982; Hiraoka et al., 1986; Manca de Nadra et al., 1986; Poolman et al., 1987).
The ADI pathway imparts LAB with enhanced tolerance to acid, primarily through
the continuous production of acid-neutralizing ammonia from arginine.
DEGRADATIVE AMINO ACID DECARBOXYLASES

Another strategy bacteria employ to maintain a favorable intracellular pH depends
on amino acid decarboxylation-antiporter reactions. These reactions involve trans-
porting an amino acid into the cell where it is decarboxylated. A proton is consumed
in the reaction, and the product is exported from the cell via an antiporter. The result
of this reaction is a decrease in intracellular acidity (Molenaar et al., 1993).
A gadC-encoded glutamate-γ-aminobutyrate antiporter and a gadB-encoded glu-
tatmate decarboxylase have been identified in L. lactis (Sanders et al., 1998). The
two genes are located in a bicistronic gadCB operon and show increased expression
during growth and acidification of unbuffered media supplemented with glutamate
(Sanders et al., 1998). According to the model proposed by Waterman and Small
(1996) for Shigella flexneri, the putative membrane protein, GadC, is involved in
the antiport of glutamate, while glutamate decarboxylase, GadB, converts the inter-
nalized glutamate to γ-aminobutyrate with the simultaneous consumption of a proton
and production of one molecule of CO2. The net result is the removal of a proton
from the cytosol, which increases the intracellular pH. Support for the amino acid
decarboxylation-antiport model is displayed in a histidine decarboxylase mutant of
Lactobacillus 30a that was unable to alkanize its environment in the presence of
histidine (Recsie and Snell, 1972).
Another function for GadCB may be production of a proton motive force and
generation of energy in the presence of glutamate, as shown for a strain of Lactoba-
cillus (Higuchi et al., 1997). A similar mechanism was described for a different
Lactobacillus strain, where the action of amino acid antiport and decarboxylation are
combined for pH regulation and energy production. The aspartate–alanine antiporter
generated ATP in Lactobacillus strain M3 (Abe et al., 1996), and histidine decarbox-
ylation coupled with electrogenic histidine–histamine antiport contributed to energy
production and intracellular acid reduction in Lb. buchneri (Molenaar et al., 1993).
CITRATE TRANSPORT SYSTEM

Citrate is present in milk at low concentrations and is co-metabolized with glucose
by many strains of LAB (Cocaign-Bousquet et al., 1996). Citrate fermentation has
been studied in detail in Lc. mesenteroides (Marty-Teysset et al., 1995, 1996) and

L. lactis (Hugenholtz, 1993). The citrate fermentation pathway is induced by citrate
in Lc. mesenteroides (Marty-Teysset et al., 1996). Alternatively, citrate utilization in
L. lactis subsp. lactis biovar diacetylactis is dependent on the rate of uptake, cata-
lyzed by the product of the citP gene (David et al., 1990), and expression of citP is
influenced by extracellular pH (Garcia-Quintans et al., 1998). Activity of CitP is
also dependent on extracellular pH, with the highest uptake rates observed at pH
4.5 (Magni et al., 1996). Accordingly, cell growth accompanied by the natural
acidification of the medium results in increased synthesis of CitP, higher citrate
transport activity, and greater flux through the citrate fermentation pathway. Upon
entering the cell, citrate is cleaved by citrate lyase, which yields acetate and oxalo-
acetate. Decarboxylation of oxaloacetate yields carbon dioxide and pyruvate, con-
sumes a proton, and results in alkalinization of the cytoplasm (Ramos et al., 1994;
Lolkema et al., 1995; Marty-Teysset et al., 1996). Pyruvate is converted to the end
product lactate, which leaves the cell through the CitP transporter in exchange for
citrate. Together, the consumption of a proton during oxaloacetate decarboxylation
and the excretion of lactate, in exchange for citrate, provide citrate-fermenting LAB
with a resistance mechanism against acid toxicity. This acid resistance mechanism
was demonstrated by the undiminished growth of L. lactis in a medium at pH 4.5
containing both glucose and citrate, whereas growth was poor in the absence of
either glucose, citrate, or CitP (Garcia-Quintans et al., 1998); glucose is required to
produce lactate that drives the exchange for citrate via the CitP transporter. The CitP
transporter is purported to be among the proteins that are synthesized de novo during
inducible adaptation to acid (e.g., acid tolerance response), shedding some light on
the poorly understood mechanisms involved in acid resistance in bacteria.
ALKALINE STRESS RESPONSE

The alkaline response is the least studied of the stress responses. Exposure to sublethal
alkaline conditions results in increased resistance to lethal alkalinization (pH 10.0 to
10.5) in E. coli (Goodson and Rowbury, 1990) and thermotolerance at 55°C in
Salmonella enteritidis (Humphrey et al., 1991). However, sublethal alkaline conditions
sensitized E. coli to acid (Rowbury et al., 1993) and, conversely, sublethal acid treat-
ment sensitized E. coli to alkaline pH (Rowbury and Hussain, 1996).
With regard to the LAB, the alkaline stress response has been investigated only
for Enterococcus species. As early as 1934, Sherman and Stark, identified E. faecalis
by its ability to grow at pH 9.6. A neutral cytoplasmic pH is not required to withstand
high alkaline pH in Enterococcus hirae (Mugikura et al., 1990); however, the alkaline
treatment amplifies the Na+-ATPase (Kakinuma and Igarashi, 1990), suggesting
modification in gene expression. This hypothesis was confirmed when whole-cell
proteins extracted from E. faecalis, viewed on two-dimensional gel electrophoresis,
showed amplification of 37 polypeptides after a 30-minute alkaline treatment at pH
10.5 (Flahaut et al., 1997). Furthermore, cells adapted to pH 10.5 were tolerant to
pH 11.9. The addition of chloramphenicol to the culture at pH 10.5 resulted in a
minor decrease in alkaline tolerance. Acquisition of acid tolerance of acid-exposed
cells treated with chloramphenicol was similar to untreated cells (Flahaut et al.,
1997). These observations suggest that protein synthesis in E. faecalis is not a

prerequisite to developing tolerance to lethal extremes in pH, similar to results found
with L. lactis subsp. lactis IL1403 (Hartke et al., 1996). Furthermore, as found in
E. coli (Rowbury et al., 1993; Rowbury and Hussain, 1996), acid-exposed E. faecalis
cultures were sensitized to alklaline pH, and alkaline-treated cells acquired sensitivity
to acid damage (Flahaut et al., 1997). The next section will highlight what has been
published to date for select genera and species of LAB relative to osmoregulation.
OSMOTIC STRESS
COMPATIBLE SOLUTE
Organisms, both eukaryotic and prokaryotic, respond to osmotic stress in essentially
the same way: by accumulating non-toxic low molecular weight compounds. These
compounds, called compatible solutes, which include sugars, polyols, amino acids
and amine derivatives, do not inhibit vital cellular functions even when present in
very high concentrations. Compatible solutes have at least three functions: 1) allow
the cell to retain positive turgor pressure which contributes to osmotic balance with
the extracellular environment; 2) enhance enzyme stability at low aw; and 3) maintain
the integrity of the cellular membrane during desiccation (Kets and de Bont, 1994).
A review on the role of compatible solutes in osmoregulation in bacteria has recently
appeared (Bremer and Kramer, 2000).
Lactobacillus acidophilus IFO 3532 is tolerant of osmotic pressures from elec-
trolytes or non-electrolytes up to an osmolality of 2.8 M. Glycine betaine was
identified some years ago as the intracellular osmolyte which protected Lb. acido-
philus from osmotic stress (Hutkins et al., 1987). Glycine betaine is a constituent
of the yeast extract present in MRS medium and upon the addition of NaCl (1 M),
glycine betaine is transported into the cells by a specific transport system. The rate
of glycine betaine transport is proportional to the osmolality of the medium. Energy
in the form of a fermentable sugar was necessary for glycine betaine transport.
Transport of glycine betaine in Lb. acidophilus appears to be activated (stimulated)
rather than induced by osmotic stress. Chloramphenicol did not inhibit glycine
betaine transport indicating that the induction of new protein synthesis was not
necessary for transport (Hutkins et al., 1987).
In a defined medium, the growth of Lb. plantarum strain P743 in 0.6 M NaCl
decreased seven-fold; however, the addition of 2 mM glycine betaine permitted
growth almost to the level of the control treatment that was lacking salt. In addition,
glycine betaine addition allowed growth at higher NaCl levels (Kets and de Bont,
1994). Survival of dried cells increased significantly when Lb. plantarum was grown
in the presence 2 mM betaine and 0.6 or 1.0 M NaCl as compared to cells grown
in the absence or presence of glycine betaine or salt (Kets and de Bont, 1994).
Similar results were obtained with Enterococcus faecium strain URL-EF1 and
Lb. halotolerans ATCC 35410 (Kets et al., 1996). Cells grown under osmotic stress
(NaCl) in the presence of glycine betaine survived drying at higher levels than did
unconditioned cells. However, Linders et al. (1997) found that Lb. plantarum strain
P743 grown in a complex medium with 1 or 1.25 M NaCl prior to drying had reduced
survival as compared to controls lacking salt. In addition, Lb. plantarum strain P743

grown in the absence of salt produced dried cells with higher levels of residual
glucose fermenting ability than cells grown in a salt-containing medium (Linders
et al., 1997). This resulted from the fact that lower cell numbers were recovered
from the NaCl-containing medium.
At 0.11 g dried cells/g sample, the residual glucose fermenting activity of
Lb. plantarum strain P743 dried cells harvested from a synthetic medium lacking
salt was approximately 12 times that of the same amount of dried cells obtained
from a salt-containing medium (Linders et al., 1998). In dried cells of Lb. plantarum
strain P743, residual glucose-fermenting activity was a function of the cell density
before drying. At the highest cell density (0.23 g dried cells/g sample), the glucose-
fermenting activity was eight times that of the lowest cell density (0.025 g dried
cells/g sample). To obtain enzymatically active dried cells of Lb. plantarum, Linders
et al. (1998) recommended growing cells in an osmotically unstressed medium. They
also suggested that harvested and washed cells should not be diluted before drying.
Lactobacillus plantarum strain P743, when grown in a complex medium (MRS
broth) with NaCl, accumulated glycine betaine and another compatible solute which
was identified as L-carnitine. No accumulation of carnitine occurred when beef
extract was eliminated from the formulation for MRS broth; similarly, glycine
betaine was not accumulated by Lb. plantarum when yeast extract was omitted (Kets
and de Bont, 1994). Addition of 0.5 mM L-carnitine to a chemically defined medium
containing 0.4 M NaCl led to a doubling of the growth rate as compared to a salt-
containing medium lacking carnitine. Thus, carnitine acts as a compatible solute
making Lb. plantarum more tolerant to salt stress (Kets et al., 1994).
In Lb. plantarum ATCC 14917 growing in a chemically defined medium con-
taining high levels of KCl, the preferred osmoprotectant was glycine betaine. How-
ever, it was necessary to add glycine betaine to the chemically defined medium,
indicating that the organism could not synthesize the compound (Glaasker et al.,
1996b). While K+ accumulates intracellularly under KCl stress, it does not act as a
osmoprotectant. In a medium in which the growth of Lb. plantarum is inhibited by
KCl or NaCl, the addition of glycine betaine or proline increased the specific growth
rate. Moreover, transport and accumulation of glycine betaine increased rapidly with
an increase in osmolarity even in the presence of chloramphenicol (Glaasker et al.,
1996b). Iso-osmolar sucrose, lactulose, NaCl, or KCl had similar effects on transport
rates and accumulation of glycine betaine. The final levels of accumulated glycine
betaine were proportional to the increase in the final medium osmolarity, but the
initial rate of uptake was similar regardless of osmolarity. Glycine betaine transport
rates (activated transport) increase when the difference between internal and external
osmolarity reaches a certain threshhold. When the osmolarities are balanced, then
net glycine betaine uptake ceases. Osmotic downshock caused an efflux of glycine
betaine, which was rapid and greatly exceeded the uptake rates (Glaasker et al.,
1996b). Efflux, too, depended on osmolarity. Metabolic energy is not necessary for
efflux, whereas uptake required ATP synthesis (Glaasker et al., 1996b).
Glycine betaine, accumulated at high osmolarity by Lb. plantarum ATCC 14917,
was released from the cell upon osmotic downshock. Efflux of glycine betaine was
biphasic, with a rapid release phase and a slower release phase (Glaasker et al.,
1996a). The rapid efflux phase was mechanosensitive channel-mediated, whereas

the slow efflux of glycine betaine was mediated through a carrier (uniporter) trans-
port system (Glaasker et al., 1996a).
Addition of equimolar amounts of KCl or lactose to a chemically-defined
medium used to grow Lb. plantarum ATCC 14917 enhanced the rate of glycine
betaine uptake to the same extent. Facilitated influx of lactose to the equilibration
level combined with uptake of glycine betaine resulted in hyperosmolarity of the
cytoplasm. As a compensatory mechanism to decrease the resultant hyperosmolarity,
there was a net exit of glycine betaine (Glaasker et al., 1998b). Cells stressed by
KCl behaved differently than cells stressed by lactose. While there was uptake of
KCl into Lb. plantarum cells, the increased cellular level of KCl did not compensate
for a decrease of turgor. Therefore, glycine betaine uptake was necessary to reverse
the cell turgor decrease imposed by the salt stress, and efflux of glycine betaine did
not occur (Glaasker et al., 1998b).
The intracellular accumulation of quaternary ammonium compatible solutes in
Lb. plantarum ATCC 14917 is mediated via a single transport system, QacT (qua-
ternary ammonium compound transporter), which has a high affinity for glycine
betaine or carnitine and a low affinity for proline (Glaasker et al., 1998a). Transport
uptake rates were inhibited by internal glycine betaine or proline; however, with an
increase in osmolarity, the inhibition by the internal osmolyte was relieved with the
rapid activation of the QacT system. Upon osmotic downshock, there was release
of glycine betaine via a system resembling that of a mechanosensitive ion channel
(Glaasker et al., 1998a). At least in Lb. plantarum, both uptake and efflux of com-
patible solutes utilize osmoregulated systems. Therefore, in Lb. plantarum ATCC
14917, compatible solutes are taken up via a single system, QacT, which is activated
by osmotic shock. The QacT uptake system is turgor-regulated; however, when cell
turgor is restored, solute uptake is diminished. Inhibition of QacT by an internal
compatible solute such as glycine betaine also acts to control excessive accumulation
of compatible solute. On hypo-osmotic shock, compatible solutes are released from
the cell in order to maintain cell turgor. Efflux occurs via two mechanisms: 1) a
rapid almost instantaneous release of solute mediated by a channel system, followed
by 2) a slow release of solute via an efflux carrier system (Glaasker et al., 1996a,
1996b, 1998a, 1998b). Osmoregulation in Lb. plantarum ATCC 14917 has been
envisioned by Poolman and Glassker (1998) as follows: under osmostasis, there is
a basal level of glycine betaine or other compatible solute which is maintained by
the combined action of efflux via the specific efflux carrier system and uptake by
QacT; the efflux channel system does not play a role in osmostasis. During hyper-
osmotic shock, QacT is activated and glycine betaine enters the cell; both efflux
systems are inhibited. Under hypo-osmotic shock, QacT is inhibited but both efflux
systems are activated (Poolman and Glaasker, 1998). Therefore, maintenance of cell
turgor is tightly regulated in Lb. plantarum.
Subjecting Lb. plantarum strain L-73 to glycerol or NaCl in a solute/water
medium at –5.6 MPa had little or no effect on the subsequent viability of the cells
when the solute was added rapidly (within 1 s) to simulate shock conditions or added
slowly, over 20 min (Poirier et al., 1998).
As another example, a commercial culture of Lactobacillus alimentarius has
been used as a bioprotectant in certain meat products (Andersen, 1997). The culture

is generally used as a “biohurdle” in conjunction with chemicals such as NaCl,
organic acids, and/or sulfite. The growth of Lb. alimentarius strain BJ33 in MRS
broth was not inhibited by 8% NaCl, 50 mM citric acid or 100 mM gluconic acid.
However, when washed cells from a medium containing one of the organic acids
were inoculated into MRS broth supplemented with 8% NaCl, there was limited
growth (OD600nm = 0.2 to 0.3 within 7 days) (Lemay et al., 2000). When the reverse
experiment was conducted, growth in 8% NaCl followed by inoculation of washed
cells into either citric acid or gluconic acid containing MRS broth, an OD600nm of
0.6 to 0.8 was reached in 3 to 4 days. Thus, Lemay et al. (2000) demonstrated that
Lb. alimentarius grown in the presence of sublethal levels of organic acids grew
poorly when subsequently inoculated into a medium containing a sublethal concen-
tration of NaCl. It is not clear why cells grown in organic acids are stressed by
sublethal concentrations of salt. It is clear, however, that if the above mentioned
strain of Lb. alimentarius is used as a biohurdle in foods, the influence of chemical
hurdles on the growth of the organism must be determined.
In the presence of 500 mM KCl, glycine betaine uptake was stimulated five-
fold in cells of L. lactis subsp. cremoris MG1363 or L. lactis subsp. lactis IL1403
(van der Heide and Poolman, 2000). In contrast to the finding that proline inhibits
uptake of glycine betaine in Lb. plantarum (Glaasker et al., 1998a), van der Heide
and Poolman (2000) found that proline did not inhibit the uptake of glycine betaine
in these two subspecies of L. lactis. Molenaar et al. (1993) previously demonstrated
that the proline uptake system in L. lactis subsp. lactis ML3 had a higher affinity
for uptake of glycine betaine than for proline; therefore, it is not surprising that
proline did not inhibit uptake of glycine betaine (van der Heide and Poolman, 2000).
Osmotic downshock in the L. lactis subspecies led to an almost instantaneous efflux
of glycine betaine from the cells. The efflux on downshock was proportional to the
decrease in osmolarity of the downshock. As such, efflux is not due to an activation
of a transport system, but rather it is mediated by a channel-like activity.
An NaCl level of 0.4 M present in a chemically-defined medium reduced the
growth rate of the bacteriocin-producing strain of L. lactis subsp. lactis ADRIA
85LO30 by about 70%, but it did not inhibit the production of lacticin 481 (Uguen
et al., 1999). Addition of 1 mM glycine betaine to the medium containing 0.4 M
salt increased the growth of L. lactis to a level comparable to the control lacking
NaCl and betaine glycine. However, glycine betaine completely eliminated produc-
tion of lacticin 481. The relief of osmolarity stress was detrimental to the production
of the bacteriocin.
Growth of eight L. lactis strains in a complex medium supplemented with NaCl
or with glucose at the same aw indicated that there were two classes of L. lactis: a
salt-tolerant group and a salt-sensitive group. L. lactis subsp. lactis strains C10, BA1
and BA2 and L. lactis subsp. cremoris NCDO 712 (and its plasmid-free derivative,
MG1363) were capable of growth at 4% NaCl, whereas the growth of strains BK5,
HP and US3 of L. lactis subsp. cremoris was inhibited by >2% NaCl (O’Callaghan
and Condon, 2000). In a chemically defined medium, the growth of the salt-tolerant
strains of L. lactis was stimulated by the addition of glycine betaine when NaCl was
present. However, glycine betaine did not stimulate the growth of the salt-sensitive
strains in the presence of NaCl. In addition, the salt-sensitive strains accumulated

very little glycine betaine, indicating that the strains had little or no glycine betaine
transport activity (O’Callaghan and Condon, 2000).
The moderately halophilic lactic acid bacterium, Tetragenococcus halophila (for-
merly known as Pediococcus halophilus), is associated with the production of soy
sauce and cured anchovies. When grown in a complex medium, T. halophila accu-
mulated glycine betaine and carnitine even in the absence of NaCl (Robert et al.,
2000). Addition of glycine betaine or carnitine to T. halophila growing in a chemically-
defined medium increased the growth rate and final yield of cells in the absence of
NaCl, as well as in saline concentrations up to 2.5 M. Unlike other LAB that have
been studied, T. halophila can oxidize choline to produce glycine betaine; thus, choline
is an osmoprotectant, since it can be enzymatically converted to glycine betaine
(Robert et al., 2000). There are two systems for the uptake of glycine betaine. One
system transports only glycine betaine, whereas the other system transports glycine
betaine, carnitine and choline. De novo synthesis of protein is not necessary for the
uptake of the osmoprotectants, since uptake was not inhibited by chloramphenicol.
In the absence of glycine betaine or carnitine, T. halophila cannot control
intracellular Na+ levels. In the presence of osmoprotectants, the intracellular Na+
level is maintained at 150 to 320 nmol/mg (dry weight of cells) in media with
salinities ranging from 0 to 2 M. Glycine betaine or carnitine, therefore, can maintain
the intracellular sodium level of T. halophila within narrow limits, regardless of the
external sodium level (Robert et al., 2000). Glycine betaine and carnitine not only
act as osmotic stabilizers in T. halophila but also act as stabilizers of intracellular
Na+ levels.
PROTEIN SYNTHESIS DURING OSMOTIC SHOCK

Exponentially growing cells of E. faecalis ATCC 19433 subjected to 6.5% NaCl or
52% sucrose for 2 h were resistant to heat (62°C for 15 min), ethanol (22%), H2O2
(45 mM), bile salts (0.3%) and SDS (0.017%) (Flahaut et al., 1996). Salt stress led
to the induction of at least 96 proteins; approximately half of these proteins were
induced 2- to 4-fold in concentration as compared to unstressed cells, and 20 of the
proteins increased at least 10-fold. Addition of chloramphenicol during the 2-hour
adaptation period to sugar or salt led to the inhibition of heat resistance, but blockage
of protein synthesis did not inhibit tolerance to the other stresses (Flahaut et al.,
1996). Thus, de novo protein synthesis is necessary for resistance to heat by osmot-
ically adapted cells, but cross-protection against ethanol, bile salts, H2O2, and SDS
does not require de novo protein synthesis.
L. lactis subsp. cremoris strain MG1363 subjected to a temperature shift of 30
to 43°C produced 17 heat shock proteins (HSPs) including GroES, GroEL and DnaK.
Eleven of these proteins (including GroES, GroEL and DnaK) also were produced
by cells stressed with 2.5% NaCl. However, seven of the eleven proteins were
produced at higher levels under heat stress. In addition, a salt shock protein (SSP;
Ssp21), produced at high levels by salt stress, was produced at low levels with heat
stress (Kilstrup et al., 1997). The data presented by Kilstrup et al. (1997) indicated
that there is an overlap in the type of stress proteins produced by heat- and salt-
shocked cells.

Smeds et al. (1998) cloned, sequenced and characterized a stress-inducible gene,
htrA, from Lb. helveticus strain CNRZ 32. The addition of NaCl (4%) to the growth
medium induced an eight-fold increase in the level of htrA transcription. Exposure
of growing Lb. helveticus cells to ethanol (5%) or puromycin (100 µg/mL) resulted
in approximately a five-fold induction of the transcription of htrA. Upshift of growing
cells from 37 to 52°C led to a doubling of htrA mRNA. The protein encoded by Lb.
helveticus htrA is a serine protease; however, its role and how its expression is
regulated are unknown (Smeds et al., 1998).
Lactobacillus sakei strain LTH681, a commercial starter culture for fermented
sausages, has a dnaK operon that consists of four heat shock genes in the order,
hrcA-grpE-dnaK-dnaJ (Schmidt et al., 1999). Transcription of the genes is induced
by heat shock (42°C), NaCl (6%) and ethanol (10%). Analysis of the transcription
start site revealed that the dnaK operon was preceded by an sA-type promoter (P2);
the transcription starting site varied depending on the type of stress. Transcription
induced with either heat or ethanol had a different start site than transcription induced
by salt (Schmidt et al., 1999). A CIRCE element was located between the transcrip-
tion and translation start sites (Schmidt et al., 1999). Schmidt et al. suggest that
under non-stress conditions, HrcA represses the expression of heat shock genes or
operons by binding to the cis-element CIRCE.
OXIDATIVE STRESS
TOLERANCE AND ADAPTATION TO OXIDATIVE STRESS
LAB are facultative anaerobes that metabolize carbohydrates via fermentation.
Although they lack a functional electron transport chain, LAB perform several
oxidation and reduction reactions during the catabolism of carbohydrates. Some of
these reactions (Table 6.2) use molecular oxygen (O2) as a substrate. The presence
of oxygen can generate partially reduced toxic intermediates of O2 such as superoxide
anion (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) (McCord et al.,
1971; Repine et al., 1981). These intermediates are also formed through a variety
of intracellular reactions. For example, H2O2 is formed through the activity of H2O2-
forming flavoprotein oxidases (Whittenbury, 1964), such as NADH oxidase and
pyruvate oxidase (see Table 6.2), and during the dismutation of O2– by superoxide
dismutase (SOD) (Britton et al., 1978). The simultaneous presence of hydrogen
peroxide and superoxide anions can lead further to the formation of hydroxyl
radicals (O2– + H2O2 → OH- + •OH + O2 [Gregory and Fridovich, 1974]), which
are particularly harmful in Lactobacillus since members of this genus lack SOD
and are unable to eliminate superoxide anions (Gregory and Fridovich, 1974).
Together, these reactive oxygen intermediates can cause severe oxidative damage
such as strand breaks in DNA (Storz et al., 1987; Teebor et al., 1988; Piard and
Desmazeaud, 1991), oxidation of membrane lipids (Meads, 1976), and inactivation
of enzymes (Wolff et al., 1986). To counter oxidative stress, LAB maintain an
inducible defense system to detoxify the oxidants and repair the damage. The
dismutation of reactive oxygen intermediates in LAB depends on the activities of
NADH oxidase, NADH peroxidase, glutathione, and thioredoxin. With the excep-

TABLE 6.2
Reactions Involving Oxygen and Oxygen Metabolites
Enzyme Reaction Gene Organism References
NADH:H2O2 NADH + H+ + O2 → NAD+ + H2O2 nox-1 S. mutans (Higuchi et al.,

oxidase 1994)
NADH:H2O oxidase 2 NADH + H+ + O2 → 2NAD+ + H2O nox E. faecalis (Ross and
Claiborne, 1992)
nox-2 S. mutans (Higuchi et al.,
1993)
NADH:peroxidase NADH + H+ + H2O2 → NAD+ + H2O npr E. faecalis (Ross and
Claiborne, 1991)
Glutathione NADPH+ + H+ + GSSG → NADP+ + gor S. thermophilus (Pébay et al.,
reductase 2 GSH 1995)
2 GSH + H2O2 → GSSG + 2 H2O S. thermophilus
Haem-dependent 2 H2O2 → 2 H2O + O2 katA Lb. sake (Knauf et al.,
catalase 1992)
Superoxide O2– + 2H+ → H2O2 sod L. lactis (Sanders et al.,
dismutase 1995)
Pyruvate oxidase pyruvate + phosphate + O2 + FAD + poxB Lb. plantarum
TPP
→ acetylphosphate + CO2 + H2O2 (Murphy and
Condon, 1984)
spxB S. pneumoniae (Spellerberg et al.,
1996)
Oxidase α-glycerophosphate + O2 → — S. faecium
α-glycerophosphate dihydroxyacetone phosphate + H2O2 (Koditchek and
Umbreit,1969)
TPP: thiamine pyrophosphate; FAD: flavine adenine dinucleotide; NAD: nicotinamide adenine dinucleotide
(Adapted from Condon, S., FEMS Microbol. Rev., 46, 269, 1987, and de Vos, W.M., Antonie Van Leeu-
wenhoek, 70, 223, 1996.)
tion of certain strains of Lactobacillus sake (Knauf et al., 1992), Lb. plantarum
(Kono and Fridovich, 1983), Lactobacillus pentosus, and Pediococcus acidilactici
(Wolf et al., 1991), LAB are notable for their inability to produce catalase. LAB
exhibiting this rare property are summarized by Hammes et al. (1990).
Enhanced tolerance to H2O2 after a sublethal treatment of H2O2 has been
described in Gram-negative bacteria such as E. coli and S. typhimurium (Demple
and Halbrook, 1983; Christman et al., 1985) and in Gram-positive bacteria such as
B. subtilis (Murphy et al., 1987; Dowds, 1994), other Gram-positive bacteria such
as E. faecalis (Flahaut et al., 1998) and L. lactis (Condon, 1987) exhibited an
inducible oxidative stress response when exposed to sublethal concentrations of
H2O2. The induced response provided enhanced protection against normally lethal
levels of H2O2. Inhibition of protein synthesis by rifampin during H2O2 pretreatment
blocked the acquisition of resistance, suggesting that de novo protein synthesis is
required (Flahaut et al., 1998).

REGULATION AND FUNCTION OF OXIDATIVE STRESS RESPONSE PROTEINS
NADH Oxidase/NADH Peroxidase
Some LAB have NADH oxidases (Anders et al., 1970; Lucey and Condon, 1986;
Condon, 1987; Smart and Thomas, 1987) that use molecular oxygen to oxidize
NADH. The NADH oxidases are thought to detoxify molecular oxygen by catalyzing
its reduction via NADH into either H2O or H2O2 (Higuchi, 1992). The H2O-forming
NADH oxidase has been proposed to function as a defense against oxidative stress,
based on the production of large amounts of H2O-forming NADH oxidase to reduce
O2 relative to smaller amounts of H2O2-forming NADH oxidase in S. mutans (Higuchi,
1992).
Streptococcus mutans has two distinct NADH oxidases, Nox-1 catalyzing the
formation of H2O2 and Nox-2 producing H2O (Higuchi et al., 1993). The two
enzymes reveal different characteristics (Higuchi et al., 1993): Nox-1 catalyzes the
two-electron reduction of O2 by NADH, whereas Nox-2 catalyzes the four-electron
reduction of O2 by NADH (see Table 6.2). Furthermore, antibodies raised against
Nox-1 or Nox-2 reacted with the corresponding antigens but did not cross-react
(Higuchi et al., 1993). Working with E. faecalis, Ross and Claiborne (1992) were
the first to identify the nox gene encoding NADH:H2O oxidase. This was followed
by the isolation of the homlogous gene for NADH:H2O oxidase from S. mutans
NCIB 11723 (Matsumoto et al., 1996). The gene encoding NADH:H2O2 oxidase has
also been identified and characterized from S. mutans NCIB 11723 (Higuchi et al.,
1994). Since the genes encoding two distinct NADH oxidases were characterized
from the same S. mutans strain (NCIB 11723), the NADH:H2O2 oxidase gene was
named nox-1 and the NADH:H2O oxidase gene was designated nox-2 (see Table 6.2)
(Higuchi et al., 1994). Also, nox-1 and nox-2 were located at different positions on
the genome and the deduced amino acid sequence of each gene showed little
homology between these enzymes (Higuchi et al., 1994; Matsumoto et al., 1996).
Recently, the NADH oxidase gene (nox) was identified in Streptococcus pneu-
moniae (Auzat et al., 1999). The growth rate of a nox mutant was similar to the wild
type under aerobic and anaerobic conditions, suggesting that NADH oxidase in this
strain does not provide resistance to oxidative stress. However, the nox mutant strain
showed decreased competence and attenuated virulence (Auzat et al., 1999). Based
on these results, the researchers concluded that Nox provides protection against
oxidative stress in two ways. First, the reduction of oxygen to water evades the
formation of any toxic intermediates (Higuchi, 1992). Second, the development of
competence through NADH oxidase activity provides an extracellular source of DNA
to aid in repairing damage to the chromosome caused by oxygen radicals (Auzat
et al., 1999).
The production of a reactive oxygen species such as H2O2 by Nox-1 to counter
oxidative damage is illogical. However, located directly upstream of the nox-1 gene
on the S. mutans chromosome is an ahpC gene encoding an enzyme homologous
with the non-flavoprotein component (AhpC) of S. typhimurium alkyl hydroperoxide
reductase. This enzyme system functions to defend cells against oxidative damage
(Jacobson et al., 1989). Because nox-1 is linked to ahpC, AhpC can reduce the H2O2

produced by nox-1 to H2O. The combined reactions of Nox-1 and AhpC are as
follows: 2NADH + 2H+ + O2 → 2 NAD+ + 2H2O (Higuchi et al., 1999). Therefore,
Nox-1 functions in combination with AhpC to form an alkyl hydroperoxide reductase
system in S. mutans (Poole et al., 1997). In S. typhimurium, alkyl hydroperoxide
reductase is composed of AhpC and AhpF and defends against oxidative damage
by reducing organic hydroperoxides and hydrogen peroxide (Jacobson et al., 1989;
Poole and Ellis, 1996). S. typhimurium and E. coli ahpCF-defective mutants showed
increased sensitivity to cumene hydroperoxide (Storz et al., 1989). Transforming an
ahpCF-defective E. coli mutant with both nox-1 and ahpC genes from S. mutans
not only restored, but also actually enhanced resistance to cumene hydroperoxide
relative to the E. coli parent strain (Higuchi et al., 1999). Surprisingly, S. mutans
lacking Nox-1 and/or AhpC did not show increased sensitivity to cumene hydro-
peroxide and H2O2, suggesting that an alternative antioxidant defense system is
functioning in response to oxidative damage (Higuchi et al., 1999).
Another flavoprotein oxidase that protects cells against the threat of H2O2-
induced oxidative stress is NADH peroxidase. In converting H2O2 to water, NADH
peroxidase plays an analogous role to Nox-2 and alkyl hydroperoxidase reductase
(Table 6.2). The gene encoding NADH peroxidase (npr) has been identified and
characterized in E. faecalis (Ross and Claiborne, 1991), and like nox-2 and ahpC
in S. mutans (Higuchi et al., 1999), the npr gene is induced in E. faecalis upon
exposure to oxygen (Rothschild et al., 1991). The upstream region of the npr gene
shares homology with the OxyR-binding site of the ahpC gene from S. typhimurium
(Ross and Claiborne, 1991). In S. typhimurium and E. coli, OxyR is a transcriptional
activator and global regulator protein (Christman et al., 1985) that mediates the H2O2-
induced oxidative stress response (Tartaglia et al., 1989; Storz et al., 1990; Storz and
Altuvia, 1994). More specifically, it controls the expression of a set of antioxidants
that detoxify reactive oxygen species and repair the damage caused by oxidative
stress (Storz and Imlay, 1999). Upon a shift in the intracellular redox potential, OxyR
binds to a specific sequence located just upstream from the promoter region of the
corresponding structural genes (Tartaglia et al., 1989). No homologue of OxyR has
been found in LAB; however, OxyR purified from E. coli binds to and retards DNA
fragments containing npr from E. faecalis in gel shift assays, suggesting that npr
from E. faecalis may be regulated by OxyR (Ross and Claiborne, 1997).
The activity of catalases that detoxify H2O2 have been detected in a limited
number of LAB consisting mostly of lactobacilli and pediococci (Hammes et al.,
1990). To date, the katA gene encoding catalase has been characterized only from
L. sake LTH677. This gene was cloned and phenotypically expressed in L. casei
(Knauf et al., 1992), which is catalase negative, illustrating the potential to enhance
adaptation to oxidative stress in LAB (de Vos, 1996).
Glutaredoxin and Thioredoxin
Glutaredoxin and thioredoxin are structurally similar, particularly in the region of

the active site (Holmgren and Aslund, 1995). The active site of these proteins
contains two conserved cysteine residues that form a disulfide when oxidized and
a dithiol when reduced (Holmgren, 1989). Aside from participating in the reduction

Thioredoxin System
thioredoxin thioredoxin
reductase (trxA)
(trxB)
NADPH
Protein
glutathione glutathione
reductase (gshA gshB)
(gor)
glutaredoxin
(grxA grxB grxC)
Glutaredoxin System
FIGURE 6.2 E. coli components of the thioredoxin system (top) and glutaredoxin system
(bottom) and the corresponding genes. (Adapted from Prinz, W.A. et al., J. Biol. Chem., 272,
15661, 1997.)
of essential enzymes, such as ribonucleotide reductase and a number of metabolic

enzymes that form a disulfide as part of the catalytic cycle (Holmgren, 1989; Rietsch
and Beckwith, 1998), glutaredoxin and thioredoxin function to repair oxidatively
damaged proteins (Holmgren, 1989; Wells et al., 1993) and maintain a favorable
intracellular redox potential by reducing disulfide bonds (Prinz et al., 1997). To
return to the functional state, these proteins must be reduced. Thioredoxin reductase
and glutathione reductase are flavoenzymes that use NADPH to reduce thioredoxin
and glutathione, respectively, and glutathione then reduces glutaredoxin. These reac-
tions are well studied in E. coli. Figure 6.2 diagrams the E. coli thioredoxin system
(consisting of thioredoxin reductase and thioredoxin) and the glutaredoxin system
(consisting of glutathione reductase, glutathione, and three glutaredoxins) (Gleason
and Holmgren, 1988; Holmgren, 1989; Prinz et al., 1997). Many recent reviews
discuss these systems in more detail (Holmgren, 1985, 1989; Holmgren and Aslund,
1995; Aslund and Beckwith, 1999; Ferrari and Soling, 1999; Mustacich and Powis,
2000). Since these systems are highly conserved, it is reasonable to assume that they
are present in most LAB.
Thioredoxin is a ubiquitous protein isolated and characterized from bacteria,
yeast, plants, and animals (Holmgren, 1985). The active site of thioredoxin contains
two cysteine residues that form a disulfide when the protein is oxidized or a dithiol
when reduced. The disulfide bond of oxidized thioredoxin is reduced by NADPH
and an enzyme called thioredoxin reductase (Moore et al., 1964). In B. subtilis,
thioredoxin is an essential protein (Scharf et al., 1998), whereas in E. coli it is non-
essential (Holmgren et al., 1978). However, E. coli mutants lacking both proteins
are non-viable (Prinz et al., 1997). In B. subtilis, thioredoxin was induced by a variety
of stresses including heat, salt, and ethanol (Scharf et al., 1998). The gene encoding
thioredoxin (trxA) identified in O. oeni was induced by hydrogen peroxide and heat

shock (Jobin et al., 1999), which is also the case in B. subtilis (Scharf et al., 1998).
In E. coli and S. typhimurium, trxA genes are not known to be regulated (Farr and
Kogoma, 1991). Although trxA in O. oeni is induced by heat, no CIRCE element
or CtsR consensus sequence was found in the promoter region.
Glutathione can provide intracellular reducing capacity and accumulation of
glutathione in LAB is dependent on the type of medium (Fernándes and Steele,
1993) and transport from the environment (Wiederholt and Steele, 1994). In some
LAB, such as L. lactis, glutathione is present in high concentrations (Fahey et al.,
1978). Glutathione reductase is involved in the oxidative stress response in S. typh-
imurium with expression regulated by OxyR (Christman et al., 1985). The glu-
tathione reductase gene (gor) was identified in S. mutans (Yamamoto et al., 1999),
S. thermophilus CNRZ368 (Pébay et al., 1995), and L. acidophilus NCFM (Girgis
et al., 2000). S. thermophilus CNRZ 368 growing aerobically showed increased gor
expression according to Northern blot analysis and glutathione reductase enzyme
activity (Pébay et al., 1995). In the presence of 2 mM diamide, a thiol-specific
oxidant, an S. mutans gor mutant failed to grow, whereas proliferation of the wild-
type strain was not significantly inhibited (Yamamoto et al., 1999). Expression of
the glutathione reductase gene in Lb. acidophilus NCFM increased in response to
500 µM H2O2 after 15 minutes or during the transition from the exponential to
stationary phase of growth (Girgis et al., 2000).
Superoxide Dismutase
Superoxide dismutase (SOD) converts superoxide anions (O2–) to molecular oxygen

(O2) and hydrogen peroxide (H2O2) (see Table 6.2) (Bannister et al., 1987). There-
fore, this enzyme provides defense against oxygen toxicity and a direct correlation
has been found between the concentration of SOD in an organism and its level of
tolerance to oxygen (Tally et al., 1977). Many LAB eliminate oxygen radicals by
superoxide dismutase or a high internal Mn2+ concentration (Archibald and Fridovich,
1981). LAB that lack SOD use Mn2+ to scavenge O2–, as demonstrated in
Lb. plantarum and many other lactobacilli and streptococci strains (Archibald and
Fridovich, 1981). However, organisms possessing SOD were more oxygen tolerant
than organisms dependent upon Mn2+ for scavenging O2– (Archibald and Fridovich,
1981). Using degenerate primers, internal regions of the gene encoding superoxide
dismutase (sod) were amplified and sequenced from L. lactis, E. faecalis, E. faecium,
Streptococcus agalactiae, S. pneumoniae, and Streptococcus pyogenes, in addition
to C. perfringens and S. aureus (Poyart et al., 1995).
The entire gene was also identified, cloned, and sequenced from S. mutans
through the complementation of a sod-deficient E. coli strain (Nakayama, 1992).
Based on N-terminal amino acid sequence, SOD was also identified in L. lactis during
a search for proteins expressed at higher levels in a medium with a low pH (Sanders
et al., 1995). Aeration was effective in increasing the transcriptional expression (Sand-
ers et al., 1995) and enzyme activity of SOD in L. lactis (Hansson and Häggström,
1984; Smart and Thomas, 1987). Oxygen radicals formed during aeration inhibited
the growth of sod-deficient strains of S. mutans (Nakayama, 1992) and L. lactis
(Sanders et al., 1995). A similar effect was observed in E. coli (Carlioz and Touati,

1986). The E. coli superoxide dismutase gene was cloned and expressed in L. lactis
and Lb. gasseri (Roy et al., 1993); since Lb. gasseri lacks SOD, this study demon-
strates the potential to increase tolerance to oxidative stress in LAB (de Vos, 1996).
recA, fpg, and DNA Damage
The recA gene is ubiquitous among bacteria and responds to DNA damage caused
by oxidative stress. In the absence of oxidative stress, RecA initiates recombination
between homologous strands of DNA (Cassuto et al., 1980) (for reviews, see Miller
and Kokjohn, 1990, and Roca and Cox, 1990). When DNA is damaged, the RecA
protein is activated upon binding to single-stranded DNA (Roberts and Devoret,
1982). The activated RecA protein induces expression of several DNA-repair genes
in the SOS pathway (Walker, 1984). Therefore, RecA serves a regulatory function
in response to oxidatively damaged DNA (Walker, 1984; Miller and Kokjohn, 1990).
Using degenerate primers, internal regions of the recA gene were amplified,
cloned, and sequenced from L. lactis subsp. lactis ML3 and IL 1403 and L. lactis
subsp. cremoris IL 736, Lb. bulgaricus, Lb. helveticus, Lc. mesenteroides, and
Streptococcus salivarius subsp. thermophilus, in addition to B. subtilis, Clostridium
acetobutylicum, L. monocytogenes, and S. aureus (Duwat et al., 1992). An L. lactis
mutant with a reduced capacity for recombination showed increased sensitivity to
UV (Anderson and McKay, 1983); however, the location of the mutation has not
been identified. Another L. lactis recA mutant exhibited a recombination frequency
about 104-fold lower than wild type and increased sensitivity to DNA damage caused
by UV light, mitomycin C, ethyl methane sulphonate, and methyl methane sulpho-
nate (Duwat et al., 1995). These compounds were effective in increasing recA expres-
sion by three- to five-fold (Duwat et al., 1995). A number of genes associated with
DNA repair have been identified in a study in which UV-sensitive mutants of L. lactis
strain MG1363 were obtained by ISS1 mutagenesis. Of the 18 mutants sensitive to
mitomycin and UV, DNA sequence analysis identified 11 insertions of ISS1 within
genes associated with DNA metabolism (polA, hexB, and deoB), cell envelope
formation (gerC and dltD), and various metabolic pathways (arcD, bglA, gidA, hgrP,
metB, and proA) (Duwat et al., 1997). The polA, hexB, and deoB mutants were more
sensitive to low doses of UV treatment than the other mutants and homologous
recombination was reduced by 10- to 300-fold in the gidA, polA, and uvs-75 mutants.
These seemingly unrelated sets of affected genes suggest that UV resistance involves
several interactive mechanisms in L. lactis.
In addition to DNA damaging agents, expression of recA was also induced in
aerated cultures. An L. lactis recA mutant was highly sensitive to aeration, as evi-
denced by a lower growth rate and reduced viability during stationary phase (Duwat
et al., 1995). As L. lactis produces hydrogen peroxide and acid in the presence of
iron, hydroxyl radicals are formed. Hydroxyl radicals can be produced by the Fenton
reaction: H2O2 + Fe2+ + H+ → •OH +H2O + Fe3+ (Fenton, 1984; Lesko et al., 1980).
It is believed that hydroxyl radical formation is the leading cause of the poor growth
of the recA aerated culture because the addition of catalase to the recA aerated growth
medium restored growth, such that the doubling time was the same as in the non-
aerated culture (Duwat et al., 1995). Furthermore, the removal of Fe2+, by adding

the Fe2+-specific chelating agent ferrozine (Artiss et al., 1981), also restored the
doubling time of the aerated recA cultures to that of non-aerated cultures (Duwat
et al., 1995). The L. lactis recA mutant had three-fold higher levels of HflB (which
down regulates expression of heat shock proteins in E. coli [Herman et al., 1995]),
and decreased levels of heat shock proteins, and it showed poor growth at 37ºC
relative to the wild type strain. These observations suggested that recA may also be
involved in the regulation of the heat shock response (Duwat et al., 1995). To further
investigate the role recA plays in the heat shock response, insertional mutants of a
thermosensitive recA-deficient strain of L. lactis were isolated based on their ability
to withstand high temperatures (Duwat et al., 1999). Eighteen trm (for thermoresis-
tant mutant) mutants were characterized that contained insertions of six genes impli-
cated in purine metabolism (deoB, guaA, tktA), phosphate uptake (pstB and pstS),
and mRNA stability (pnpA), and in one uncharacterized gene (trmA). A deoB
insertional mutant conferring UV sensitivity was isolated previously (Duwat et al.,
1997). Furthermore, insertional mutations in four genes — deoB, guaA, pstB and
pstS — were obtained by Rallu et al. (2000), using a similar procedure was followed
to isolate acid-tolerant insertional mutants of L. lactis. See section on “acid adapta-
tion.” The inability to synthesize purines and import phosphate from the extracellular
environment simulated a starvation-like physiology in the corresponding mutants
that conferred resistance to multiple forms of stress. This finding suggests that stress
response mechanisms in L. lactis are interactive and are intimately associated with
metabolic pathways (Duwat et al., 1999; Rallu et al., 2000).
Upstream of the L. lactis recA gene is a region of DNA with strong homology
with the gene encoding the DNA repair enzyme formamidopyrimidine DNA glyc-
osylase (fpg), found in E. coli and Bacillus firmus (Boiteux et al., 1987; Boiteux and
Huisman, 1989). In E. coli, the fpg gene product is involved with DNA repair
associated with oxidative stress (Czeczot et al., 1991) and is unlinked to recA
(Boiteux and Huisman, 1989). L. lactis fpg is co-transcribed with recA, and Duwat
et al. (1992) suggest the proximity of recA and fpg in L. lactis may indicate over-
lapping regulation linking recombination and DNA repair. The L. lactis Fpg protein
is structurally and functionally similar to the Fpg protein in E. coli. In both organ-
isms, Fpg protects DNA against the mutagenic action of 8-oxoguanine (Michaels
and Miller, 1992; Grollman and Moriya, 1993; Duwat et al., 1995).
STARVATION
Bacterial cells enter the stationary phase upon depletion of essential nutrients from
the growth medium. During nutrient starvation, there is a gradual decrease in the
growth rate which eventually approaches zero. To survive, bacteria must make an
orderly transition into the stationary phase in such a manner that DNA replication
is not terminated prematurely, that viability is maintained, and that cells can return
to exponential growth when starvation is relieved. In non-sporulating bacteria during
starvation, there occur a number of changes in cellular protein composition that are
characterized by degradation of some previously synthesized proteins, increased
synthesis of some proteins common to exponential phase growth and de novo protein

synthesis. Starvation also induces resistance to a number of environmental stresses
without prior exposure to those stresses (Kolter et al., 1993).
The proteins synthesized during starvation are probably involved in maintenance
of cell viability and in resistance to numerous stresses. When the synthesis of
starvation proteins is completed, metabolic activity is greatly diminished; however,
the cells are not dormant like bacterial spores. The starved cells do maintain some
level of metabolic activity (Kolter et al., 1993), because when fresh nutrients are
added, the cells respond rapidly. Synthesis of RNA starts almost immediately, but
protein synthesis lags for a short period. Increases in cell mass, in rate of DNA
synthesis, and in cell number follow the reinstating of RNA and protein synthesis,
but there is a progressive loss of the enhanced resistance to environmental stresses
that was induced during starvation (Kolter et al., 1993). The responses to starvation
of some LAB are described in this section.
Enterococcus faecalis JH2-2 cells from the exponential growth phase are less
tolerant to a number of stresses than cells from the stationary growth phase (i.e.,
starved cells). Glucose-starved cells of E. faecalis are more resistant to ethanol
(17%), acid (pH 3.7; adjusted with lactic acid), H2O2 (20mM) and heat (62°C for
30 min) than cells from the exponential phase (Giard et al., 1996). In general, stress
resistance increased up to 24 h after entrance into the stationary phase. However,
ethanol resistance was established early in the stationary phase and did not increase
over time during starvation. Starvation did not increase the resistance of E. faecalis
to UV irradiation (Giard et al., 1996). Utilizing chloramphenicol as a protein syn-
thesis inhibitor, Giard et al. (1997) found that early stationary-phase protein synthesis
was necessary for the acquisition of resistance against heat, acid, and oxidative
stresses, but not against ethanol stress. After 24 h of starvation, approximately one-
third of the proteins typically observed in exponential cells were reduced in con-
centration or were completely absent, indicating that protein degradation had taken
place. Glucose starvation induced significant increases in the synthesis of 42 proteins
(Giard et al., 1996, 1997). The synthesis of these proteins was time dependent and
different proteins were seen at different stages of starvation. Proteins synthesized
between 6 and 24 h into the stationary phase were crucial for the development of
maximal resistance to heat, H2O2 and acid. Some of these proteins may be involved
in the development of alternate pathways of energy production that permit survival
of E. faecalis under stress conditions (Giard et al., 1996, 1997).
Lactococcus lactis subsp. lactis IL1403, upon glucose-starvation, demonstrated
augmented resistance to heat (52°C for 30 min), ethanol (20%), pH 4.0 (adjusted
with lactic acid), NaCl (3.5 M), and H2O2 (1.5 mM) in the absence of prior exposure
to these stresses (Hartke et al., 1994). The starvation-induced cross-protection to stress
was evident at the beginning of the stationary stage. The acquisition of these stress
resistances was initiated during the period of transition from growth to non-growth,
and reached a maximum upon entrance into stationary phase. Interestingly, addition
of chloramphenicol or rifamycin during the transitional growth phase did not inhibit
acquisition of stress resistance, but rather it increased resistance to a level comparable
to that seen in cells in the stationary stage (Hartke et al., 1994). When the authors
compared stress adapted, exponentially grown L. lactis subsp. lactis IL1403 cells
with starved cells, they found that adapted cells had comparable resistance to heat,

acid or osmotic stresses as starved cells. However, adapted cells were not as resistant
as starved cells to ethanol, but were more resistant than starved cells to H2O2 stress.
During glucose starvation of L. lactis subsp. lactis ML3 cells, at least 45 polypep-
tides which are present in exponentially growing cells were no longer synthesized
(Kunji et al., 1993). In addition, there was significant degradation of protein during
the first 60 to 90 min of starvation, and protein degradation during starvation was
non-selective (Kunji et al., 1993). After the initial degradation period, the polypep-
tide pool remained stable for up to 40 h. During the first hour of starvation, the
synthesis of at least two proteins was induced and as starvation continued, an
additional 14 or 15 proteins were synthesized. It is likely that the amino acids
produced by degradation of exponential growth proteins were used to synthesize the
starvation-specific proteins. These newly synthesized proteins probably play a role
in the increased stress resistance shown by the starving bacteria.
Unlike the wild type, recA mutants of L. lactis subsp. cremoris MG1363 do not
grow at 39.3°C. By subjecting the recA strain to insertional mutagenesis and selecting
those mutants that grew at 39.3°C, Duwat et al. (1999) isolated several double
mutants that could grow at that temperature. These double mutants also displayed
resistance to heat shock (55°C for 15 min), H2O2 (1 mM) and carbon starvation.
The following are the mutations in the recA double mutant strains: deoB (involved
in purine and pyrimidine salvage and nucleoside degradation), guaA (involved in
synthesis of GMP from XMP), tktA (involved in transformation of xylose-5-P to
ribose-5-P), or pstB and pstS (involved in phosphate transport) induced multiple
stress resistance during both the exponential and stationary stages (Duwat et al.,
1999). Expression of multiple stress resistances during the exponential stage by these
double mutants suggests that mutations leading to the reduction of the guanine
nucleotide pool or phosphate pool can induce a starvation-like physiology in the
cells (Duwat et al., 1999). In a similar fashion, Rallu et al. (2000), using insertional
mutagenesis to isolate acid-resistant mutants of L. lactis subsp. cremoris MG1363,
found that mutations in pstS and pstB, guaA, deoB or RelA (involved in both synthesis
and degradation of [(p)ppGpp]) not only induced increased resistance to acid (pH
3.7 or 3.0 adjusted with HCl), but also increased resistance to hydrogen peroxide
(1 mM for 30 min) and heat shock (55°C for 15 min).
When GMP synthase (coded for by guaA) of the wild type strain of L. lactis
subsp. cremoris MG1363 was inhibited, there was induction of the stress-resistance
phenotype (Duwat et al., 2000). Addition of guanine to the growth medium of the
RecA-guaA double mutant abolished the stress-resistant phenotype. Similarly, addi-
tion of phosphate to the RecA-pstS double mutant eliminated stress resistance (Duwat
et al., 2000). The work of Duwat et al. (1999 and 2000) and Rallu et al. (2000)
suggests that a decreased internal phosphate concentration, a decreased guanine
nucleotide pool, and/or an increased (p)ppGpp concentration may be perceived by
cells of L. lactis subsp. cremoris as intracellular stress signals leading to tolerance
to a number of stresses.
Stationary phase cells of L. lactis subsp. lactis strains LL-40-1, LL-41-1 and
LL43-1 were resistant to pH 2.5 (adjusted with HCl), bile salts (0.1%) and freezing
at –20°C for 24 h (Kim et al., 1999). Cells of L. lactis subsp. cremoris strains LC-10-1,
LC-11-1 and LC-12-1 from the stationary phase were resistant to pH 3.0 (adjusted

with HCl) and 0.04% bile salts. Strains LC-10-1 and LC-11-1 were also resistant
to freezing, but strain LC-11-1 was not. Subjecting the L. lactis subsp. cremoris
strains to pH 2.5 or 0.1% bile salts resulted in death of the cells (Kim et al., 1999).
Thus, L. lactis subsp. cremoris strains are typically less stress tolerant than L. lactis
subsp. lactis strains.
OVERLAPPING REGULATORY NETWORKS

AND CROSS-PROTECTION
Aside from synthesizing a specific set of proteins in response to an individual stress,
many microorganisms induce a stress regulon consisting of an overlapping set of
general stress response proteins which may confer general protection to a variety of
deleterious conditions. The universal induction of many of the same stress proteins
following exposure to a variety of different mild stresses has been demonstrated in
E. coli (Jenkins et al., 1991), B. subtilis (Hecker and Völker, 1990; Völker et al.,
1992), E. faecalis (Flahaut et al., 1996), and L. lactis (Hartke et al., 1994, 1995,
1997). For example, a significant overlap between acid- and heat-inducible polypep-
tides was observed in L. lactis subsp. lactis. Of the 12 heat shock proteins detected
in this strain, 9 were also induced by acid treatment, including DnaK and GroEL
(Hartke et al., 1996). This suggests a relationship between the mechanisms respon-
sible for the heat shock response and the acid tolerance response. In E. faecalis, a
considerable number of heat shock proteins were also expressed in response to
ethanol (Boutibonnes et al., 1993) and bile (Flahaut et al., 1996). Similar results
have been found in E. coli (Heyde and Portalier, 1990) and S. typhimurium (Foster,
1991) in which heat shock proteins were among the proteins induced by acid
adaptation. Furthermore, acid was effective in inducing members of the SOS and
H2O2 stimulons of L. lactis (Hartke et al., 1995). This production of overlapping
stress response proteins due to a variety of different environmental stresses may be
responsible for the phenomenon known as cross-protection, which is observed when
cells survive an otherwise lethal exposure to one form of stress after adapting to a
different sublethal condition.
Cross-protection has been demonstrated in E. coli (Jenkins et al., 1990),
S. typhimurium (Leyer and Johnson, 1993), and B. subtilis (Völker et al., 1992).
Among the LAB, cross-protection has been described in L. lactis, E. faecalis, and
Lactobacillus collinoides. For example, carbohydrate-starved cultures of L. lactis
are significantly more resistant to heat, ethanol, acid, and osmotic stress than nour-
ished, exponential-phase cells (Hartke et al., 1994). Thermotolerance in L. lactis was
developed after exposures to ethanol (Boutibonnes et al., 1991), puromycin
(Boutibonnes et al., 1992), or chemicals such as cadmium chloride, mercury chlo-
ride, sodium azide, and β-mercaptoethanol (Boutibonnes et al., 1995). Furthermore,
heat-induced cross-protection against freezing and lyophilization was achieved in L.
lactis. Cross-protection was abolished in the presence of erythromycin, indicating
that protein synthesis is required for tolerance (Broadbent and Lin, 1999).
Enterococcus faecalis showed enhanced tolerance to lethal doses of hydrogen
peroxide after a 30-minute incubation in acid or NaCl, or after thermal treatment

(Flahaut et al., 1998). Heat-adapted cells of E. faecalis showed significant cross-
protection against bile (Flahaut et al., 1996) and ethanol (Boutibonnes et al., 1993),
and bile-adapted cells provided resistance against heat challenge (Flahaut et al.,
1996). As stated above, the overlap in the number of heat shock proteins expressed
during exposure to heat, bile, and ethanol, suggesting similar mechanisms of
response, may be the basis for cross-protection. Preconditioning E. faecalis with
heat or bile failed to induce acid tolerance, and acid-adapted cells displayed slight
resistance to heat and no resistance to bile challenge (Flahaut et al., 1996). Appar-
ently, the response of E. faecalis to acid treatment is distinct from the method of
adaptation to bile or heat. Adaptation to ethanol and heat in Lb. collinoides conferred
homologous resistance and enhanced tolerance to acid. However, adaptation to acid
did not provide protection from ethanol or heat (Foster and Hall, 1990). As observed
in E. faecalis (Flahaut et al., 1996), acid treatment elicits a specific response relative
to treatments with heat or ethanol.
THE FUTURE
LAB used as starter cultures are normally stored and distributed in liquid, spray-
dried, frozen, or lyophilized forms (Porubcan and Sellars, 1979; Sandine, 1996).
Such preparations drastically reduce population numbers and severely damage their
capacity for growth, fermentation, or survival upon passage through the gastrointes-
tinal tract. Furthermore, during the production of fermented food products, lactic
starter cultures are typically subjected to extremes in temperature, pH, and osmo-
larity. As knowledge regarding stress response systems of LAB accumulates, meth-
ods will inevitably be developed to engineer strains that are more resistant to routine
industrial practices.
An enormous volume of knowledge will be provided through the sequencing of
microbial genomes. In 1999, L. lactis IL1403 became the first LAB to have its entire
genome sequenced and published (Bolotin et al., 1999). Within the year, the genomes
of another 23 industrially important LAB will be sequenced. This group includes
Lb. acidophilus, Lb. plantarum, Lb. johnsonii, L. lactis subsp. cremoris, Lb. delbrueckii
subsp. bulgaricus, Lb. sakei, Lb. casei, Lb. helveticus, Lb. rhamnosus, S. thermophilus,
O. oeni, Lb. gasseri, Lactobacillus brevis, L. lactis subsp. cremoris, Lc. mesenteroi-
des, and P. pentosaceus. The information gathered from whole-genome sequencing
combined with new technologies designed to analyze genomic data, such as microar-
rays, will inevitably provide a global view of the genetic mechanisms which con-
tribute to the observed physiological responses of the LAB to environmental stress.
CONCLUSIONS
A microorganism’s ability to grow and survive depends largely on its capacity to
adapt to changing environments. LAB are constantly subjected to harsh conditions
that can affect their performance in food fermentations. Adaptation to adverse
environments is usually associated with the induction of a large number of genes,
the synthesis of stress response proteins, and the development of cross resistance to

a variety of stresses. The regulation of these responses is complex, interactive, and
sometimes intimately related to metabolic pathways.
The further elucidation of molecular genetic mechanisms involved in the regu-
lation of the stress responses of these organisms will provide fundamental informa-
tion regarding the development of stress adaptation and tolerance. Information gath-
ered on stress adaptation will ultimately raise the possibility of enhancing tolerance
to adverse environmental conditions and consequently improve viability and perfor-
mance of these organisms in food systems.
TABLE 6.3
Genes Induced by Environmental Stress in LAB
Stress Gene Function of Protein Organisms References
Heat groEL chaperone Lb. johnsonii (Walker et al., 1999)

Lb. acidophilus (Girgis et al., 1999; Girgis et al.,
2000)
L. lactis (Kim and Batt, 1993; Hartke et al.,
1997)
E. faecalis (Flahaut et al., 1997)
Lc. mesenteroides (Salotra et al., 1995)
Lb. helveticus (Broadbent et al., 1998)
groES chaperone Lb. johnsonii (Walker et al., 1999)
Lb. acidophilus (Girgis et al., 1999; Girgis et al.,
2000)
L. lactis (Kim and Batt, 1993; Hartke et al.,
1997)
Lb. helveticus (Broadbent et al., 1998)
hrcA heat shock regulator S. mutans (Jayaraman et al., 1997)
Lb. sakei (Schmidt et al., 1999)
Lb. acidophilus (Girgis et al., 2000)
L. lactis (Eaton et al., 1993)
dnaK chaperone L. lactis (Eaton et al., 1993; Barril et al.,
1994)
S. mutans (Jayaraman et al., 1997)
E. faecalis (Flahaut et al., 1997)
Lc .mesenteroides (Salotra et al., 1995)
grpE chaperone L. lactis (Eaton et al., 1993)
S. mutans (Jayaraman et al., 1997)
dnaJ chaperone L. lactis (van Asseldonk et al., 1993)
ctsR heat shock regulator L. lactis (Derre et al., 1999)
S. salivarius (Derre et al., 1999)

S. pneumoniae (Derre et al., 1999)

S. pyogenes (Derre et al., 1999)
S. thermophilus (Derre et al., 1999)
E. faecalis (Derre et al., 1999)
Lc. oenos (Derre et al., 1999)
Lb. sake (Derre et al., 1999)
clpB protease L. lactis (Ingmer et al., 1999)
clpC protease L. lactis (Ingmer et al., 1999)
clpE protease L. lactis (Ingmer et al., 1999)
Lb. sake (Stentz et al., 1997)
clpX chaperone/protease O. oeni (Jobin et al., 1999)
clpP protease L. lactis (Frees and Ingmer, 1999)
S. salivarius (Giffard et al., 1993)
ftsH heat shock regulator L. lactis (Nilsson et al., 1994)
hsp18 membrane maintenance O. oeni (Jobin et al., 1997)
Cold cspA RNA stabilization L. lactis (Wouters et al., 1998)
cspB RNA stabilization L. lactis (Chapot-Chartier et al., 1997;
Wouters et al., 1998)
cspC RNA stabilization L. lactis (Wouters et al., 1998)
cspD RNA stabilization L. lactis (Wouters et al., 1998)
cspE L. lactis (Wouters et al., 1998)
cspF L. lactis (Wouters et al., 2000)
cspG L. lactis (Repine et al., 1981)
cspL RNA stabilization Lb. plantarum (Mayo et al., 1997)
cspP RNA stabilization Lb. plantarum (Mayo et al., 1997)
Acid gadCB acid stress protection L. lactis (Sanders et al., 1998; Small and
Waterman, 1998)
citP acid stress protection L. lactis (Garcia-Quintans et al., 1998)
atp acid stress protection Lb. acidophilus (Kullen and Klaenhammer, 1999)
arcABCTD acid stress protection Lb. sake (Zuniga et al., 1998)
Oxidative sodA O2– scavenging L. lactis (Poyart et al., 1995; Sanders et al.,
1995)
E. faecalis (Poyart et al., 1995)
E. faecium (Poyart et al., 1995)
S. agalactiae (Poyart et al., 1995)
S. pneumoniae (Poyart et al., 1995)
S. pyogenes (Poyart et al., 1995)
S. mutans (Nakayama, 1992)
recA DNA repair L. lactis (Duwat et al., 1995)
Lb. bulgaricus (Duwat et al., 1992)
Lb. helveticus (Duwat et al., 1992)

Lc. mesanteroides (Duwat et al., 1992)

S. salivarus (Duwat et al., 1992)
fpg DNA repair L. lactis (Duwat et al., 1992; Duwat et al.,
1995)
fnr O2– scavenging L. lactis (Gostick et al., 1999)
nox H2O2 reducing S. mutans (Higuchi, 1992; Matsumoto et al.,
1996; Higuchi et al., 1999)
S. pneumoniae (Auzat, 1999)
Lb. delbreuckii (Marty-Teysset et al., 2000)
npr H2O2 reducing E. faecalis (Ross and Claiborne, 1991; Ross
and Claiborne, 1992)
katA H2O2 reducing Lb. sake (Knauf et al., 1992)
gor H2O2 reducing S. thermophilus (Pébay et al., 1995)
S. mutans (Yamamoto et al., 1999)
trxA H2O2 reducing O. oeni (Jobin et al., 1999)
Osmotic htrA stress protection Lb. helveticus (Smeds et al., 1998)
Paper No. FSR-0043 of the Journal Series of the Department of Food Science,
NCSU, Raleigh, NC 27695-7624. The use of trade names in this publication does
not imply endorsement by the North Carolina Agricultural Research Service, or the
US Department of Agriculture, of the products named nor criticism of similar ones
not mentioned. Work on the stress response of lactobacilli, conducted at NCSU is
supported by grants from the Southeast Dairy Foods Research Center, Dairy Man-
agement Inc., and Rhodia, Inc.
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Relationship between
7 Stress Adaptation and
Virulence in Foodborne
Pathogenic Bacteria
Cormac G. M. Gahan and Colin Hill
CONTENTS
Introduction
Infection and the Need for Environmental Sensing
Infection with Salmonella spp.
Infection with Listeria monocytogenes
Two-Component Systems and Environmental Sensing
Environmental Stresses Encountered by Bacteria during Infection
Body Temperature, Heat-Shock and the General Stress Response
Acid Tolerance and Virulence
Oxidative Stress Response
Osmotic Stress
Starvation Stress
Methods to Detect Genes Transcribed in Vivo
In Vivo Expression Technology (IVET)
Green Fluorescent Protein (GFP) Technology
Signature-Tagged Mutagenesis
Conclusion
Acknowledgments
References
INTRODUCTION
Bacteria capable of causing foodborne infections must negotiate a long and tortuous
passage from the environment to the site of infection in the susceptible host. Food-
borne pathogens may encounter stressful environments during the production, prep-
aration and storage of food. Following consumption they are exposed to the low pH
of the stomach and survivors subsequently encounter volatile fatty acids, bile and

low oxygen in the small intestine. Bacteria that survive to this point must compete
with established gut flora for niches and nutrients and must overcome, among other
insults, antimicrobial peptides produced by their competitors (Dunne et al., 1999).
Those organisms capable of invasion subsequently penetrate the gut epithelium and
are internalized within phagosomes, specialized organelles which prevent bacterial
multiplication by means of acidic pH, and through the production of defensins
(oxygen-independent mechanisms), hydrogen peroxide and superoxide radicals
(oxygen-dependent mechanisms).
To survive and grow in these inhospitable environments, foodborne pathogens
possess mechanisms to overcome these stresses, and thus are capable of colonization
resulting in either clinical or sub-clinical infection. In this chapter, the mechanisms
employed by foodborne pathogens to adapt to the host environment and cause disease
have been considered. Our primary focus is the pathogenesis of Listeria monocyto-
genes and Salmonella enterica serovar Typhimurium as examples of Gram-positive
and Gram-negative foodborne pathogens capable of causing invasive disease in a
mouse model of infection.
INFECTION AND THE NEED FOR

ENVIRONMENTAL SENSING
INFECTION WITH SALMONELLA SPP.
The invasiveness of various Salmonella serovars varies greatly. S. enterica serovar

Typhimurium (hereafter referred to as S. Typhimurium) causes a localized gas-
trointestinal infection in humans with symptoms of vomiting and diarrhea.
S. Cholerasuis can cause more severe symptoms, while S. Typhi are responsible for
the serious invasive disease typhoid fever. However, S. Typhimurium infection of
mice results in a disease with similarities to human typhoid fever and, as a conse-
quence, murine S. Typhimurium infection is extensively used as a model for invasive
disease. The usefulness of this model and the ease with which S. Typhimurium can
be manipulated at the genetic level have led to extensive studies of this pathogen
and phenomena uncovered first in S. Typhimurium are now being investigated in
other foodborne pathogens.
Studies indicate that S. Typhimurium invade primarily via M cells of the small
intestine (Jones et al., 1994). However, the pathogen can adhere to and invade
numerous mammalian cell lines (Figure 7.1A). Adherent Salmonella induce a specific
membrane ruffling effect at the surface of appropriate host cells that leads to rear-
rangement of host actin in the vicinity of the bacterial cell (Finlay and Ruschkowski,
1991; Hardt et al., 1998). Membrane ruffling is dependent upon bacterial expression
of SopE, a protein which is required for efficient invasion of cultured cells (Hardt
et al., 1998). SopE is a substrate of a protein secretion system (type III) that trans-
locates bacterial proteins into the host cell. The genes encoding this type III secretion
system and other invasion-associated loci are clustered on a pathogenicity island —
Salmonella pathogenicity island 1 (SPI1) — and are expressed prior to invasion.
Bacteria are subsequently engulfed by the host cell and are internalized within host
cell phagosomes, whereupon SPI1 genes are repressed (Pegues et al., 1995; Bajaj

A B
6
1 1
2
2
3
5
FIGURE 7.1 (A) Intracellular pathogenesis of Salmonella Typhimurium. 1. Membrane ruf-

fling and invasion predominately mediated through expression of genes in Salmonella patho-
genicity island 1 (SPI1); 2. Existence within the phagosome requires expression of SPI2 genes
with downregulation of SPI1 genes. SPI2 genes involved in preventing maturation of the
phagosome to phagolysozome. Genes involved in sensing low pH, low Mg2+ and low osmo-
larity (PhoP-PhoQ and OmpR-EnvZ) are also implicated in promoting survival. (B) Genes
involved in intracellular pathogenesis of Listeria monocytogenes. 1. invasion (invA/invB);
2. entrapment in the phagosome; 3. escape (hly/plcA); 4. growth; 5. actin polymerization
(actA); 6. cell to cell spread (plcB).
et al., 1996; Cotter and Miller, 1998). S. Typhimurium can survive within this hostile
environment by preventing the maturation of phagosomes to lethal phagolysosomes.
Recent evidence suggests that S. Typhimurium may interfere with trafficking of
oxidase-containing vesicles to the phagosome through expression of components of
Salmonella pathogenicity island 2 (SPI2) (Vazquez-Torres et al., 2000). However,
many other bacterial genes are involved in the survival of the bacterium in the host
phagosome, including genes involved in acid tolerance and responses to low iron,
carbon starvation, oxidative stress and high osmolarity.
INFECTION WITH LISTERIA MONOCYTOGENES
The genus Listeria comprises both avirulent and virulent species. Although Listeria
seeligeri and L. ivanovii are capable of causing human or animal disease, it is
L. monocytogenes that is the most common cause of infection (listeriosis) in humans
(Farber and Peterkin, 1991; Gahan and Collins, 1991). The potentially high mortality
rates associated with outbreaks of listeriosis highlight the serious nature of
L. monocytogenes infection and eliminating the organism from ready-to-eat foods
remains an imperative for the food industry. Infection of mice with L. monocytogenes
has long been accepted as a suitable model for the study of pathogenesis and resulting
immunity to this Gram-positive organism.
L. monocytogenes, like S. Typhimurium, invades tissue culture cells by inducing
its own phagocytosis. However, it does so by using a so-called trigger mechanism
rather than the membrane-ruffling (zipper mechanism) exhibited by Salmonella spp.
(Isberg and Tran Van Nhieu, 1994; Cossart and Lecuit, 1998). Invasion is mediated

by a number of genes including invA and invB, although other genes may also be
necessary for adherence prior to invasion (Gaillard et al., 1991; Dramsi et al., 1995;
Milohanic et al., 2000). During murine infection, L. monocytogenes does not seem
to have a preference for invasion of a specific cell type and will invade a wide variety
of cell types in vitro (Gaillard et al., 1987; Kathariou et al., 1990). The organism
can penetrate the small intestine through either M cells or enterocytes and, imme-
diately following invasion, the phagocytic cells of the Peyers patches provide a focus
of bacterial accumulation (Pron et al., 1998). Subsequently the spleen and liver
become foci of infection, possibly seeded by migrating macrophages containing
internalized bacteria. In humans, bacterial infection of the meninges causes a poten-
tially fatal meningitis; in pregnant women, infection of the fetus may result in
spontaneous abortion.
The genetic loci contributing to intracellular pathogenesis of L. monocytogenes
have been well characterized (reviewed by Cossart and Lecuit, 1998). Survival of
L. monocytogenes within host cells is mediated by production of virulence factors
including a hemolysin (listeriolysin) encoded by hly, and a phospholipase encoded
by plcA. These may act singly or in concert to lyse the phagosomal membrane
releasing the bacterium into the host cell cytoplasm where bacterial division can
occur (see Figure 7.1B). In this environment L. monocytogenes can mobilize host
actin filaments for motility using the virulence factor actin polymerase (ActA), and
can infect neighboring cells without an extracellular phase. Release from a double-
membrane bound vesicle into the neighboring cell is mediated in part by another
virulence factor encoded by plcB. Coordinate regulation of virulence factors is
mediated by the transcriptional activator, PrfA (positive regulatory factor A), together
with a putative PrfA-binding factor (Bockmann et al., 1996). Expression of the PrfA
regulon is in turn regulated by environmental signals encountered during the infec-
tious cycle.
TWO-COMPONENT SYSTEMS AND ENVIRONMENTAL SENSING

It should be apparent, given the complexities of the pathogenic cycles of foodborne
pathogens, that an ability to sense and react to extracellular stimuli is essential for
adaptation to new host environments. Two-component regulatory systems provide a
means to detect perturbations in growth conditions and to respond with the synthesis
of gene products which facilitate adaptation. These systems involve a sensor mole-
cule which is often present in the bacterial cell membrane to detect environmental
signals, and a cytoplasmic response regulator which functions to effect intracellular
changes in response to the initial stimulus (Parkinson and Kofoid, 1992; Russo and
Silhavy, 1993). Two-component systems in various bacteria control diverse functions
including chemotaxis, sporulation and responses to nitrogen, phosphate or carbon
source availability. In pathogenic bacteria, two-component systems have emerged
as mechanisms signalling host–pathogen interactions and play a dynamic role in
bacterial adaptation to the host environment.
In S. Typhimurium the membrane sensor EnvZ and its companion response
regulator, OmpR, represent a two-component system which responds to changes in
osmolarity. The EnvZ-OmpR system plays a major role in signalling entry of Salmonella

into the host cell and in triggering bacterial acid adaptation (Lee et al., 2000; Bang
et al., 2000). Similarly, the PhoP-PhoQ two-component system responds to acid
stress and low Mg2+ conditions encountered in the macrophage phagosome and
regulates genes involved in stress adaptation and virulence (García Véscovi et al.,
1994; Bearson et al., 1998). In addition, a recent study has identified a two-compo-
nent system in L. monocytogenes (LisR-LisK) which plays a role in pH homeostasis
and is required for full virulence of the pathogen (Cotter et al., 1999). These roles
of these sensor-regulators will be discussed in later sections.
ENVIRONMENTAL STRESSES ENCOUNTERED

BY BACTERIA DURING INFECTION
During infection, bacterial pathogens encounter stressful conditions that range from
sub-optimal to potentially lethal. Non-lethal stresses often induce the expression of
bacterial genes whose function is to protect against further stress. Gene products in
this category include molecular chaperones and heat shock proteins as well as
ATPases and other systems responsible for maintaining cellular homeostasis in
adverse conditions. In addition, environmental stresses in host microenvironments
can act as stimuli for the regulation of genes which play a specific role in colonization
and pathogenesis. For instance, the production of cholera toxin by Vibrio cholera is
activated by pH and temperature in the small intestine, a process mediated through
the ToxR-ToxS two-component system (DiRita, 1992).
BODY TEMPERATURE, HEAT-SHOCK AND THE GENERAL STRESS RESPONSE

The sudden shift in temperature from environmental/ambient temperatures to body
temperature at the outset of infection has the potential to directly influence regulation
of genes involved in bacterial virulence. However, bacteria also possess the ability
to react to even higher temperatures by inducing the expression of genes in the heat-
shock stimulon. The products of many of these genes are capable of protecting cells
against numerous stresses and can be considered general stress proteins.
S. Typhimurium demonstrates a classical heat-shock response when exposed to
elevated temperature (Bunning et al., 1990). Components of this response include
the chaperonins GroEL, GroES, DnaK and DnaJ, whose function is to maintain the
integrity of cellular proteins under certain stress conditions (Langet et al., 1992).
The temperature-dependent expression of the heat-shock response in Salmonella is
dependent upon the alternate sigma factor σH (also referred to as RpoH or σ32) (Yura
et al., 1993). Considerable overlap exists between oxidative stress responses and the
heat-shock response in E. coli and S. Typhimurium. Deletions in rpoH render E. coli
extremely sensitive to peroxide and superoxide stress as well as heat stress (Farr
and Kogoma, 1991) while S. Typhimurium cells adapted to peroxide stress exhibit
greatly increased resistance to heat-shock (Christman et al., 1985; Morgan et al.,
1986).
Since bacterial cells may be exposed to elevated temperatures as well as oxidative
stress during infection, researchers have examined the expression of heat-shock
proteins by bacteria during internalization by macrophages. Two separate studies

have used two-dimensional gel electrophoresis to examine the expression of heat-
shock proteins by S. Typhimurium during infection of macrophage cell cultures.
Infection of J774 macrophages resulted in the expression of 34 proteins at signifi-
cantly increased levels relative to Salmonella grown in laboratory media (Buchmeier
and Heffron, 1990). Many of these proteins seem to be specific for macrophages
and were not induced in cultured epithelial cells. In one study, both DnaK and GroEL
were shown to be synthesized by macrophage-internalized bacteria but were not
produced by bacteria grown in epithelial cells (Buchmeier and Heffron, 1990).
Interestingly a similar study examining infection of U937 macrophage cells failed
to demonstrate induction of DnaK and GroEL by intracellular S. Typhimurium but
did illustrate the synthesis of proteins from other stress stimulons (Abshire and
Neidhardt, 1993). The use of different macrophage cell lines may explain the lack
of correlation between studies, but both give an indication of the extent of bacterial
adaptation which must take place as Salmonella cells struggle to maintain homeo-
stasis during infection.
Listeria monocytogenes has the capacity to elicit a heat-shock response and also
induces synthesis of DnaK and GroEL homologues following sub-lethal heat shock
(Bunning et al., 1990; Hanawa et al., 1995). An analysis of those proteins produced
by Listeria grown within J774 macrophages revealed that none were involved in
in vitro responses to heat shock or oxidative stress responses (Hanawa et al., 1995).
Similarly, a separate study showed that synthesis of DnaK and GroEL actually
decreases during growth in mouse phagocytes (Hevin et al., 1993). It has been
suggested that rapid escape from the phagosome prevents expression of stress pro-
teins by L. monocytogenes during macrophage infection (Hanawa et al., 1995).
However, an increase in the expression of GroEL mRNA was detected recently in
macrophage-internalized L. monocytogenes using both reverse transcriptase-poly-
merase chain reaction (RT-PCR) and green fluorescent protein technology
(Figure 7.2), indicating that cells may respond with the synthesis of heat-shock
proteins but at levels which may not be detectable on protein gels (Gahan et al.,
2001). Recently the dnaK gene of L. monocytogenes has been cloned and sequenced
(Hanawa et al., 1999). A deletion mutant in dnaK is not phagocytosed efficiently by
macrophages, although the mutant grows as efficiently as the wild-type once inter-
nalized. The authors suggest a possible role for DnaK in the synthesis, folding and/or
translocation of surface proteins involved in adhesion (Hanawa et al., 1999). Inter-
estingly, exposure of the Gram-positive pathogen Enterococcus faecalis to bile salts
has been shown to elicit expression of some heat-shock genes including GroEL and
DnaK (Flahaut et al., 1996). In addition, heat shock of E. faecalis induced increased
protection against bile salts in vitro (Flahaut et al., 1996). These findings suggest
that heat-shock proteins may play an essential role in bacterial survival of bile salts
encountered during colonization of the small intestine.
In S. Typhimurium and L. monocytogenes, a number of other proteins associated
with thermotolerance and general stress resistance may play a role in vivo. For
example, in Salmonella and E. coli several genes involved in high-temperature resis-
tance (htr) have been identified (Delaney et al., 1993). Of these the best characterized
is htrA, encoding a serine protease (HtrA) required for survival of E. coli at high
temperatures (>42°C) and for the resistance of S. Typhimurium to oxidative stress

FIGURE 7.2 Use of green fluorescence protein (GFP) technology to determine gene expres-
sion in Listeria monocytogenes during infection of J774 mouse macrophage cells. (a) J774
cells infected with L. monocytogenes carrying promoterless copy of GFP (negative control).
(b) J774 cells infected with L. monocytogenes expressing GFP from the promoter of the heat-
shock operon groESL.
(Strauch et al., 1989; Johnson et al., 1991). HtrA is thought to assist in the degra-
dation of denatured proteins which may accumulate under stress conditions (Strauch
et al., 1989). In Gram-negative organisms, the expression of htrA is not regulated
by σH but by σE, a sigma factor synthesized under extreme stress conditions (Hiratsu
et al., 1995; Humphreys et al., 1999). Deletion of htrA in S. Typhimurium greatly
reduces ability to survive within macrophages and significantly attenuates virulence
for mice (Chatfield et al., 1992; Baumier et al., 1994). Elimination of the σE regulon
by deletion of rpoE has a greater effect on attenuation of virulence than deletion of
the htrA gene alone (Humphreys et al., 1999). Since rpoE mutants are rapidly
eliminated from host tissues they demonstrate reduced immunogenicity in mice and
fail to work as effective vaccines. In contrast htrA mutants retain the ability to
proliferate in host tissues and represent excellent vaccine candidates (Humphreys
et al., 1999). Homologues of htrA have been identified in Gram-positive organisms
including Bacillus subtilis (Noone et al., 2000) and play a role in stress resistance.
To date no work has focused upon HtrA homologues in L. monocytogenes.
Other heat-shock proteins involved in proteolysis include the family of Clp
proteases which play a role in heat tolerance of both Gram-negative and Gram-
positive organisms (Squires and Squires, 1992; Hecker et al., 1996). In Listeria, this
family of proteases is evidently of vital importance in governing stress responses
during infection and subsequent survival in the host. The gene encoding ClpC in
L. monocytogenes was identified in a transposon mutant displaying sensitivity to
low iron conditions (Rouquette et al., 1995). Disruption of clpC results in reduced
thermotolerance and increased sensitivity to high salt and low iron growth conditions.
ClpC mutants also display significantly attenuated virulence for mice and reduced
ability to grow in cultured macrophages (Rouquette et al., 1996, 1998). Examination
of macrophages infected by ClpC mutant strains using electron microscopy suggests

that the ability of these mutants to escape from the macrophage phagosome is
impaired (Rouquette et al., 1998).
Identification of clpE in L. monocytogenes has also been described (Nair et al.,
1999). Mutation of clpE impaired prolonged survival at high temperatures and
significantly reduced virulence for mice. Interestingly, clpE is not induced under
stress conditions but is upregulated in a clpC mutant, indicating cross regulation of
these Clp ATPases (Nair et al., 1999). Another member of the family of Clp pro-
teases, ClpP, is required for growth under stress conditions and for virulence of L.
monocytogenes. Evidence suggests that ClpP may be required for full activity of the
essential hemolysin, listeriolysin (Gaillot et al., 2000). Regulation of Clp protease
expression is mediated by CtsR, the product of the first gene in the ClpC operon
(Nair et al., 2000). This regulator is homologous to the B subtilis CtsR repressor of
stress responses; accordingly, deletion of the gene in L. monocytogenes results in
increased stress tolerance. Constitutive expression of CtsR in L. monocytogenes
results in significant attenuation of virulence for mice, most likely as a consequence
of the repression of the stress response.
In L. monocytogenes, the transcriptional regulator PrfA coordinates virulence
factor expression in response to environmental changes experienced within the host.
Virulence genes encoding internalins (inlA, inlB and inlC), hemolysin (hly), ActA
(actA) and phopholipases (plcA and plcB) are all under PrfA regulation and numerous
other genes uncovered by the Listeria genome sequencing project are postulated to
be PrfA-regulated (Glaser et al., 2001). Increased temperature provides a key signal
for the increased expression of certain PrfA-regulated genes and L. monocytogenes
cells are hemolytic at temperatures of 37°C and higher but show reduced hemolysis
below 30°C (Datta, 1994). Virulence genes are coordinately regulated in sequence
at specific stages of intracellular pathogenesis, such that genes required for escape
from the phagosome are expressed at an earlier stage than genes required for
intracellular motility and cell-to-cell spread; see Figure 7.1B (Bubert et al., 1999;
Freitag and Jacobs, 1999). The bacterium evidently has evolved the ability to sense
specific aspects of the host cell environment and to respond with the synthesis of
appropriate virulence factors. While PrfA undoubtedly plays an important role in
coordinating this response, other factors must play a role in detecting extracellular
signals and a putative PrfA-binding factor is thought to enhance the sensitivity of
the system (Böckmann et al. 1996).
ACID TOLERANCE AND VIRULENCE

Invasive foodborne pathogens will encounter low pH and/or organic acids during
passage through the stomach, during transient colonization of the small intestine
and during residence within the host cell phagosome. In order to survive in these
sub-optimal environments, bacteria have evolved mechanisms which allow adapta-
tion to low pH. The adaptive response, termed the acid tolerance response (ATR),
involves the acquisition of acid tolerance following a brief exposure to mildly acidic
growth conditions and involves a significant shift in patterns of protein synthesis as
bacteria are subjected to the reduction in pH of the growth media (Foster, 1991;
O’Driscoll et al., 1997). This response is shared by a number of foodborne pathogens

including S. Typhimurium (Foster and Hall, 1991) and L. monocytogenes (O’Driscoll
et al., 1996; Davis et al., 1996) and contributes to the survival of these pathogens in
low pH foods (Leyer and Johnson, 1992; Gahan et al., 1996). Studies have also been
performed to determine whether the ability to survive at low pH can contribute to
virulence potential.
A number of studies have attempted to determine whether acid adaptation of
pathogens prior to use in virulence studies can affect the outcome of infection. In
S. enterica serotype Enteritidis PT4, acid tolerant bacterial cells (stationary phase)
demonstrate similar virulence potential to acid sensitive, chilled log-phase cells
(Humphrey et al., 1998). Similarly, an increase in acid tolerance following acid
adaptation of L. monocytogenes fails to alter the virulence of the pathogen (Gahan
and Hill, 1999). It was suggested that this may simply be a consequence of the
ability of Listeria cells to naturally develop acid tolerance following uptake by
macrophages, such that natural stress adaptation during infection eliminates any
advantage of prior adaptation (Gahan and Hill, 1999). In support of this view, we
have demonstrated that a mutant of L. monocytogenes which is incapable of inducing
an adaptive acid tolerance response is significantly impaired in its virulence potential
for mice, relative to the parent (Marron et al., 1997). Furthermore, a spontaneous
acid tolerant mutant of L. monocytogenes displaying increased acid tolerance without
prior adaptation, demonstrates increased survival potential in mice relative to the
wild type (O’Driscoll et al. 1996). This may be a result of an increased ability to
survive the initial exposure to low pH encountered by internalized Listeria cells.
Indeed, a recent study demonstrates that both acid-adapted Listeria and a constitu-
tively acid tolerant mutant are capable of increased survival and growth following
uptake by macrophage (J774.A1) or enterocyte-like (Caco-2) cell lines (Conte et al.,
2000).
Similarly, an acid tolerant S. Typhimurium mutant displays moderately elevated
virulence potential in the mouse typhoid model (Wilmes-Riesenberg et al., 1996)
and an acid tolerant strain of S. Enteritidis is more virulent than a phenotypically
normal reference strain (Humphrey et al., 1996). Collectively, these data provide
evidence that acid tolerance plays a role in the virulence potential of these foodborne
pathogens and that further molecular characterization of the response is warranted.
In S. Typhimurium the development of acid tolerance is regulated in part by the
starvation/stationary phase alternate sigma factor σs encoded by rpoS. The avirulent,
acid-sensitive laboratory strain S. Typhimurium LT2 harbors a mutation in rpoS (Lee
et al., 1995; Swords et al., 1997; Wilmes-Riesenberg et al., 1997). Introduction of
wild-type rpoS from virulent S. Typhimurium strains into the LT2 strain restores
both virulence potential and acid tolerance (Lee et al., 1995). Similarly, the live oral
typhoid vaccine S. Typhi Ty21a, in common use, is an rpoS mutant which is
susceptible to a variety of environmental stresses (Robbe-Saul et al., 1994).
Expression of rpoS in S. Typhimurium is clearly induced following a shift to
low pH (Lee et al., 1995) and during macrophage infection (Chen et al., 1996) and
deletion mutants in rpoS are avirulent (Wilmes-Riesenberg et al., 1997). However,
since σs regulates the virulence plasmid-associated spv operon (Kowarz et al., 1994)
and is involved in oxidative stress and starvation stress responses, it is difficult to
attribute loss of virulence simply to loss of acid tolerance. Indeed, one study

demonstrated that in a plasmid-cured background both rpoS+ and rpoS- strains
survive well during the first 5 days of infection, but that at 21 days postinfection
the rpoS+ strain reaches significantly higher numbers than the mutant strain (Kowarz
et al., 1994). In another study utilizing infection of mice by the oral route, a plasmid-
cured rpoS– strain demonstrated a reduced ability to colonize Peyers patches relative
to its rpoS+ counterpart (Nickerson and Curtiss, 1997). The results suggest a role
for σs in bacterial persistence in vivo, even in the absence of plasmid-borne spv
genes (Kowartz et al., 1994; Nickerson and Curtiss, 1997).
The acid induction of σs is controlled by the product of the mouse virulence
gene mviA (Bearson et al., 1996; Benjamin et al., 1996). MviA has significant homol-
ogy to bacterial response regulator proteins and is most likely involved in environ-
mental sensing, in turn leading to induction of the σs regulon. This occurs through
an mviA dependent reduction in proteolytic turnover of σs, resulting in increased
levels of the protein (Foster, 1999). Therefore, both mviA and rpoS contribute to
virulence potential of S. Typhimurium.
While the σs regulon appears to respond primarily to organic acids, responses
to low pH (elevated H+ concentration) are influenced by PhoP, the regulatory element
of the PhoP-PhoQ two-component system (Bearson et al., 1998). This two-compo-
nent system senses and responds to conditions encountered within the host cell
phagosome including low Mg2+ (Soncini et al., 1996) and inorganic acid stress
(Bearson et al., 1998). Deletion of this response mechanism significantly attenuates
virulence of S. Typhimurium for mice (Miller et al., 1989) with mutant strains
demonstrating an impaired ability to survive within macrophages (Fields et al.,
1986), impaired growth in low Mg2+ media (Soncini et al., 1996) and reduced
resistance to bile (van Velkinburgh and Gunn, 1999), low pH (Bearson et al., 1998)
and host defense antimicrobial peptides (Groisman et al., 1992, 1997). Constitutive
expression of the phoP regulon also attenuates virulence, suggesting that dynamic
regulation of PhoP-activated genes (pag) and PhoP-repressed genes (prg) during
infection is necessary for full virulence (Miller and Mekalanos, 1990).
Examination of genes regulated by PhoP is ongoing. An important prg group
encode elements of the Salmonella pathogenicity island 1 (SPI1) type III secretion
system. This locus is required for entry of Salmonella into host cells but is repressed
following internalization as a result of activation of the PhoP-PhoQ system (Pegues
et al., 1995; Bajaj et al., 1996; Cotter and Miller, 1998). Many pag products show
no similarity to proteins in the database, while others encode genes with diverse
functions in many aspects of bacterial physiology (Gunn et al., 1998). One gene,
pagC, shows homology to a Yersinia enterocolitica invasion protein (Miller et al.,
1989) while another, pagO, encodes a product similar to that of the Yersinia virulence
plasmid (Gunn et al., 1998). Other pags play a role in resistance of Salmonella to
antimicrobial peptides present within the macrophage phagosome (Gunn and Miller,
1996; Soncini and Groisman, 1996).
Interestingly, the important virulence locus, Salmonella pathogenicity island 2
(SPI2), contains genes that are necessary for virulence and are induced by low Mg2+
in a cascade that is modulated by PhoP-PhoQ (Deiwick et al., 1999). However, the
regulation of SPI2 is further complicated by the fact that SsrAB, a two-component

regulatory system encoded on SPI2 also plays a role in expression of other SPI2
genes (Deiwick et al. 1999). Furthermore, yet another two-component regulatory
system, OmpR-EnvZ, is essential for SPI2 gene expression in response to low pH
(Lee et al., 2000). The PhoP-PhoQ regulon is obviously extremely complex; how-
ever, it is clear that this two-component system plays a major role in coordinate
regulation of virulence genes in response to environmental changes. Indeed, in a
recent study of genetic loci induced during infection it was found that, out of seven
genes subject to pH or Mg2+ mediated regulation, all were part of the PhoP-PhoQ
regulon (Heithoff et al., 1999). In another study, eight out of fourteen S. Typhimurium
genes identified as in vivo inducible were demonstrated to be regulated by PhoP-
PhoQ (Valdivia and Falkow, 1997).
Finally, the iron regulator protein Fur also plays a role in acquisition of acid
tolerance in S. Typhimurium (Foster, 1991). As a repressor of genes involved in iron
acquisition, Fur down-regulates transcription from appropriate promoters when intra-
cellular Fe(II) concentrations are high. However, Fur also influences the expression
of several acid shock proteins in an iron-independent manner. It is low pH rather
than iron concentration that influences Fur regulation of proteins involved in acid
adaptation (Foster and Hall, 1992). In addition, a specific mutation in fur can block
responses to iron but has no effect on acid tolerance (Hall and Foster, 1996). Knock-
out mutants in fur demonstrate an obvious virulence defect when analyzed in the
mouse typhoid model (Wilmes-Riesenberg et al., 1996). However, this effect may
be pleiotrophic and may affect iron metabolism as well as acid tolerance during
infection. The recent construction of acid-blind/iron-sensing and iron-blind/acid-
sensing mutants in fur will allow a more precise determination of the in vivo require-
ment for iron and acid-regulated Fur-dependent proteins (Foster, 1999).
In L. monocytogenes, relatively little is known of the circuits involved in regu-
lation of stress responsive genes. The alternative sigma factor, σB, has been identified
and sequenced in L. monocytogenes (Wiedmann et al., 1998; Becker et al., 1998).
This sigma factor appears to regulate the synthesis of a number of stress responsive
proteins. Mutation of the σB gene eliminates the ability to tolerate acid stress and
reduces the ability to respond to high salt environments (Wiedmann et al., 1998;
Becker et al., 1998). Elimination of sigma B in L. monocytogenes also reduces the
starvation stress response (Herbert and Foster, 2001) and decreases resistance to bile
salts (Begley, Hill and Gahan, manuscript in preparation). However, the mutation
does not appear to influence the virulence potential of this strain (Wiedmann et al.,
1998). Similarly, mutating the σB homologue in Staphylococcus aureus fails to
influence virulence of the pathogen in a mouse abscess model of infection (Chan
et al., 1998). To date, the data indicate that σB-influenced gene expression may not
play a significant role in the in vivo survival of Gram-positive pathogens. Nonethe-
less, further studies will be required to investigate any potential role for σB in
virulence. Another sigma factor, σH, is induced in L. monocytogenes following a
shift to acidic growth conditions (Phan-Thanh and Mahouin, 1999; Phan-Thanh
et al., 2000). In Bacillus subtilis, this sigma factor plays a major role in environmental
adaptation; however, a mutant in σH is not yet available in L. monocytogenes.
As mentioned previously, recent work has identified an operon in L. monocyto-
genes with significant sequence homology to two-component regulatory systems of

A orfA lisR lisK orfD
Region deleted
in ∆lisK
B
7 Wild-type
Log CFU Listeria / spleen
∆lisK
6
3
0 1 2 3 4
Day
FIGURE 7.3 (A) Genetic map of Listeria monocytogenes genes encoding the stress respon-
sive two-component regulatory system LisR-LisK. (B) Survival of knockout mutant in LisR
during infection of mice relative to the wild-type strain (LO28). (From Cotter, P.D. et al.,
J. Bacteriol., 181: 6840–6843, 1999. With permission.)
Group A streptococci, Lactococcus lactis and B. subtilis (Cotter et al., 1999). This
two-component signal transduction system, designated LisR-LisK, appears to play
a role in the regulation of acid resistance in L. monocytogenes. Mutation of either
the histidine kinase component (lisK) or the response regulator (lisR) results in a
significant attenuation of virulence potential, as evidenced by an inability to survive
during the early stages of infection in the mouse model (Figure 7.3). Interestingly,
a mutation in the Enterococcus faecalis homologue of lisR also results in a virulence
defect, suggesting a general role for this two-component system in virulence of
Gram-positive pathogens (Teng et al., 2002).
In addition to environmental sensors and regulators, some information is avail-
able concerning effectors which play a direct role in maintaining intracellular pH
homeostasis during shifts in external pH. A mutation in the major proton translo-
cating ATPase (atp) in virulent S. Typhimurium increases acid sensitivity, eliminates
the ability to induce an ATR and significantly decreases virulence in the mouse
typhoid model (Garcia del-Portillo et al., 1993). As mentioned previously, virulent
S. Typhimurium strains contain wild-type rpoS. Inactivation of single genes known

to contribute to acid tolerance in the attenuated strain LT2 has only a marginal effect
on acid tolerance in virulent Salmonella (Wilmes-Riesenberg et al., 1996). In a
virulent S. Typhimurium background, mutation of two or more genes was required
to eliminate acid resistance and the ability to induce an ATR. Double and triple
mutants containing a polA (DNA polymerase I) mutation lacked an ATR and were
highly attenuated in mouse and macrophage tissue culture models.
A portion of the F0-F1 ATPase of L. monocytogenes has recently been charac-
terized (Cotter et al., 2000). Disruption of ATPase activity eliminates the ability to
induce an acid tolerance response. However, further work is required to establish
the role, if any, of the F0-F1 ATPase in the virulence of the pathogen. The role of
the glutamate decarboxylase system in L. monocytogenes acid tolerance has also
been investigated (Cotter et al., 2001). In this system, cells grown in appropriate
media accumulate glutamate via a specific antiporter (encoded by gadC). Glutamate
is converted to γ-amino butyrate (GABA) by the enzyme glutamate decarboxylase
(encoded by gadB) with the net consumption of a single proton, thereby reducing
intracellular pH. GABA is then exported from the cell via the glutamate antiporter.
Initial evidence suggested that L. monocytogenes possesses two glutamate decar-
boxylase genes, gadA and gadB, with different roles in regulating pH homeostasis.
Mutation of gadB renders cells acid sensitive while deletion of gadA only marginally
reduces acid tolerance (Cotter et al., 2001).
The role of a third glutamate decarboxylase gene, uncovered as a result of the
Listeria genome sequencing project (Glaser et al., 2001), remains unknown. Addi-
tion of glutamate to an in vitro model of gastric acid (pH 2.5) significantly improves
survival of wild-type L. monocytogenes. In addition, a double mutant in gadA/gadB-
is exquisitely sensitive in the gastric acid model even when glutamate is added. The
results suggest a role for glutamate in aiding gastric survival of the pathogen, a
phenomenon mediated by the GAD system (Cotter et al., 2001). In a recent study,
knock-out of the lysine decarboxylase system (cad) in V. cholerae greatly reduced
acid tolerance but did not affect ability to colonize the small intestine in mice (Merrell
and Camilli, 1999). However, in this study the authors did not add lysine to the
inoculum prior to feeding. Collectively these studies suggest that these decarboxylase
systems are essential for full acid tolerance in complex media but further studies
are required to determine their roles in protecting against gastric acid.
OXIDATIVE STRESS RESPONSE

Bacterial pathogens encounter oxidative stress as a result of normal aerobic metab-
olism but are exposed to severe oxidative stress following uptake by macrophages
which generate phagosomal superoxide anion (O2–) and hydrogen peroxide (H2O2)
as well as the reactive nitrogen intermediate, nitric oxide (NO). S. Typhimurium
reacts to superoxide and peroxide with increased expression of two different, but
overlapping, sets of proteins. As mentioned previously, some overlap also exists
between responses to oxidative stress and heat shock, and the heat shock proteins
GroES and GroEL are induced by both peroxide- and superoxide-mediated oxidative
stresses (Farr and Kogoma, 1991). However, well defined regulons exist in S. Typh-
imurium which only react to peroxide stress (oxyR) or superoxide stress (soxRS).

OxyR is a transcriptional regulator with similarity to the LysR family (Christman
et al., 1989) and activates nine genes in E. coli in response to peroxide stress (Christ-
man et al., 1985). These genes include one of two catalase genes (katG), a gene
encoding glutathione reductase (gorA), and an NADPH-dependent alkyl hydroper-
oxidase (ahpFC) (Morgan et al., 1986; Tartaglia et al., 1990; Michan et al. 1999).
An OxyR-regulated locus (ahpC) is clearly upregulated in Salmonella during mac-
rophage infection (Francis et al., 1997). However, despite an obvious requirement
for OxyR in resistance to in vitro peroxide stress, deletion of either oxyR or katG
has no effect on ability of S. Typhimurium to resist neutrophil bactericidal activity
(Papp-Szabo et al., 1994). Similarly, a double mutant lacking KatE and KatG activity
is not affected in virulence for mice or sensitivity to macrophage killing (Mahan
et al., 1996).
SoxR and SoxS regulate the expression of a number of genes in response to
superoxide stress. The SoxR-SoxS regulon includes genes encoding the manganese-
containing Mn-cofactored superoxide dismutase (sodA), glucose-6-phosphate dehy-
drogenase (zwf), oxidation resistant fumarase (fumC), ferredoxin-NADPH oxi-
doreductase (fpr), an inner membrane efflux pump (acrAB), and at least five other
genes involved in superoxide resistance (Liochev and Fridovich, 1992; Liochev et al.,
1994; Fang et al., 1997). SoxR is activated by exposure to superoxide and in turn
activates expression of soxS, the product of which is a transcriptional regulator. SoxS
is necessary for resistance of S. Typhimurium to redox-cycling agents such as
paraquat, which generate intracellular superoxide (Fang et al., 1997). However, SoxS
is not required for resistance to macrophage killing or for virulence in mice (Fang
et al., 1997). Surprisingly, mutation of a SoxS-regulated gene, zwf, which encodes
a glucose 6-phosphate dehydrogenase, significantly reduces virulence potential in
the mouse model as well as compromising resistance to oxidative stress (Lundberg
et al., 1999). However, this locus is not exclusively regulated by SoxR-SoxS and
may be expressed at basal levels in the absence of SoxS (Fawcett and Wolf, 1995).
Deletion of SoxS-regulated sodA in S. Typhimurium does not significantly attenuate
virulence for mice, even though mutants do exhibit reduced resistance to macrophage
killing in vitro (Tsolis et al., 1995). A distinct Cu/Zn-cofactored superoxide dismu-
tase (SodC) exists outside of the SoxR-SoxS regulon and appears to have been
acquired by S. Typhimurium through bacteriophage-mediated horizontal transfer.
Curing of the prophage or deletion of the sodC gene dramatically reduces virulence
potential for mice, suggesting that sodC plays a significant role in Salmonella
pathogenesis (Farrant et al., 1997; De Groote et al., 1997; Figueroa-Bossi and Bossi,
1999).
In addition to SoxR-SoxS and OxyR regulons, another transcriptional regulator,
SlyA, has been implicated as a regulator of resistance to both peroxide and super-
oxide stress in S. Typhimurium. A slyA mutant is highly sensitive to both hydrogen
peroxide and paraquat, is avirulent in mice and is unable to replicate in mouse
macrophages (Libby et al., 1994; Buchmeier et al., 1997). In addition, transcription
of this gene is significantly enhanced following uptake by macrophages, suggesting
an important role in coordination of the bacterial response to oxidative stress during
infection (Buchmeier et al., 1997).

Iron-containing compounds are particularly prone to damage by oxidative stress
(Farr and Kogoma, 1991). In addition, hydrogen peroxide reacts with iron to form the
extremely reactive hydroxyl radical, which can cause further damage to cellular com-
ponents. Under conditions of oxidative stress, uptake of ferric iron is therefore reduced.
Recent evidence indicates that in E. coli both OxyR and SoxS activate the expression
of Fur, the global repressor of iron uptake (Zheng et al., 1999). The data indicate that
oxidative stress responses and iron metabolism are coordinately regulated.
Activated macrophages are capable of producing toxic nitrogen intermediates
(e.g., nitric oxide) in addition to peroxide and superoxide. Nitrosative stress, imposed
by nitric oxide donors such as S-nitrosothiols, has the ability to activate OxyR at
the transcriptional level in E. coli (Hausladen et al., 1996). In S. Typhimurium, a
component of the SoxR-SoxS regulon, the zwf gene product, is involved in resistance
to nitrosative stress as well as oxidative stress (Lundberg et al., 1999). In addition,
SodC plays a significant role in protecting bacteria from both nitric oxide and
superoxide both in vitro and during macrophage infection (De Groote et al., 1997).
However, further pathways exist which are specific for nitrosative stress and not
shared by oxidative responses. A gene encoding a hemoglobin homolog, flavohe-
moglobin (hmp) is required for resistance to S-nitrosothiols and acidified nitrite, but
not resistance to oxidative stress (Crawford and Goldberg, 1998a). Transcriptional
activation of hmp is independent of SoxS and OxyR but requires inactivation of Fur.
Interestingly, other Fur-repressed genes were also activated by nitric oxide, suggest-
ing that Fur plays a role not only in coordination of iron metabolism and responses
to acid stress, but also responses to nitric oxide (Crawford and Goldberg, 1998b).
Another gene contributing to nitric oxide resistance in S. Typhimurium is metL
encoding a protein involved in homocysteine biosynthesis. Deletion of metL in
S. Typhimurium dramatically reduces resistance to S-nitrosothiols and significantly
attenuates virulence for mice (De Groote et al., 1997).
The superoxide dismutase and catalase genes of L. seeligeri and the superoxide
dismutase gene from L. monocytogenes have been cloned and sequenced (Haas et al.,
1991; Hess et al., 1997). However, there is some evidence to suggest that neither
catalase nor superoxide dismutase play a role in the virulence of L. monocytogenes.
For instance, catalase negative strains of L. monocytogenes have been isolated from
listeriosis patients, indicating that catalase production by the pathogen is not neces-
sary to cause human disease (Swartz et al., 1991; Bubert et al., 1997). In addition,
transposon-induced mutants lacking catalase activity do not exhibit a virulence defect
(Leblond-Francillard et al., 1989). Also, unlike salmonellosis, listeriosis is not a
common opportunistic infection of individuals with chronic granulomatous disease,
a condition in which phagocytes fail to produce reactive oxygen intermediates (Gallin
et al., 1983; Safe et al., 1991).
Analyses of the mechanisms of bacterial killing by macrophages have failed to
determine whether reactive oxygen intermediates are absolutely required. Studies
have demonstrated that murine macrophages can kill L. monocytogenes by a mech-
anism which depends predominately upon nitric oxide, rather than superoxide or per-
oxide (Beckerman et al., 1993; Boockvar et al., 1994). However, a more recent study
indicates that reactive oxygen intermediates may be most important for bactericidal

activity (Ohya et al., 1998). Further studies are evidently required to determine the
extent to which L. monocytogenes encounters reactive oxygen species during infec-
tion and the mechanisms used to counteract this potentially lethal stress.
OSMOTIC STRESS
During the pathogenic cycle, foodborne pathogens encounter a range of environ-
ments with differing osmolarities. Following foodborne infection, pathogens are
exposed to an osmolarity in the intestinal lumen equivalent to 0.3 M NaCl, while
the osmolarity of blood is about 0.15 M NaCl (Chowdhury et al., 1996). S. Typh-
imurium and L. monocytogenes differ in their ability to naturally survive exposure
to high salt environments with Listeria spp. capable of surviving salt concentrations
as high as 30%, while the tolerance of Salmonella spp. for high salt environments
is much lower. However, both organisms respond to increases in external osmolarity
by synthesis and/or uptake of osmoprotectants, substances which counterbalance
external pressure, prevent water loss from the cell and thereby maintain cell turgor.
Osmoprotectants capable of being transported into the cell during periods of osmotic
stress include glycine betaine, proline and carnitine; proline, glutamate and trehalose
can be synthesized internally when required (Csonka and Hanson, 1991; Foster and
Spector, 1995).
In S. Typhimurium, the proU and proP systems govern uptake of both glycine-
betaine and proline in response to shifts in external osmolarity (Csonka et al., 1994)
while the putP system is a high affinity proline transporter (Liao et al., 1997). The
proU uptake system is clearly regulated by environmental stresses, including osmotic
stress and low pH stress, but no information is available concerning a possible role
during infection (Foster and Spector, 1995). In E. coli, a strain capable of causing
urinary tract infections and pyelonephritis demonstrates an abnormally high level of
proP activity, while deletion of proP dramatically reduces ability of this strain to
colonize mouse bladders (Culham et al., 1998). Similarly, inactivation of putP in
S. aureus significantly reduces virulence in an experimental endocarditis model
(Bayer et al., 1999). Studies analyzing the role of osmoprotectant uptake and syn-
thesis systems in virulence of foodborne pathogens are awaited with interest.
A glycine betaine uptake system (BetL) linked to the salt tolerance of L. mono-
cytogenes has recently been characterized (Sleator et al., 1999). This system is one
of three known mechanisms for the uptake of glycine betaine during periods of
osmotic stress. Other systems comprise the gbuABC operon and a system homolo-
gous to OpuC in Bacillus subtilis (Ko and Smith, 1999; Sleator et al., 2001a). A
betL mutant is significantly impaired in its ability to survive salt stress when glycine
betaine is the most abundant osmoprotectant, but does not differ from the wild-type
during intraperitoneal infection of mice (Sleator et al., 2000). The region upstream
of betL contains a putative σB promoter binding site and transcription of betL appears
to be up-regulated at high osmolarity (Sleator et al., 1999, 2000). Given that σB
mutants are not attenuated for mice (Wiedmann et al., 1998), it is not surprising that
mutation of this putatively σB-regulated gene does not result in a virulence defect.
In L. monocytogenes a homologue of the B. subtilis carnitine transporter, OpuC
plays a major role in carnitine transport (Fraser et al., 2000) and is also capable of

glycine betaine transport in an ATP-dependent manner (Sleator et al., 2001a). Car-
nitine is predicted to be the most important osmolyte in foods of animal origin
(Smith, 1996). Indeed, an OpuC mutant in L. monocytogenes demonstrates signifi-
cantly impaired survival during manufacture of fermented sausage and may therefore
play a significant role in facilitating growth of the pathogen to high levels in meat
products (Sleator et al., unpublished data). Recent work has demonstrated that
knockout of opuC in L. monocytogenes strain LO28 can reduce the virulence poten-
tial of this strain following intraperitoneal infection. Interestingly, this effect is strain
specific and was not seen in a knockout mutant in L. monocytogenes ScottA. How-
ever, elimination of opuC in both strains significantly reduced the ability to colonize
the upper small intestine in mice following peroral administration. This suggests
that this uptake system plays an important role in growth and survival in the osmot-
ically challenging environment of the gastrointestinal tract (Sleator et al., 2001a).
Finally, it is possible that systems for synthesis of osmoprotectants may play a
role in maintainance of homeostasis during infection, reducing the importance of
uptake systems. Recent evidence indicates that L. monocytogenes posesses a glycine
betaine synthesis system (Phan-Thanh and Mahouin, 1999; Glaser et al., 2001).
However a role for this system in maintainance of cell turgor has not yet been
examined. In addition, a Listeria proline synthesis system has been characterized
which is homologous to a locus in Salmonella (proBA) known to play a role in salt
tolerance (Sleator et al., 2001b). Knockout of this locus in L. monocytogenes reduces
salt tolerance in complex broth but does not appear to affect virulence potential
when administered to mice by the intraperitoneal or peroral routes. This finding
supports an earlier study which suggested that proline auxotrophs do not demonstrate
impaired virulence and suggests that host tissues contain a relatively abundant source
of free proline (Marquis et al., 1993). Furthermore, manipulation of the system to
induce overproduction of proline fails to alter the virulence potential in L. monocy-
togenes (Sleator et al., 2001c).
In S. Typhimurium, a two-component system, OmpR-EnvZ which responds to
perturbations in external osmolarity, has emerged as a global regulator of virulence
potential. EnvZ is the inner membrane sensor component that signals changes in
specific environmental signals via phosphorylation of OmpR, a transcriptional reg-
ulator. The system was originally identified as a regulator of the outer membrane
proteins, OmpF and OmpC. Mutation of ompR dramatically reduces virulence of
both Shigella flexineri and S. Typhimurium for mice, suggesting a major role in both
pathogens (Dorman et al., 1989; Bernardini et al., 1990). In S. Typhimurium, OmpR
mutants fail to lyse infected macrophages and so fail to induce a key step in
pathogenesis (Lindgren et al., 1996). Other studies have demonstrated that double
mutation of ompF and ompC also results in attenuation of virulence but not to the
same extent as ompR deletion, indicating that other components of the OmpR regulon
are important for infection (Chatfield et al., 1991). One theory is that the osmolarity
of the intestine favors expression of OmpC, a porin with small pore size that should
exclude harmful molecules such as bile salts. OmpF is most likely expressed outside
of the host environment (Russo and Silhavy, 1993; Foster and Spector, 1995). A
role for these proteins in intestinal survival is supported by the fact that double
mutants in ompC and ompF are severely attenuated when administered to mice by

the oral route but only marginally affected when administered intravenously (Chat-
field et al., 1991).
As indicated above, the influence of EnvZ-OmpR on virulence potential extends
beyond the regulation of outer membrane proteins. Other genes regulated by OmpR
include aas, a gene encoding 2-acylglycerolphosphoethanolamine acyltransferase,
which is induced within macrophages and is influenced by low pH (Valdivia and
Falkow, 1997). However, mutation of aas has no significant effect on virulence
potential (Lee et al., 2000). In addition, a gene responsible for the formation of
Salmonella-induced filaments within HeLa cells (sifA) is regulated by OmpR (Mills
et al., 1998). Deletion of this gene results in partial attenuation of virulence indicating
some requirement for filament formation during infection (Stein et al., 1996). Since
mutations of individual components of the OmpR regulon have only a marginal
effect on virulence potential, researchers have continued the search for the key
component of the regulon. In this regard, the most interesting recent discovery is
the fact that OmpR regulates the two-component system SsrA-SsrB in SPI2, which
in turn regulates a type III secretion system required for replication in macrophages
and infection of mice (Lee et al., 2000). DNA footprinting studies demonstrated that
OmpR binds directly to the promoter region in ssrA. In addition, OmpR was nec-
essary for SPI2 gene expression when cells were grown at low pH (Lee et al., 2000).
Evidence suggests that EnvZ responds to the low pH and low osmolarity of the
phagosome and activates OmpR, which in turn stimulates rapid expression of ssrA
and ssrB. SsrA-SsrB then detects another signal (possibly mediated by PhoP-PhoQ),
and in turn activates expression of the SPI2 type III secretion system (Deiwick et al.,
1999; Lee et al., 2000).
That OmpR is involved in low pH-dependent stimulation of SsrA-SsrB expres-
sion is interesting, given recent evidence implicating OmpR as the major regulator
of stationary-phase acid tolerance responses (Bang et al., 2000). Mutants in OmpR
are defective for an inducible stationary-phase acid tolerance response, yet can still
induce acid tolerance in log phase. It appears that OmpR activation by low pH and/or
low osmolarity following host cell invasion leads to induction of acid tolerance, as
well as stimulation of SPI2 genes and other genes which may play a role in patho-
genesis (e.g., sifA).
STARVATION STRESS
During residence in the host cell phagosome and during colonization of the small
intestine, invasive foodborne pathogens may struggle to accumulate adequate
amounts of phosphate, carbon and nitrogen. Starvation for such nutrients represents
a stress for the bacterium and results in a distinct physiological response. Moreover,
in Salmonella the starvation stress response induces potent cross resistance against
acid stress, heat stress, oxidative stress and osmotic challenge (Foster and Spector,
1995; Spector et al., 1999). Starvation stress may therefore represent a mechanism
to induce resistance to a number of in vivo stresses.
In S. Typhimurium, starvation resistance requires a number of genes including
rpoS and the starvation survival genes, stiA, stiB and stiC (Spector and Cubitt, 1992;

O’Neal et al., 1994). Mutants deleted in rpoS fail to exhibit carbon-starvation-
induced cross-protection and stiA, stiB and stiC are also required for the development
of cross-protection (Foster and Spector, 1995). Recent evidence suggests that stiA
encodes a nitrate reductase and is required for carbon-starvation-inducible thermo-
tolerance and acid tolerance (Spector et al., 1999). Interestingly, this locus is signif-
icantly induced during infection of Madin-Darby canine kidney epithelial cells
suggesting responsiveness to intracellular conditions. In addition, a deletion mutant
in stiA demonstrates reduced infectivity for mice by the oral route (Spector et al.,
1999).
Relatively little is known of starvation stress responses in L. monocytogenes.
However, it is clear that synthesis of virulence factors, such as hemolysin and
phospholipase, is repressed in the presence of rapidly metabolizable carbon sources,
with the result that a shift from complex to minimal growth conditions may act as
a trigger for the synthesis of virulence factors (Bohne et al., 1994, 1996; Milenbachs
et al., 1997). It is likely that limitation of nutrients experienced in vivo serves to
induce virulence factor expression. Furthermore, the global regulator of virulence
gene expression, PrfA, is required for an efficient starvation stress response, sug-
gesting a further role for this factor in maintaining homeostasis under starvation
conditions (Herbert and Foster, 2001).
METHODS TO DETECT GENES TRANSCRIBED IN VIVO

Traditionally, the analysis of the role of stress genes in virulence experiments has
followed from in vitro studies. Using this approach, the physiological role of a gene
(e.g., groEL) is well established before tests are performed to analyze its function,
if any, in bacterial virulence. However, a new range of techniques have been devel-
oped to identify genes, many of them previously unknown, which are expressed
during infection but not during growth on laboratory media. Many of these genes
expressed as bacteria attempt to adapt to the stresses of the new host environment.
IN VIVO EXPRESSION TECHNOLOGY (IVET)

Mahan et al., (1993) have developed an elaborate strategy for the selection of in vivo
induced (ivi) genes in S. Typhimurium. This strategy, termed in vivo expression
technology (IVET), was initially based on the use of an avirulent purine auxotroph
(pur–) mutant of S. Typhimurium. Random integration of the pur gene back into the
mutant resulted in a bank in which integration of pur downstream of properly posi-
tioned promoters resulted in expression of pur and complementation of virulence.
Mutants of interest were those that expressed pur in vivo and therefore survived
screening in a mouse model of infection, yet were Pur – in vitro. These represented
fusions of pur to promoters that were induced exclusively during infection. Further
IVET strategies in Salmonella utilized a promoterless cat gene encoding chloram-
phenicol resistance. Mice and/or tissue cultured macrophages inoculated with a bank
of clones expressing cat from random promoters were subsequently treated with
chloramphenicol to select for promoters that are active in vivo (Mahan et al., 1995).

The use of these IVET approaches has led to an appreciation of the array of
genes expressed during host infection, but not during growth in laboratory media.
Some of these genes were already closely associated with virulence and included
the regulator phoP and the plasmid-encoded virulence gene spvB. Other genes that
are evidently expressed as bacteria adapt to intracellular stresses and include a
catalase cofactor (hemA), a gene involved in acid tolerance (cadC), a gene involved
in recombination/repair (recA) and genes involved in iron acquisition (entF, fhuA)
and Mg2+ uptake (mgtA/mgtB) (Heithoff et al., 1997). The IVET system has been
instrumental in determining members of the PhoP-PhoQ regulon as recent charac-
terization of S. Typhimurium ivi genes has revealed many loci that are regulated by
low pH and low Mg2+ in a PhoP-dependent manner (Heithoff et al., 1999).
Other foodborne pathogens analyzed by IVET include V. cholerae and Y. entero-
colitica. Analysis of ivi genes in V. cholerae revealed genes involved in amino acid
and carbon metabolism, including a gene encoding a component of the TCA cycle
enzyme succinase (sucA). In addition, genes involved in motility and a gene desig-
nated hlyC encoding a secreted lipase were also isolated (Camilli and Mekalanos,
1995). The Y. enterocolitica study revealed a number of ivi genes, including five
loci involved in the iron starvation response, a gene involved in DNA repair (mutL),
a gene encoding a stress response regulator (acrR) and the malate synthase gene
(aceB) (Young and Miller, 1997).
A recent IVET approach for L. monocytogenes utilizes the hemolysin gene, hly,
as both a reporter of gene expression and as a means of selection of promoter
elements expressed in vivo (Gahan and Hill, 2000). Hemolysin functions in vivo to
allow escape of the bacterium from the phagosome into the cytoplasm of host cells.
Hemolysin negative mutants of L. monocytogenes are avirulent for mice and do not
produce zones of hemolysis on blood agar plates. A hly- host was used to create a
bank of clones in which the hemolysin gene is expressed from random promoter
elements (Figure 7.4). Infection of mice with this bank allowed the selection of ivi
clones that express hly in vivo but not on blood agar plates. Using this procedure it
was determined that L. monocytogenes selectively induces a number of genes in
response to the hostile host environment. These loci include a gene encoding the
TCA cycle enzyme fumarate hydratase (fum), a gene involved in DNA supercoiling
and putatively in gene regulation (DNA topoisomerase, topB), and a gene involved
in transport of a cellobiose analogue (celB) (Gahan and Hill, 2000).
GREEN FLUORESCENT PROTEIN (GFP) TECHNOLOGY

GFP from the jellyfish Aequorea victoria will cause bacteria to fluoresce if the gene
is placed downstream of an active promoter. This fluorescence can be readily
detected by fluorimetry, fluorescence microscopy or by flow-cytometry. Since no
cofactors are required for fluorescence, this reporter system represents a useful
means of monitoring expression of in vivo expressed genes. Recently, the system
has been used to screen for ivi promoters in S. Typhimurium (Valdivia and Falkow,
1997). Integration of promoterless gfp was used to create a bank of S. Typhimurium
cells in which expression of the protein is dependent on random promoter elements.

A
Em r or i
pCOR2
hly
MCS
Fragments of LO28
chromosomal DNA
B P hly
X Y
P
X Y LO28 ∆ hly
P P
X Y hly Emr X Y
INFECTED MICE
FIGURE 7.4 An IVET system to detect in vivo induced genes in Listeria monocytogenes.
(A) The pCOR2 IVET suicide vector comprises a promoterless copy of the hemolysin gene
downstream of the multiple cloning site (MCS). A gene bank is created by cloning random
fragments of Listeria DNA into the MCS. (B) The vector then integrates into the chromosome
in a hemolysin negative (∆hly) L. monocytogenes host at the point of homology provided by
the cloned DNA. The IVET bank represents clones expressing hly from random promoter
elements (P). This bank is then used to infect mice. Survivors of murine infection represent
clones expressing hly in vivo. (C) Plating of clones onto blood agar plates is used to determine
in vitro expression. (Adapted from Gahan, C.G.M. and Hill, C., Mol. Microbiol., 36, 498,
2000.)
This bank was used to infect macrophages which were then sorted based on fluo-
rescence intensity using a fluorescence-activated cell sorter (FACS). Clones of inter-
est were fluorescence positive during infection but negative in laboratory media.
Using this system eight members of the PhoP-PhoQ regulon were identified, as well
as a gene encoded on SPI2 (Valdivia and Falkow, 1997). A benefit of this approach

is that the GFP fusion strains can subsequently be used to analyze the kinetics of
gene expression in response to environmental stimuli.
Green fluorescent protein technology has been applied to L. monocytogenes to
study the sequential expression of various virulence factors during host cell infection
(Bubert et al., 1999; Freitag and Jacobs, 1999). Recently, the approach has been
used to detect other genes induced during residence in macrophages (Wilson et al.,
2001). A number of ivi genes were identified by the screening procedure, including
a mannose phosphotransferase system, a xylose repressor and a hemolysin-like
protein (yhdP). Deletion of the yhdP gene resulted in a virulence defect, suggesting
a role for this locus in pathogenesis (Wilson et al., 2001).
SIGNATURE-TAGGED MUTAGENESIS
Signature-tagged mutagenesis involves the creation of a transposon bank in which
each inserted transposon is marked with a unique DNA sequence tag. This allows
identification of transposon mutants in the bank which fail to survive mouse infection
(Hensel et al., 1995). The system results in the detection of genes that are absolutely
required for infection, as opposed to genes that are simply expressed in vivo, and
thereby isolates ready-made mutants which can then be subjected to further analysis.
This system resulted in the discovery of SPI2 and its type III secretion system (Shea
et al., 1996; Hensel et al., 1997).
Signature-tagged transposon mutagenesis has recently been applied to L. mono-
cytogenes (Autret et al., 2001). The study identified ten distinct loci essential for
murine infection, including genes involved in cell wall decoration, a transcriptional
regulator and membrane proteins.
CONCLUSIONS
It is becoming increasingly evident that, while some gene products can be classified
as “true” virulence factors (those encoding toxins or invasins, for example), there exists
a large class of proteins involved in stress management strategies which are necessary
if a bacterium is to mount a successful infection. These “stress” proteins may be
absolutely required or may only play a minor role in virulence, but collectively they
are a necessary part of the arsenal of pathogenic bacteria. The dissection of the role
of each protein within the complex orchestration of overlapping regulons is difficult,
much more so than for the “true” virulence factors, and represents a significant
challenge to researchers. The advent of elegant and imaginative techniques for detect-
ing genes expressed in vivo, allied to the completion of entire genome sequences,
offers the possibility that a more complete understanding of the relationship between
stress and virulence is within reach. In particular, the use of gene chip technology and
proteomics can be expected to reveal more of the strategies employed by pathogenic
bacteria to overcome host defenses. A thorough understanding of these strategies can
be expected to lead to the development of more effective control measures for the food
industry, and in preventing or interrupting colonization of susceptible hosts.

ACKNOWLEDGMENTS
The authors wish to thank the Health Research Board (Ireland), The Irish Department
of Agriculture, Food and Forestry, and BioResearch Ireland.
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Physiology and Molecular
8 Basis of Stress Adaptation, with
Particular Reference to the
Subversion of Stress
Adaptation, and to the
Involvement of Extracellular
Components in Adaptation
Robin J. Rowbury
CONTENTS
Introduction
Stresses Likely to Be Encountered by Bacteria in Food Preparation and
Likely Responses
Stresses Important in Food, Food Processing and Preparation, and in
Cooking
Stress Due to External Acidity
Stress Due to Internal Acidity
Heat Stress
Cold Stress
Osmotic Stress and Salt Stress
Irradiation Stress
Starvation Stress
Enhancing Effects of Metabolites on the Levels of Lethality of Some
Stresses
Lethal Sites Affected by Stresses
Gradual Build-Up of Stressing Agents and Relevance to Stress Tolerance in
Foods
Factors Influencing Stress Tolerance
The Basis for Enhanced Inherent Stress Tolerance
Growth Phase and Stress Tolerance
The author’s research on the role of ESC/EIC pairs in stress tolerance induction is funded by the Royal
Society, and he would like to express his thanks for this support.

Filamentation as a Factor in Survival and as a Major Potential
Problem in Food Microbiology
Induced Tolerance to Stress
Cross Tolerance and Sensitization Responses
Methods of Studying Mechanisms of Inducible Stress Tolerance
Studies of Proteins Synthesized de novo on Response Induction or in
Increased Levels
Isolation of Mutants Altered in Stress Tolerance
Examination of the Role of Established Cellular Components in
Tolerance Induction
Studying Involvement of Regulatory Components
Involvement of Specific Enzymes and Metabolites in Response
Induction
Predicting the Likely Components and Stages Involved in Stress
Responses
Stages in the Induction of Stress Responses
Switching-On of Stress Responses
The Likely Occurrence of Both Intracellular and Extracellular Stress
Sensors for Some Responses
Stress Sensors: Nature and Location
Some Stress Responses Probably Have Intracellular Sensors
Responses to Specific Stresses
Responses to Acidity and Acid Tolerance Responses Induced by Other
Conditions
Inducible Acid Tolerance Responses Induced by Mild Acidity
Mutants Altered in Acid Habituation
Dissecting the Early Stages of the Process
An Extracellular Acidity Sensor Formed at Neutral pH
Stationary-Phase Acid Tolerance Responses
Acid Tolerance Induced by Weak Acids at Neutral pH
Acid Tolerance Induced by Amino Acids, Sugars and Salts at
Neutral pH
Heat-Induced Acid Tolerance
Other Responses Which Affect the Level of Acid Tolerance
Alkali Sensitization at Acidic pH
Responses to Alkalinity
Heat Tolerance Induced by an Alkaline Shift
Responses to Heat in Food Preparation and Cooking
On the Nature of the Thermal Sensor
Ribosome
Membrane Components
The Medium
DNA
DnaK Chaperone

Activation of the Thermotolerance ESC by Other Stresses
On the Factors Governing the Heat-Shock Response and Its
Possible Relation to Thermotolerance
On the Involvement of Chaperones in Regulation
Responses to Cold
Sensing of Cold in Bacteria
Proteins Synthesized after Triggering of the Cold-Shock
Response by Sensor Activation
Responses Induced by Changes in Osmotic Pressure of Media
Do Both Intracellular and Extracellular Osmosensors Occur?
Responses to Salt Stress
Sensing of Salt Stress
Regulation of Salt-Induced Responses
Regulation of Responses to Irradiation
Sensing of Irradiation Stress
Stages in the Switching-On of Responses to Irradiation
Following Sensor Activation
Starvation Stress
Stress Tolerance Induction by Killed Cultures
Acid Tolerance Induction by Killed Cultures
Other Stress Responses Induced by Killed Cultures
Biochemical Changes Leading to Stress Tolerance
Role of Chaperones in the Biochemistry of Stress Tolerance
Biochemical Changes in Habituated Organisms Making Them
Acid-Tolerant
Biochemical and Physiological Changes Making Organisms Inducibly
Alkali-Tolerant
Biochemical Changes Leading to Heat Tolerance in Phenotypically
Thermotolerant Organisms
Physiological and Biochemical Changes in Organisms Inducibly
Tolerant to Cold
Osmotic Stress Tolerance: Physiological and Biochemical Changes
Involved in Inducible Responses
Biochemistry of Tolerance to Salt
Biochemical Changes Following Starvation
Counteracting the Induction of Stress Tolerance Responses
Subverting Stress Responses Using Metabolites
Subverting Other Stress Responses
Alkali Tolerance Induction
Acid-Induced Alkali Sensitization
Cu2+-Induced Thermotolerance
Extracellular Alarmones and Cell-to-Cell Communication
Future Experiments and Conclusions
Glossary
References

INTRODUCTION
This chapter will first briefly list and review stresses likely to be faced by contam-
inating organisms in food, and in food production and preparation processes. Sec-
ondly, it will discuss the likely tolerances that can arise when organisms are exposed
to those stresses. Thirdly, it considers the mechanisms involved in the induction of
stress responses and, finally, the biochemical bases for the tolerances outlined.
STRESSES LIKELY TO BE ENCOUNTERED BY BACTERIA IN

FOOD PREPARATION AND LIKELY RESPONSES
The ability of contaminating organisms to survive in foods and food production and
preparation procedures, during cooking and in domestic situations related to food,
depends on whether or not such organisms show tolerance towards the significant
number of stresses found in these situations. Table 8.1 shows the major stresses
likely to be faced by contaminating bacteria in foods or in other situations where
the exposed organisms may subsequently enter foods; with respect to all stresses,
inherent tolerance levels will be important but the major factor governing survival
will be whether or not inducible responses are put in place. Accordingly, Table 8.1
also shows responses likely to be induced by stress exposures.
STRESSES IMPORTANT IN FOOD, FOOD PROCESSING AND PREPARATION,

AND IN COOKING
The stresses to be considered initially here are as follows:
Stress Due to External Acidity
Exposure to acidic pH is of major importance in food microbiology because con-

taminating organisms commonly face low external pH in many acidic or acidulated
foods and at some stages in food production. Organisms may also face low pH in
the aquatic environment and this will be significant for survival in food, if contam-
inated water from such acidified environments is subsequently used in food produc-
tion or in the domestic environment.
Stress Due to Internal Acidity
Very commonly, organic acids are present in foods and often these foods are acidic
in pH also. In this case, the stress is due both to external acidity and to the internal
acidity arising because organic acids frequently collapse ∆pH (Salmond et al., 1984).
Heat Stress
Heat is not only involved in cooking but in numerous stages in food production also.
It should be noted that cooking can involve a wide range of temperatures, especially
with respect to the interior of the food, which will kill all organisms, down to those
which will act merely to induce thermotolerance.

TABLE 8.1
Stresses in the Environment, in Foods, in Food Preparation and Cooking
and Responses to Such Stresses
Stress Responses, Cross Tolerances
Site of Stress Responses or Gene Products
Stress Relevance to Food Damage Induced on Stress Exposures
Acidity Occurs in aquatic DNA, OM Acid tolerance1; alkali sensitivity, UV

environment* and in food resistance, salt tolerance, thermotolerance,
production H2O2 tolerance, resistance to polymixin B,
induction of Hyd genes2, of RpoS, PhoP,
HSPs and lysine3 or arginine
decarboxylase,
Acid pH + In acidic or acidulated DNA, OM, and CM Acid tolerance1, induction of Fur, AhpC and
weak acids foods HSPs
Weak acids In certain foods OM, CM Acid tolerance1
at pH 7. 0
Alkalinity In egg-white DNA, OM, and CM Alkali tolerance, thermotolerance,
resistance to UV, acid sensitivity, AHP
tolerance, AhpC, HSP and NhaA
induction
Heat During food production, DNA, OM, Thermotolerance; acid tolerance,1 alkali
preparation, and cooking ribosomes tolerance, UV tolerance, induction of
HSPs
Cold During refrigeration RNA Induction of CSPs
Osmotic In many foods with high OM, transport Osmo-tolerance, tolerance to oxidative
stress levels of sugars or salt processes components, thermotolerance, induction
of HSPs, ProP, ProU etc
Salt shock Foods containing high Effects on enzyme Acid sensitization, NhaA induction, PhoE
[NaCl] activity and induction
on protein synthesis
Irradiation In foods irradiated for DNA UV-tolerance, thermotolerance, acid
preservation tolerance4 alkali tolerance, induction of
HSPs, induction of UvrA, B, C, RecA,
LexA
Starvation5 In contaminated waters, if Proteins6 Thermotolerance, tolerance to acid, alkali,
used for food processing salt and H2O2, osmotolerance, induction of
proteases, HSPs
Exposure to In contaminated waters DNA, proteins Cu2+-tolerance, thermotolerance tolerance
Cu2+ to acid, alkali and Cd2+
* Relevant to food if contaminated water is used in production or preparation.

HSPs = heat shock proteins. AHP = alkylhydroperoxide.
1 Tolerance to inorganic acid.
2 Anaerobiosis and formate also needed.
3 Lysine and anaerobiosis needed.
4 Irradiation induces acid tolerance and UV can activate an acidity ESC.
5 For carbon.
6 A major effect of starvation is degradation of many proteins.

TABLE 8.1
Stresses in the Environment, in Foods, in Food Preparation and Cooking
and Responses to Such Stresses
Stress Responses, Cross Tolerances
Site of Stress Responses or Gene Products
Stress Relevance to Food Damage Induced on Stress Exposures
Cold Stress
There are also several types of cold that can be faced by contaminating organisms,
from exposure to relatively mildly cold temperatures inducing acclimatization pro-
cesses, down to conditions needing the formation of a whole range of cold-induced
proteins to survive freezing and thawing.
Osmotic Stress and Salt Stress
Osmotic stress occurs in many foods due to the presence of very high concentrations
of sugars or salts. It should be noted that at lower NaCl concentrations, which have
no major osmotic effect, there can be a specific salt stress.
Irradiation Stress
This results from the use of some forms of irradiation, such as those used for food
preservation; irradiation is also, on occasions, used to sterilize foods such as shellfish.
Starvation Stress
Organisms starved in natural waters can gain stress tolerance; if such organisms
subsequently enter foods, they may resist stresses because they have induced cross-
protection responses.
ENHANCING EFFECTS OF METABOLITES ON THE LEVELS OF LETHALITY

OF SOME STRESSES
Frequently, if the effects of potentially lethal agents or conditions are incomplete,

i.e., if a proportion of organisms survive these effects, then normally harmless
metabolites or other molecules may have an enhancing effect on lethality, and if
added with the stress, may greatly reduce or abolish survival of contaminating
organisms. Studies on this effect have been made, particularly on cultures exposed
to sublethal doses of heat stress, since reducing heating can in many situations
improve the organoleptic properties of foods. First, bacteriocins like nisin and pedi-
ocin, although alone unable to kill organisms like E. coli and Salmonella spp,
enhance killing by sublethal heat. The heating damages the outer membranes and
allows the bacteriocin through to degrade the cytoplasmic membranes. Heat killing
is also enhanced by polyphosphates, by cysteine and glutathione, by citrate and by

some sugars (Doyle and Mazzotta, 2000). Interestingly, killing by heat can also be
increased by low, normally sublethal, doses of other stresses, such as irradiation.
LETHAL SITES AFFECTED BY STRESSES

It is likely that there are at least four major kinds of damage to enteric bacteria like
Escherichia coli and Salmonella spp caused by stresses and, strikingly, many of the
stresses mentioned above cause several or all of these. Damage to DNA is almost
certainly the most critical lethal effect of stresses and many chemical and physical
stresses act primarily on this macromolecule, although they may damage other
molecules or structures as well. Certainly, irradiation, thermal stress, stress by
extreme acidity and alkalinity, and oxidative stress by, for example, hydrogen per-
oxide, all damage DNA, with this being the likely primary cause of death in each
case. Second, the ribosome is often targeted by lethal agents, with many leading to
inhibition of translation of m-RNA molecules by the ribosomes. Third, the outer
membrane is damaged by heat, acidity, alkalinity and oxidative components, and
even if the organisms are not killed by the damage, it allows other lethal agents (for
example, nisin), which are normally unable to penetrate, to pass through to the
cytoplasmic membrane and destroy the organism. Fourth, individual enzymes can
be damaged by several stresses and, where these are essential, this can also be lethal.
GRADUAL BUILD-UP OF STRESSING AGENTS AND

RELEVANCE TO STRESS TOLERANCE IN FOODS
In many situations, levels of toxic chemicals vary depending on amounts entering
the environment (Rowbury et al., 1989). There can be periodic rises in concentration,
possibly to potentially lethal levels, followed by falls due to dilution by unpolluted
medium, with such changes sometimes being repeated several times. The slow build-
up of toxic stresses means that there will be times when levels are non-lethal, but
induce tolerance responses which allow polluting or contaminating organisms to
survive potentially lethal levels, which build up later. The locations where gradual
build-ups of chemical stressing agents can occur include foods. In some foods,
acidity builds up gradually, eventually leading to potentially lethal pH values.
Because the pH falls gradually, organisms will habituate during early stages of H+
accumulation and, therefore, survive (Rowbury et al., 1989).
FACTORS INFLUENCING STRESS TOLERANCE

There is a range of factors which influence stress tolerance. First, levels of inherent
tolerance vary widely between natural isolates, e.g., in one study with S. enteritidis
PT4, it was striking to observe that organisms isolated from clinical situations had
higher levels of inherent tolerance to heat, acidity, oxidative stress and surface stress
than non-clinical ones (Humphrey et al., 1995). Such increased tolerances probably
make clinical isolates better able to resist host defenses, since the stress resistant
isolates also show enhanced virulence (Humphrey et al., 1996).

THE BASIS FOR ENHANCED INHERENT STRESS TOLERANCE
The differences in inherent stress tolerances between isolates depend, first, on RpoS
differences. RpoS is a sigma factor and presumably allows induction of certain stress-
related components in tolerant (rpoS+) isolates which cannot form or form at lower
levels in rpoS mutants. Second, envelope mutations alter acid tolerance (Bielicki
et al., 1982), due to changes in proton penetration through the outer or cytoplasmic
membranes (OMs or CMs). Since surface changes are known to occur during growth
in vivo, these could lead to altered penetration of chemical stressing agents and
changed stress tolerance.
TABLE 8.2
Resistance of Free and Attached Escherichia coli
to Chemical and Biological Inhibitory Agents
% Survival (or % Growth*)
Inhibitory Agent after Incubation of Attached
(concentration, where or Free Organisms with Inhibitor
applicable) Attached Organisms Free Organisms
Inorganic acid (pH 2.5)* 82.0 0.0

Trans-cinnamic acid (30 mM)* 75.9 0.8
Lactic acid (30 mM)* 95.5 7.5
Sorbic acid (30 mM)* 117.0 17.7
Citric acid (30 mM)* 81.5 36.4
Propionic acid (30 mM)* 100.0 69.4
Phage T4 50.9 0.1
Colicin V 74.0 4.0
Acrylate (1 mgml–1) 87.1 0.04
Chlorine (11 µgml–1)* 94.9 3.8
H2O2 (42 mM)* 65.4 6.0
Cu2+ (5 µgml–1) 90.3 0.5
Results are given for single representative experiments but each was repeated
with consistent results.
* Organisms were incubated with the inhibitor and, after removal of organisms
from the surface, if required, and removal of inhibitor, growth in pH 7.0 broth
followed, with results being compared to those of the control without inhibitor.
Weak acids were tested at pH 3.5; at this pH without weak acid, there were
only slight effects on either free organisms (9.8% inhibition of subsequent
growth) or attached organisms (0.4% inhibition). Treatments with inorganic
acid and weak acids were on E. coli strain P678-54ColV; all other treatments
were with E. coli strain 1829ColV except that colicin V-sensitivity was tested
with the sensitive strain P678-54. Some of these results are from the Ph.D.
thesis of G.C. Whiting (University of London, 1990).

Another factor affecting inherent stress tolerance is the finding that some isolates
but not others attach to surfaces. This often results from the presence of specific
surface appendages e.g., pili. Strikingly, attached but not free E. coli are resistant to
many agents relevant to survival in the environment, in foods and food preparation-
production, in domestic, hospital and commercial situations and in the body. Thus,
on attachment, E. coli becomes resistant to Cl2, fatty acids, acidity and alkalinity, to
acrylate, metal ions, detergents and antibiotics (Table 8.2). For these, levels of agents
which kill most free organisms allow survival of most attached ones (Hicks and
Rowbury, 1986). Several of the above agents are relevant to survival in foods or in
food preparation or production. Attached bacteria in shellfish may resist chemical
and physical agents, e.g., weak fatty acids, acrylate and irradiation, in natural waters
and agents (e.g., chlorine and UV irradiation) used to decontaminate the product
after harvesting. It is not clear how attachment protects, but it may be because such
chemicals cannot penetrate surface layers. Of interest is the finding that attachment
protects organisms from weak acids at acidic external pH, pHo (see Table 8.2), e.g.,
trans-cinnamate is lethal at pH 3.5 on free but not on attached organisms.
Attached organisms are also less affected than free ones by biological agents.
Thus, attachment allows organisms to tolerate both bacteriophages and bacteriocins
(see Table 8.2); some bacteriocins appear to play a role in protecting certain foods
from potentially pathogenic bacteria, so attachment of the latter might allow them
to avoid being killed. Attachment may also enhance resistance to physical conditions;
e.g., it has recently been shown that attachment can protect Salmonella spp from
heat (Humphrey et al., 1997), with organisms attached to muscle tissue being pro-
tected; this clearly has major implications for food safety.
GROWTH PHASE AND STRESS TOLERANCE

Another factor playing a major role in inherent stress tolerance levels and likely to
influence survival in foods is the growth stage of the contaminating organism. It is
known that stress tolerance is generally at its lowest in midlog phase and increases
in late log phase and in the stationary phase. There is no doubt that processes induced
via RpoS play a major role in this stationary phase tolerance response. Published
studies show that RpoS acts in two ways, first by inducing transcription of systems
which lead to increased levels of components which protect from stress damage,
e.g., molecules which protect proteins or DNA-binding components which protect
DNA, i.e., there is primarily induction of damage limitation components. Second,
RpoS is likely to be involved in induction of systems which repair damage to both
proteins and DNA and possibly other molecules.
Another factor must be emphasized. During entry into stationary phase, rounds
of chromosome replication in progress continue but no new rounds begin because
protein synthesis and mass increase are needed for initiation of replication
(Donachie, 1968). Accordingly, after a short time in stationary phase, rounds of
replication have terminated and there are no replication forks. In this situation,
organisms tolerate many stresses which damage DNA by acting at replication forks.
For example, starvation and entry into stationary phase lead to tolerance to heat,
H2O2 (Jenkins et al., 1988), osmotic stress (Jenkins et al., 1990), irradiation (Row-

bury, 1972), acid (Matin, 1991) and alkali. Each of these damages DNA and part
of the stress tolerance in stationary phase, therefore, is likely to result from chro-
mosome termination; this has been overlooked. It is easy to prove whether comple-
tion of rounds of replication per se leads to stress tolerance by comparing effects
of passage into stationary phase (or of starvation) with effects of completion of
rounds of replication without starvation. Such a situation occurs if a strain with
temperature-sensitive DNA synthesis initiation is shifted to restrictive temperature
(Rowbury, 1972). After the shift, organisms rapidly produce completed chromo-
somes. Analysis of stress tolerances in this situation would be of interest as to
whether such organisms show enhanced tolerance coinciding with completion of
rounds; they do show increased tolerance to UV irradiation (Rowbury, 1972). In
view of the above, one cannot state confidently the basis for inherent stationary
phase stress tolerance but the above two factors are likely to play a role.
FILAMENTATION AS A FACTOR IN SURVIVAL AND AS A MAJOR POTENTIAL

PROBLEM IN FOOD MICROBIOLOGY
Another factor in stress tolerance has been emphasized by pioneering work of
Humphrey and his group (Phillips et al., 1998; Mattick et al., 2000), who have found
that two stresses, low aw and low temperature, lead to filament formation in con-
taminating organisms. These filaments frequently survive for long periods, and are
able, given changed conditions, to fully divide rapidly. Only these two stresses have
been shown to lead to filament formation so far, but others could do so. What is
important is that both stresses can be significant in foods. It is striking that the
filaments can, under suitable conditions, divide to give as many as 100 organisms
per filament. For this reason, foods that appear, on the basis of cfu, to have as few
as 1 organism mL–1, may, within a short period have one hundred times as many, if
conditions have ensued that allow division. Accordingly, it is critical that foods are
entirely free of viable organisms. It is striking that for the organisms exposed to
cold, filament formation was much more marked for S. enteritidis isolate E (rpoS+)
than for isolate I (rpoS); the fact that E is more pathogenic (Humphrey et al., 1996),
means that the possibility of filaments in cold medium dividing to give many cells
on warming up, will be highly significant. The effects of high osmolality on cell
size are also of interest although there was no enhanced filamentation in rpoS+
strains, compared to rpoS ones.
INDUCED TOLERANCE TO STRESS

A third major factor affecting tolerance is whether organisms have induced any
specific log-phase tolerances, i.e., tolerances against a specific stress induced by low
levels of the same stress. Exposure of E. coli to micro-molar levels of hydrogen
peroxide, for example, leads to tolerance to milli-molar H2O2 levels (Demple and
Halbrook, 1983) and mild heat treatment induces tolerance to potentially lethal heat
stress (Mackey and Derrick, 1986). Such tolerance inductions usually occur very
rapidly and are generally dependent on de novo protein synthesis.

CROSS TOLERANCE AND SENSITIZATION RESPONSES
Cross-tolerance or sensitization responses are ones (Tables 8.1 and 8.3) where expo-
TABLE 8.3
Some Characteristics of Cross Responses Leading to Increased Sensitivity
or Tolerance to Stresses: Role of Extracellular Components
and Regulatory Molecules
Involvement of
Extracellular
Involvement Induction
Inducer of Response Regulatory and of Extracellular Component Inhibitors of
Cross Response Induced Other Components Sensor (ESC) (EIC) Response
pH 9.0 Acid H-NS Yes Yes; two EICs?

KCl, Amil,
sensitivity Fe3+
NaCl Acid H-NS, RelA1, N.T. Yes, dialyzable Glucose,
sensitivity cAMP2, PhoE non-protein FeCl3,
EIC acetate
L-leucine Acid RelA*, H-NS, Fur, N.T. Yes; small Glucose, Fe3+,
sensitivity CysB, OmpA protein EIC Fe2+ NaCl,
Nal, Tet
pH 5.5 Alkali IHF, H-NS, Lrp, Fur, Yes, small heat- Yes, small heat- Nal,
sensitivity CysB, NhaA stable protein stable protein phosphate,
ESC EIC NaCl, Fe2+
Shift up to 45°C Acid H-NS Yes, protein ESC Yes, protein EIC Nal
tolerance
N.T. = not tested
* Appreciable effect.
1 envZ lesions reverse effect of relA on this response.
2 Glucose represses this response but cAMP reverses this repression and allows induction.
The responses shown here were induced by pH 9.0, NaCl 300mM, L-leucine 50 µgml–1, pH 5.5 and a
temperature shift up respectively. Nal = nalidixic acid; amil = amiloride; Tet = tetracycline. The salt-induced
acid sensitization response was only partially inhibited by chloramphenicol, rifampicin and tetracycline
whereas the L-leucine-induced acid sensitization response was abolished by tetracycline but only slightly
inhibited by chloramphenicol and rifampicin.
sure to one stress induces tolerance or sensitivity to another; several are relevant to
survival of contaminating organisms in food. For example, mild heat shock induces
acid tolerance (Humphrey et al., 1993) in both S. enteritidis and E. coli. This
response could allow contaminating organisms, which had survived heating during
food production or preparation, to pass through the stomach because of their heat-
induced acid tolerance. Another major cross response is the heat tolerance induced
by alkaline pH (Humphrey et al., 1991). Organisms from egg-white, which has an
alkaline reaction, would, therefore, survive normally lethal heat shocks, allowing

such heated organisms to go on to cause disease. Shifts to acidic pH also induce
cross responses, organisms becoming tolerant to heat (some strains), salt, oxidative
components (Leyer and Johnson, 1993) and irradiation (Goodson and Rowbury,
1991). Accordingly, contaminating organisms exposed to acid might later resist
irradiation, heat or salt in foods or salt and oxidative components in the body.
One interesting response is thermotolerance induced by Cu2+. Organisms
exposed to copper in natural waters and then entering foods or food components
might survive cooking because of this process; Cu2+-exposed organisms also gain
acid and alkali tolerance. Cross responses can also induce stress sensitivity, e.g.,
E. coli on growth at alkaline external pH or plus salt or L-leucine (see Table 8.3)
becomes acid sensitive (Rowbury, 1997) while incubation at pH 5.5 induces alkali
sensitization (Rowbury, 1997). As stated above, several stresses induce cross toler-
ance against potentially lethal heat. It has been shown, in this laboratory, that glycerol
(2 M) strongly induces thermotolerance and smaller effects occur with phosphate
(10 mM), FeCl3 (1 mM)and FeSO4 (1 mM).
METHODS OF STUDYING MECHANISMS

OF INDUCIBLE STRESS TOLERANCE
STUDIES OF PROTEINS SYNTHESIZED DE NOVO ON RESPONSE INDUCTION
OR IN INCREASED LEVELS
The primary method of studying regulatory mechanisms and biochemical bases for
inducible stress tolerance has been to examine and analyze labelled proteins imme-
diately induced on exposure to the stress. This technique has led to useful findings
but two major factors have been overlooked.
First, it is unlikely that information can be obtained by this method about the
most important aspect of the process, namely the switching-on of the response. This
is because the sensors which detect the stress must be present in unstressed cells or
they cannot detect it. Accordingly, the sensor is unlikely to show enhanced synthesis
on exposure to the stress. It is, therefore, unlikely that studying proteins labelled at
high levels on stress exposure will throw light on stages needed to switch on the
response. It is not that regulatory proteins are not induced by stress, only that those
involved in the initiating or switching-on stages are unlikely to be.
Secondly, studies of labelled proteins have thrown light on many of the compo-
nents involved in the biochemistry of stress tolerance, e.g., temperature up-shifts
lead to the enhanced synthesis of several chaperones which are involved in repair
of heat-damaged proteins. Nonetheless, many of the proteins showing enhanced
synthesis may be red herrings as they appear not to be directly related to the tolerance
response.
ISOLATION OF MUTANTS ALTERED IN STRESS TOLERANCE

Mutants totally unable to induce a response are likely to have lesions in regulation,
because mutants altered in the biochemistry of tolerance will probably show loss of
only one aspect of tolerance, and this would usually lead to organisms with reduced

but not abolished tolerance. Thus, most mutants which have lost a response entirely
are probably regulatory ones, and useful results throwing light on how responses are
switched on have been obtained by studying such mutants; e.g., work on ompB mutants
led to elucidation of the processes governing OmpF/OmpC synthesis regulation, while
studies of oxyR mutants altered in oxidative responses (Kullick et al., 1995) involving
peroxides, and of soxRS mutants involved in oxidative responses relating to superoxide,
have thrown light on these responses. Similarly, studies of fur mutants, which appear
to be altered in switching-on of some acid tolerance responses in Salmonella spp,
suggest that Fur may, by analogy with its iron sensing role, act as a proton sensor
(Foster and Moreno, 1999), detecting intracellular H+, just as an extracellular sensing
component (ESC) detects extracellular H+ (Rowbury and Goodson, 1999a).
Analysis of mutants with reduced tolerance induction can throw light on bio-
chemical bases for tolerance. This is because when organisms lose one tolerance
component, analysis can indicate the nature of individual components involved e.g.,
if a DNA repair enzyme is absent from a mutant with low stress tolerance, DNA
damage is a likely effect of the stress, and repair of this damage a feature of tolerance.
EXAMINATION OF THE ROLE OF ESTABLISHED CELLULAR COMPONENTS

IN TOLERANCE INDUCTION
Studying Involvement of Regulatory Components
Another approach to studying the molecular biology of stress responses has been to
examine whether a response or individual components of it are aberrant if specific
regulatory gene products are absent, or if specific regulatory metabolites are added.
One study involved examining induction of the L-lysine and L-arginine decarbox-
ylases. Bennett and his group have shown that CysB and IHF are essential for AdiA
synthesis while H-NS interferes with induction (Shi et al., 1993; Shi and Bennett,
1994). A similar approach establishes that only CysB (of a range of components)
is needed for acid tolerance induction at pH 5.0, and that cyclic AMP interferes with
such induction (Rowbury and Goodson, 1997).
Involvement of Specific Enzymes and Metabolites

in Response Induction
Stress tolerance studies in strains altered in enzymes or components known to be

involved in specific protection or repair can throw light on tolerance responses; e.g.,
RecA, PolA and UvrA gene products are involved in DNA repair and Sinha (1986)
found that E. coli mutants altered in these genes are acid-sensitive. This, however,
applies to inherent acid tolerance and the levels of inducible tolerance are unaffected
by lesions in these genes (Goodson and Rowbury, 1991), suggesting that a novel
DNA repair process appears in acid-habituated organisms.

PREDICTING THE LIKELY COMPONENTS AND STAGES INVOLVED
IN STRESS RESPONSES
This is a new approach for studying stress responses induced by extracellular

stresses, which involves predicting likely components involved, their properties and
how cultural conditions may influence their structures and functioning. The first
work was by Nikolaev (1996, 1997a, b) who argued that organisms exposed to lethal
extracellular chemicals might secrete “protectants” to neutralize or inactivate the
stress and demonstrated agents which had these properties.
More comprehensive studies came from a proposal (Rowbury and Goodson,
1998) that in the presence of toxic extracellular chemical agents, E. coli might
produce extracellular induction components (EICs) which induce stress tolerance,
EIC production acting as an early warning against stress. These EICs have been
found to function for most stress responses where the stressing agent is extracellular.
It was then proposed that EICs are not secreted on exposure to stress, but arise from
extracellular sensing components (ESCs), already present in the medium (Rowbury
and Goodson, 1999a). The proposal is that these ESCs are produced in the absence
of stress, and are activated by stress (i.e., the ESC is a stress sensor). The proposal
was made because presence of the sensor in the medium would allow an immediate
response to stress. Recent studies confirm that many stress responses have ESC–EIC
pairs functioning. To function efficiently, these agents need to diffuse from the site
of formation to influence organisms nearby. These agents are, therefore, usually
small. This predictive approach has also been used to propose how stress sensors
might anticipate changes in stress levels.
Another useful prediction relates to killed cultures. It has been found that,
although ESCs and EICs are highly sensitive to reversible activation or inactivation
by very low levels of stress, they are insensitive to irreversible inactivation. Because
of this, another major prediction can be made, namely that killed cultures can confer
stress tolerance and this has been shown to occur (see “Stress Tolerance Induction
by Killed Cultures,” later in this chapter).
STAGES IN THE INDUCTION OF STRESS RESPONSES

The stages in induction of a stress response can be defined as 1) interaction of the
stress with the stimulus (stress) sensor, and 2) production of a signal or component
from the above interaction, which can set in train a series of reactions which generally
lead to increased transcription.
SWITCHING-ON OF STRESS RESPONSES

The first stage of induction involves activation by the stress (stimulus) of a specific
sensor. Sensors are defined as components which are produced in the presence or
absence of the stress, and which are activated by the stress to an induction compo-
nent, which sets in train the initial stages of induction. To be certain that a particular
component is the sensor, and that the induction component produced by activation

of this sensor by the stress leads to response induction, several other conditions need
to be fulfilled:
1. That agents or mutations which stop synthesis of the proposed sensor

ESC or its conversion to EIC also stop induction of the response.
2. Agents or conditions which destroy or remove the sensor or induction
component block induction.
3. Where more than one stressing agent produces the same response, each
of the stressing agents should interact with the proposed sensor to produce
the same induction component.
4. If the proposed sensor and component produced by its activation are
indeed involved in induction of the response, then addition of the induction
component should induce the response.
Some other conditions may apply if the sensor and induction component are
extracellular. Where the sensor is intracellular, the product of sensor activation
produces an internal signal which leads to a series of internal reactions which
culminate in increased transcription of stress response genes. Sensors for many
inducible and repressible non stress-related processes have been known for many
years to be integral CM proteins, and when studies of osmotic stress showed that a
sensor which detects osmotic shock (Igo and Silhavy, 1988) is a CM, it was assumed
that all stress sensors would be intracellular components. The location of such
sensors has become of interest, however, because it is now known that many, if not
most, are extracellular.
THE LIKELY OCCURRENCE OF BOTH INTRACELLULAR AND EXTRACELLULAR

STRESS SENSORS FOR SOME RESPONSES
Where organisms are exposed to stresses, it appears to be the rule that where the
stressing agent is initially in the medium, it is sensed by an ESC, whereas when it
is produced internally (e.g., as for some toxic electrophiles, Ferguson, 1999), it is
sensed by an intracellular sensor. If the stressing agent is present in the medium and
also produced intracellularly, then dual sensors are likely. It is important for an
organism to immediately detect a rise in the level of a stressing agent, since any
delay could lead to lethal effects. For a primarily external agent, the sensor would
need to be extracellular, so that there would be no delay while the agent penetrated
to an intracellular site. Conversely, if the agent were to be formed or released in the
cell, an extracellular sensor might not be activated with the needful response not
occurring at all. Since many lethal agents occur in media and can also be formed
in the cell, for many responses there may be dual sensors for a stress.
Stress Sensors: Nature and Location
It is now well established that several reponses induced by osmotic stress involve
sensing by CM proteins. Although the above sensors are intracellular, it can be
argued that where the stress is by a chemical agent, intracellular sensing may delay

response induction if the agent is in the medium. Such delay may make theoretically
resistable levels of the chemical stress lethal in practice. It seems likely that extra-
cellular stress sensors would have evolved to prevent delayed responses to external
toxic chemicals.
The first extracellular sensing component (ESC) reported was the acidity sensor
involved in induction of acid tolerance at pH 5.0. This sensor is found in medium
filtrates from cultures grown under a range of conditions, and is converted to an
extracellular induction component (EIC) on exposure to acidity. The EIC then
induces acid tolerance. This ESC/EIC pair exemplify a large group of extracellular
response induction pairs. It should be noted that the EIC is not formed by the cells;
it arises by chemical activation of ESC at acidic pH, organisms not being needed.
SOME STRESS RESPONSES PROBABLY HAVE INTRACELLULAR SENSORS

Some toxic chemicals are produced intracellularly. For these, it is important that the
sensor is also intracellular. For agents which can be present in the medium and formed
in the cell, e.g., H+, OH–, hydrogen peroxide and electrophiles, there may be both
intracellular and extracellular sensors. There is some evidence for such dual sensing
systems. Thus, as stated above, extracellular acidity is sensed by an extracellular sensor
(Rowbury and Goodson, 1999a). There is, however, evidence that the Fur gene
product acts as an intracellular acidity sensor. Firstly, fur mutants of S. typhimurium
fail to induce some acid habituation responses (Foster and Hall, 1992). The evidence
that protons interact with Fur to switch on some responses is indirect but compelling.
Thus, ferric ions are known to interact directly with Fur to switch off some responses
to low iron, and various mutants are refractory to this Fe3+ effect. Foster and
colleagues have now established (Hall and Foster, 1996; Foster and Moreno, 1999)
that one class of mutants, unable to bind and respond to ferric iron, also fails to
induce some responses by H+. Foster calls this group “iron-blind” and suggests that
they fail to bind both H+ and Fe3+.
Dual (intracellular and extracellular) sensing may also apply to hydrogen per-
oxide and other peroxides. Thus, OxyR is an intracellular component activated by
peroxides (Kullick et al., 1995); it presumably has evolved to detect intracellularly
produced peroxides, and after activation induces transcription. Recent studies have
established that H2O2 oxidizes OxyR to form an intramolecular bond between Cys199
and Cys208 of this protein. This is both the sensing mechanism and the activating
one, the OxyR form with the intramolecular bond enhancing transcription of the
oxyR regulon. Peroxides can also be produced extracellularly and detection may
then involve an ESC, since a non-protein extracellular sensor is known to be involved
in alkylhydroperoxide tolerance induction (details are given below) and an ESC/EIC
pair is used for tolerance induction by extracellular H2O2 (Rowbury, 2001). Almost
certainly, the mode of O2– sensing will involve the functioning of the intracellular
SoxR gene product, and detection of NO involves an intracellular sensor, if these
lethal agents are produced intracellularly, although whether extracellular O2– or NO
(e.g., from the phagolysosome) activates ESCs is not known.

RESPONSES TO SPECIFIC STRESSES
Many reviews have considered induction of stress responses, but where the switch-
ing-on of the process and the stages in induction have been considered, there has
often been a failure to consider satisfactorily how the stress is sensed, and how
activation of the sensor switches on the response. This section will redress the
balance, with the main emphasis being on sensing of chemical and physical stresses.
RESPONSES TO ACIDITY AND ACID TOLERANCE RESPONSES INDUCED

BY OTHER CONDITIONS
There is a very wide range of conditions which lead to acid tolerance induction;
these and several other responses related to the extent of acid tolerance will be
considered here.
Inducible Acid Tolerance Responses Induced by Mild Acidity
On a shift to acidic pH, Escherichia coli and Salmonella spp gain acid tolerance
(become acid habituated). One approach to the response has been to look for acid
shock proteins (ASPs) induced at acidic pH, and attempt to establish how their
synthesis is regulated and their identity and the basis for their synthesis. Many ASPs
have been found (>50 in S. typhimurium) and some progress has been made in
identification. The problem has been to understand why specific proteins are induced
by acidity. For several, it is not possible to understand the value that induction of
particular proteins has for acid-induced bacteria. Studies of regulation have been
interesting even if most identified regulated proteins have no obvious relevance.
Thus, in Salmonella typhimurium, synthesis of a group of eight proteins is regulated
by RpoS — four also being controlled by Fur and four by PhoPQ (Foster and Moreno,
1999).
It is likely that Fur has an acidity-sensing role. Obviously, if Fur does have such
a sensing role, this would apply to internal sensing whereas, when enteric bacteria
first detect acidity in the medium, the sensor is an extracellular protein ESC (Row-
bury and Goodson, 1999a). If Fur acts as an intracellular sensor, it is likely that the
proton-activated form would be a positive regulator of synthesis of one class of
ASPs; since some fur mutants are acid-sensitive, it can be assumed that some of
this group of ASPs (or other unidentified components) are essential for some aspects
of acid tolerance, either acting as regulatory components governing acid tolerance
component synthesis, or actually functioning in the tolerance processes themselves.
Another set of ASPs is controlled by PhoPQ. One of these, namely ASP29, is
PhoP itself; i.e., PhoP, like the acid-tolerance regulator RpoS, is acid-induced. How-
ever, whereas RpoS functions in tolerance to both organic and inorganic acids, the
PhoPQ system is only involved in inorganic acid tolerance. Foster proposes that the
PhoQ component senses acid (presumably protons), thus inducing PhoP; certainly
PhoP-LacZ can be induced by high proton concentrations, so possibly PhoQ senses
H+. In view of the role of ESCs in early warning against acidity (Rowbury and
Goodson, 1999a), one must note that sensing of protons here relates to internal H+.

As indicated above, mildly acidic pH, which induces an rpoS-dependent acid
tolerance, also induces RpoS synthesis. It is proposed that an activated protease, in
the absence of stress, degrades RpoS rapidly. In contrast, during stress exposure, the
activator loses its ability to enhance protease and RpoS becomes more stable.
Mutants Altered in Acid Habituation
Another approach to studying habituation has been to attempt to isolate mutants

altered in acid tolerance. These could be altered in response induction or in the
components which actually make the cells tolerant. Studies have also been made of
how mutations in known major regulatory components affect acid habituation.
Dissecting the Early Stages of the Process
One approach to studying induction of acid habituation has been to look for ASPs.
The problems with this approach have already been indicated, namely that sensors
and most other regulatory components might not be detected by this approach, as
these components would all have to be present in unstressed cells, or the response
could not be switched on. The above applies whether the sensor is extracellular or
intracellular or whether regulation involves functioning of two components or more;
if they are needed to switch on the response, all would need to be present when the
stress appears, i.e., would need to have been present under non-stressing conditions.
In view of the above, another approach has been to consider what are likely to
be the early stages of acid tolerance induction, and look for components which
appear likely to occur. We opined that, since most acidic challenges to bacteria are
due to external acidity, it was likely that responses would be more rapid if early
regulatory intermediates were in the medium and interacted there with organisms
to produce tolerance. We therefore looked for components in media from acidified
cultures, which were essential for tolerance of such cultures and induced acid
tolerance in non-stressing conditions. Filtrates from neutralized pH 5.0-grown cul-
tures contained such a component. Evidencing this were two initial findings:
1) procedures that removed or destroyed ECs, e.g., continuous filtration or protease
treatment, prevented tolerance induction at pH 5.0, and 2) neutralized filtrate from
pH 5.0 cultures induced acid tolerance in organisms at pH 7.0. The latter filtrate
was inactivated if ECs were removed or if proteins in it were destroyed by protease
(filtrate is not inactivated by RNase or DNase) or by incubation in a boiling water
bath, but not at 75°C (Rowbury and Goodson, 1998). Other studies suggest that this
extracellular induction component (EIC) is a fairly small protein.
An Extracellular Acidity Sensor Formed at Neutral pH
Accordingly, neutralized filtrates from pH 5.0-grown cultures contain an EIC, but,
as stated earlier, if the acidity-detecting system is to function rapidly, there ought
to be an extracellular acidity sensor, able to detect acidity and converted by it to an
EIC. There is indeed such an extracellular sensing component (ESC), and it is present
in culture filtrates from organisms grown at neutral or alkaline pH. The ESC can be
removed from such filtrates, if the filtrate-containing vessel is immersed in a reservoir
containing a large volume of the same medium without organisms, with the filtrate
vessel being separated from the medium reservoir by a 0.2 µm pore filter. Because

of the large volume in the reservoir, the ESC passes from the filtrate vessel, through
the membrane, into the surrounding medium. This ESC is synthesized at pH values
from 4.5 to 9.0 and is secreted to the medium.
The properties and nature of the ESC synthesized at pH 4.5 to 6.0 are not readily
studied as such sensor is immediately activated (chemically or enzymically) to EIC,
leading to induction of tolerance. In contrast, the ESCs formed at pH values from
6.5 to 9.0 can be easily examined. Incubation of ESC-containing filtrates with several
proteases and with RNase or DNase, establishes the protein nature of the sensor,
while its ability to resist exposure to 75°C (but not to 100°C) establishes its heat
stability (Table 8.4). This protein passes through 30 K nominal molecular weight
TABLE 8.4
Induction of Acid Tolerance by Acidity and at Neutral pH by Amino Acids,
Salts and Other Components; Involvement of Extracellular Sensing
Components and Extracellular Induction Components
Extracellular Components and
Acid Tolerance Switching-On of Acid Tolerance by Inducer
Inducer Involvement of ESC Involvement of EIC
pH 4.5 to 6.0 Yes; heat-stable non-dialyzable protein Yes; heat-stable non-dialyzable protein
ESC EIC
L-glutamate Yes; protein ESC senses L-glutamate Yes; non-dialyzable protein EIC
L-aspartate Yes; protein ESC senses L-aspartate Yes; non-dialyzable protein EIC
L-proline Yes; non-protein ESC senses L-proline Yes; non-dialyzable, non-protein EIC
L-glutamine N.T. Yes; non-protein EIC
Glucose Yes, ESC senses glucose* Yes, small (ca. 10 kda) EIC*
Glucosamine N.T. N.T.
FeCl3 Yes; ESC senses Fe3+ Yes; non-protein EIC
KCl N.T. Yes, small (ca. 10 kda) non-protein EIC
NH4Cl N.T. Yes, dialyzable non-protein EIC
Glycerol N.T. N.T.
N.T. = not tested.
* Probably protein components.
(NMW) membranes but is retained by 5 NMW membranes and is not removed from
filtrates by dialysis. This sensor formed at pH 7.0 is, therefore (like the EIC which
it gives rise to), a rather small heat-stable protein. Incubation of the sensor formed
at pH 7.0 under acidic conditions, i.e., at pH 4.5 to 6.0 (but not at 2.0) converts it
to the EIC; organisms are not required for this conversion, but if the activated filtrate
is neutralized and pH 7.0-grown organisms added, the EIC induces them to acid
tolerance rapidly at pH 7.0, i.e., the EIC can induce acid tolerance in unstressed
organisms.
Thus, the ESC is converted in the medium at acidic pH to EIC. One possibility is
that this is a chemical activation. The alternative is that acidity unmasks an auto-enzyme

activity in the ESC which converts it to the EIC. The ESC can be activated by other
stresses. Of particular interest is the finding (Rowbury and Goodson, 1999b) that
transfer of ESC-containing filtrates from low temperature to 42 to 55°C also activates
the ESC to EIC. This probably explains why organisms grown at >37°C are more
acid-tolerant than those grown at 25 to 37°C (Humphrey et al., 1993).
Stationary-Phase Acid Tolerance Responses
Stationary-phase organisms are more acid tolerant than log-phase ones, and at least
three tolerance responses are induced in stationary phase, with induction generally
requiring acidification. The so-called oxidative system appears during aerobic
growth to stationary phase, pH 5.5 being needed for induction. This is glucose-
repressed and generally needs Cya and Crp; it also requires glutamate or glutamine
for activation, these functioning by a protein synthesis-independent mechanism. If
organisms grow without these acids, this system is non-functional, but brief exposure
to glutamate or glutamine without protein synthesis activates the system, i.e., all
components are formed in the absence of glutamate/glutamine but one component
needs activation by one of these. How organisms induced for this system are pro-
tected from acid is not known (Castanie-Cornet et al., 1999).
The other processes are fermentative with functioning of amino acid decarbox-
ylases during acid challenge; one needs arginine (Arg) during challenge (but not
added to induction media) and is AdiA+-dependent, the other needs L-glutamate
during challenge, but not added during induction. The Arg-dependent system is
RpoS-independent and an rpoS lesion has only a small effect on the glutamate-
dependent one (Castanie-Cornet et al., 1999). The AdiA-dependent process evidently
uses Arg during challenge to produce agmatine, which is transported out of the cell
in protonated form, keeping pHi from falling. This system is dependent on CysB,
as expected, since AdiA synthesis needs this component. The final system needs
glutamate during challenge; it is assumed that γ-aminobutyric acid (GABA) produced
by decarboxylation leads to pHi rise on passage of protonated GABA to the outside.
This system is absent from gadC mutants, since these cannot transport GABA out
of the cell.
Other mutant studies show that either of the glutamate decarboxylase isoforms
(two are present, encoded by gadA and gadB) can function to produce GABA. Little
is known of how the above are switched on. Presence of weak fatty acids in
stationary-phase cultures suggests that intracellular sensors and induction compo-
nents might be involved. Two processes need decarboxylases, however; by analogy
with decarboxylase-dependent glutamate-induced acid tolerance (see below), some
or all may show involvement of ESCs and EICs.
Acid Tolerance Induced by Weak Acids at Neutral pH
E. coli normally fails to show acid tolerance induction at pH values greater than 6.0
unless metabolites are present. Guilfoyle and Hirshfield (1996), however, induced
tolerance at pH 6.5 in the presence of butyric and propionic acids, while recently
Kwon and Ricke (1998) induced tolerance in S. typhimurium grown at neutral pH

on addition of propionate. The latter induction process was protein synthesis-depen-
dent and was enhanced in anaerobic conditions. No studies have been made on
sensing of the weak acids but since EICs specific for such a process could give early
warning of exposure to lethal weak acid concentrations, it is likely that an ESC
detects the weak acids, and that its conversion to an EIC leads to acid tolerance
induction.
Acid Tolerance Induced by Amino Acids, Sugars and Salts

at Neutral pH
Three amino acids induce such tolerance without any pH change during induction.
Induction by L-glutamate, L-aspartate and L-proline requires EICs (Rowbury, 1999).
The EICs for the first two are proteins whereas that for the L-proline response is
not. For each, an ESC present in media from cells grown without inducer senses
inducer and is activated by it (Rowbury and Goodson, 1999b) to give an EIC (see
Table 8.4); ESC closely resembles EIC in properties but cannot induce the response.
A few other amino acids also induce acid tolerance at pH 7.0.
Glucose induces acid tolerance at pH 7.0 in E. coli (see Table 8.4). On incubation
of organisms with pH 7.0 broth, tolerance appears on addition of glucose, with no
fall in pH, and an EIC (able to convert organisms in pH 7.0 broth to acid tolerance)
is formed in the medium. This EIC is a protein and arises from an extracellular
sensing component (ESC) which is activated by glucose (Rowbury and Goodson,
1999b). Such tolerance induction by glucose (it occurs with other sugars also) has
probably evolved because medium acidification results from glucose degradation, and
the response protects organisms from anticipated acidity. Several salts induce acid
tolerance at pH 7.0 in E. coli (see Table 8.4). In each case, an EIC appears in media
during induction, and the EIC (in filtrates dialyzed to remove the salt) induces tolerance
in organisms in broth at pH 7.0. For the one salt tested further, FeCl3, EIC is formed
by interaction of an ESC with Fe3+ (Table 8.4 and Rowbury and Goodson, 1999b).
Heat-Induced Acid Tolerance
E. coli and Salmonella spp transferred from low temperatures to, e.g., 45°C become
more acid-tolerant by a protein synthesis-dependent process (Humphrey et al., 1993).
These findings are of medical and applied importance since contaminating organisms
in food which have survived cooking could, on ingestion, resist gastric acidity and
go on to cause disease. The histone-like regulatory component H-NS may be involved
in the control of this response since hns mutant organisms are acid-tolerant after
growth at 25°C (Rowbury, 1997). Such acid tolerance arises on exposure of cultures
to elevated temperatures, because the acid tolerance-related ESC is activated not
only by acidity, but also by elevated temperatures.
One germane finding is that, although salt normally reduces acid tolerance when
added to media, organisms grown at low temperature and shifted to 44°C become
much more acid-tolerant if salt is present in the medium. Thus organisms surviving
in partially cooked salty foods might, on ingestion, survive gastric acidity (Rowbury,
1997).

Other Responses Which Affect the Level of Acid Tolerance
There are three responses which reduce acid tolerance. It is known that H+ passage
across the OM is impeded and that some OM lesions cause acid sensitivity due to
enhanced H+ penetration into the periplasm (Bielecki et al., 1982), while introduction
or modification of certain OM pores directly or indirectly increases OM penetration
of protons and acid sensitivity. This is true for two responses considered here and
probably for a third. Acid sensitization induced by salt involves induction (Table 8.5)
TABLE 8.5
Conditions and Responses Inducing Acid Sensitization; Do These Result
from Porin Derepression?
Is a Porin or Other
OMP Derepressed or Is Porin or OMP
Response, Condition Modified by Response, Change Responsible
or Mutation Leading Mutation or Culture Directly or Indirectly
to Acid Sensitivity Condition? for Acid Sensitivity?
Phosphate starvation Yes, PhoE Yes, sensitization lost in phoE mutant

Mutation in phoS Yes, PhoE Yes, sensitization lost in phoS, phoE strain
Mutation in phoR Yes, PhoE Not proven so far
Mutation in phoT Yes, PhoE Not proven so far
Introduction of F′128 into Yes, PhoE appears Yes, loss of F′128 leads to loss of PhoE and
phoE mutant acid resistance
Growth at alkaline pH None shown so far N.A.
Growth with NaCl Yes, PhoE Yes, sensitization abolished by phoE lesion
Growth with L-leucine OmpA protein modified Yes, sensitization abolished by ompA lesion1
N.A. = not applicable.

1 ompA deletions and certain point mutations abolish response. (See “responses to specific stresses,”
in this chapter, and Rowbury, R.J., Lett. Appl. Microbiol., 24, 319, 1997.)
of the PhoE OM pore (see “Regulation of Salt-Induced Responses” later in this

chapter) and response induction involves an EIC (see Table 8.3). In addition, transfer
of organisms to pH 9.0 induces sensitization (acid sensitivity induction, ASI), a
process independent of alkali habituation which also occurs at this pH. No OM pore
induction has been linked to this response, but one probably occurs. This response
has two parts, one protein synthesis-dependent, the other protein synthesis-indepen-
dent. Both sensitization components are switched on by EICs. It is likely that the
first component of ASI is switched on by a protein EIC, the second by a non-protein
EIC (Rowbury, 1999). The third sensitization response is switched on by L-leucine;
an EIC is involved, although an ESC has not been tested for.
The later stages of induction are not well studied, but several regulatory compo-
nents are needed (see Table 8.3) and ompA deletion mutants lack this response; some
ompA point mutants have the response, others do not. The finding that only loss of

an acidic amino acid from a surface loop of the OmpA protein abolishes the response
suggests that leucine modifies OmpAp to give it pore activity (Rowbury, 1999), thus
directly or indirectly allowing H+ to cross the OM (see Table 8.5). Cells exposed to
leucine become less sensitive to phage K3 (receptor is OmpAp), in accord with
leucine altering OmpAp surface properties (Rowbury, 1997, 1999).
Alkali Sensitization at Acidic pH

Organisms transferred from pH 7.0 to pH 5.5-6.0 become alkali-sensitive by a
process distinct from the acid tolerance response. Induction depends (Tables 8.3 and
TABLE 8.6
Regulatory Components and Extracellular Components Involved in Alkali
Habituation at pH 9.0 and in Alkali Sensitization at pH 5.5
Regulatory or Needed for Alkali Needed for
Induction Component Tolerance Induction Alkali Sensitization
PhoE Yes Yes

NhaA Yes Yes
NhaB No Yes
Fur Yes Yes1
IHF Yes Yes
H-NS No Yes
CysB Yes Yes
RelA No No
TonB Yes No2
Extracellular sensing component (ESC) Yes, possibly 2 ESCs Yes, dialyzable heat-stable protein
Extracellular induction component (EIC) Yes, possibly 2 EICs Yes, dialyzable heat-stable protein
1 fur mutants have reduced response
2 tonB mutants are constitutively alkali-sensitive
8.6) on the functioning of regulatory components, antiporters and PhoE (Rowbury,

1997). Sensitization only occurs if an EIC is present (Rowbury, 1999). This EIC
derives from an ESC which is synthesized at a range of pH values and activated to
EIC at acidic pH in the absence of organisms. Formation of this ESC needs H-NS,
IHF and Fur; but involvement of other components has not been tested. The EIC
and ESC are very heat-stable proteins or peptides (survive exposure to boiling water
for 15 min) of less than 5000 Da.
RESPONSES TO ALKALINITY
Switching-on of inducible alkali tolerance (alkali habituation) involves functioning
of ECs. Thus, a sterile cell-free filtrate from a pH 9.0-grown culture can, after
neutralization, induce alkali tolerance in organisms at pH 7.0, whereas a filtrate from
a pH 7.0-grown culture cannot. Accordingly, the pH 9.0 culture contains an alkali-

tolerance-inducing EIC. Although the pH 7.0 filtrate is ineffective in inducing the
response, transfer to pH 9.0 in the absence of organisms rapidly activates it. This
pH 7.0 → pH 9.0 process converts an alkali tolerance ESC to the corresponding
EIC and the ESC behaves as an alkali sensor. The filtrates containing either EIC or
ESC are only partially inactivated by proteases; possibly there are two EICs and
two ESCs, one of each being a protein, the other being a non-protein component
(see Table 8.6). Although it is not clear how the EIC induces the response, there is
some information on which regulatory components function; NhaA, IHF, TonB and
Fur (Rowbury, 1997) are needed, whereas NhaB and H-NS are not (see Table 8.6).
These studies were with NaOH as inducer, but KOH was almost as effective in
inducing alkali habituation. The alkali sensor above is an ESC. In contrast, Padan
et al., (1999) consider NhaA an alkali sensor because its activity is greatly increased
at alkaline pH.
Heat Tolerance Induced by an Alkaline Shift
In pioneering studies, Humphrey et al. (1991) showed that a shift from neutral to
alkaline pH induces thermotolerance in S. enteritidis PT4. Accordingly, organisms
grown in the alkaline egg-white will be thermo-tolerant and this will allow survival
on cooking. It is essential to examine the regulation of this response, especially its
switching-on. It is now known that alkaline pH functions by activating the thermo-
tolerance-related ESC; exposure of this component to pH 9.0 at 30°C, in the absence
of organisms, converts this ESC to the corresponding EIC, with concomitant ther-
motolerance induction. Activation can also occur at other alkaline pH values.
RESPONSES TO HEAT IN FOOD PREPARATION AND COOKING

When considering the survival of contaminating organisms during stresses likely to
be encountered in food production, preparation or cooking, heat is likely to be the
major stress challenge, and numerous studies have been made on the influence of
prior exposure conditions on survival during heating, and on the effects that a wide
range of components present during heating, have on survival (Doyle and Mazzotta,
2000). In contrast, until now, little has been known of how exposure to heat switches-
on processes likely to aid survival.
Clearly, survival of contaminating organisms in partially cooked foods will result
partly from their ability to induce thermotolerance on temperature up-shift, with
tolerant organisms surviving exposure to 50 to 55°C, and some resisting higher
temperatures for short periods. It is important to know how thermotolerance is
induced, and what biochemical changes occur as a result of induction, which make
the organisms thermotolerant, especially as knowledge of the process could make
it possible to subvert it. There are two major matters to be considered with respect
to response of organisms to increased temperature: 1) the role of responses in
allowing organisms to grow and multiply better at high temperatures, and 2) the role
of responses in preventing killing by potentially lethal temperatures, i.e., their role
in thermotolerance.

It is essential first to establish how increased temperature is sensed in enterobac-
teria. Two approaches have been taken to establishing the sensor. First, the possible
ways in which organisms change their major metabolic pathways at higher temper-
atures have been considered, as well as the way in which a sensor could function to
ensure that a switch-over could be rapidly achieved. This has led to the proposal that
the ribosome senses temperature. In contrast, when considering damage by heat and
how this can be avoided, the approach has been to consider as likely sensors com-
ponents damaged by heat; if these are altered in some way by the damage so as to
lead to a response, this could give early warning of potential lethality, and allow
organisms to prepare to avoid death. The major components damaged by heat appear
to be membranes, DNA and ribosomes, so proposals on heat sensing for thermotol-
erance induction have usually suggested involvement of one of these components.
On the Nature of the Thermal Sensor
Five components have been proposed to function as the thermal sensor.

Ribosome
Van Bogelen and Neidhardt (1990) suggested that the ribosome senses temperature
and switches on the heat-shock response. This proposal was based on the finding
that so-called H (heat-shock response) antibiotics (e.g., kanamycin), which lead to
empty ribosomal A sites, induce a heat-shock response. They argued that at high
temperatures, there may initially be a fall in charged t-RNA, leading to some or all
A-sites being empty; this will indirectly induce the heat-shock response and
H antibiotics mimic this. It was proposed that the so-called H ribosomal state may
induce the response by causing accumulation of (p)ppGpp, which is known to be
associated with effects of H antibiotics and with high temperatures. Since organisms
must change protein synthesis rates after temperature shift-up, the ribosome would
appear to be an ideal thermal sensor.
The above is rather indirect evidence, so we must ask whether the ribosome fits
the criteria for stress sensors and whether the agent resulting from interaction of
stress and proposed sensor ((p)ppGpp) fits the criteria for a heat-shock induction
component. First, obviously ribosomes are present in both heat-stressed and
unstressed cells. Second, sudden temperature rises do affect the ribosome but do
they lead to empty A-sites? Whether they do or not, there is indeed a rise in (p)ppGpp
levels as temperature increases (Pao and Dyess, 1981). Finally, does (p)ppGpp induce
the heat-shock response?
As to other criteria where the proposed sensor is an essential component, one
cannot achieve inhibitory conditions, or mutations which stop sensor synthesis or
conditions which remove or destroy it. It is, however, possible to inhibit chemically
(or mutationally block) synthesis of (p)ppGpp; does this stop heat-shock response
induction on thermal stress and does addition of (p)ppGpp at low temperatures
induce? Additionally, where several stresses produce a response, each stress should
interact with the proposed sensor to produce the same induction component and so
one must ask whether ethanol exposures, alkaline shifts and amino acid analogue
exposures (all these induce the response) lead to a fall in charged t-RNA levels, to

empty A-sites and high (p)ppGpp levels, and whether lesions that stop (p)ppGpp
synthesis stop response-inducing effects of ethanol, and the other stresses mentioned
above. Results relating to some of the above questions support functioning of
(p)ppGpp, whereas others do not.
Membrane Components
Secondly, it is possible that a membrane component acts as thermal sensor. I will
mention two findings here. The first is the finding that thermal sensing for thermo-
taxis involves activation of a CM component (Nishiyama et al., 1999); although this
response does not involve altered transcription and induction of a new pathway, it
does involve thermal activation of a CM component, and similar interactions could
have evolved to give response induction.
The second is the finding that induction/repression of the OmpF/OmpC proteins,
in response to altered osmolality, involves sensing of stress by a CM component,
namely EnvZ (activation of this protein leading to altered auto-phosphorylation) and
these changes affect the phosphorylation and activity of the DNA-binding protein
OmpR. Clearly, therefore, a CM component can act as a stress sensor and switch-
on stress responses.
The Medium
Recently, there has been evidence suggesting that thermal sensing takes place in the
medium. There would appear to be no advantage in having a sensor in the medium
because if temperature increases, the inside of the cell and the outside will be at the
same temperature. In fact, however, a thermal ESC can allow early warning of a likely
heat shock. This is because an EIC arising from an ESC can diffuse to regions not yet
facing raised temperature, and unstressed organisms can be given early warning and
be prepared to face thermal damage; i.e., there would be “cross-talk” involving EICs
leading to intercellular communication between heat-stressed and unstressed organisms.
Several evidences now suggest that ECs function in thermal sensing. First, a
thermotolerance-inducing EIC is present in filtrates from cultures grown at 45°C.
This EIC, which induces thermotolerance at 25 or 30°C, is cleaved by protease, but
passes only poorly through dialysis membranes, implying that the EIC is a protein
of ca. 10,000 Da (Rowbury and Goodson, 2001). This EIC is now known to arise
from an ESC, which has similar properties to the EIC, but cannot induce thermo-
tolerance in unstressed cells, unless first activated to the EIC (Rowbury and Goodson,
2001). It is now clearly established that this ESC functions as a biological thermom-
eter, detecting temperature rise and inducing thermotolerance. I believe that activa-
tion of this ESC may induce the heat-shock response also, and propose to examine
synthesis of HSPs at 25 or 30°C in the presence of the corresponding EIC, following
the induction of β-galactosidase and alkaline phosphatase from HSP–LacZ and
HSP–PhoA fusions.
A second finding relates to heat-induced acid tolerance. It is well proven that
on transfer of E. coli or S. enteritidis from 25, 30 or 37°C to 42 or 45°C, acid
tolerance induction occurs (Humphrey et al., 1993; Rowbury, 1997). The basis for
this has now been established. Growth at 37°C produces an acid-sensing ESC.
Although this ESC normally senses and is activated by acid, it can also be activated
to the acid tolerance-inducing EIC at 40, 42, 45, 50 or 55°C, i.e., this ESC is a

thermal sensor as well as an acidity sensor (Rowbury and Goodson, 1999b), ESC
activation leading to formation of an acid tolerance-inducing EIC. The extent of
activation of the ESC increases with increasing temperature at least within the range
37 to 55°C and, accordingly, this component behaves like a thermometer, at least
over a limited temperature range. The temperature for thermal activation of this
sensor depends on the temperature during ESC synthesis; the ESC formed at 25°C
is activated to EIC at 30 or 37°C, whereas that synthesized at 37°C is not; i.e., this
sensing component occurs in more than one form, the form synthesized depending
on the culture conditions. These different forms could be oligomers of the EIC that
arises from them, or the EIC and various forms of the ESC could simply differ from
each other in conformation. A third ESC can act as a thermometer, as the alkali
tolerance ESC also shows gradual activation as the temperature is raised from 37°C
to 50°C.
DNA
Some have considered DNA as a likely thermal sensor. Heat damages the DNA and
so, by analogy with RecA/SS DNA as SOS sensor, an SS region (or some other
exposed region) deriving from heat-damaged DNA could act with a cellular com-
ponent in sensing; a similar lesion could also be detected following other stresses,
since many of them damage DNA. An alternative is that damage alters the confor-
mation of the DNA, releasing bound regulatory components and derepressing oper-
ons transcriptionally blocked by them. It is striking that H-NS represses many stress
responses by binding to their operons. If the binding of H-NS to these operons were
particularly weak, damaging stress treatments might lead to general stress response
induction by altering DNA conformation in such a way as to release H-NS only
from operons to which it is poorly bound.
DnaK Chaperone
A very exciting suggestion is that the HSP70 DnaK chaperone acts as a thermal
sensor, detecting either rises in temperature or changes in protein structure which
occur at high temperature. For example, McCarty and Walker (1991) suggested that
DnaK can rather precisely sense increases in temperature in the range 30 to 53°C.
This proposal was based on the finding that both the ATPase activity of DnaK and
its autophosphorylation at threo-199 are massively enhanced in the temperature range
responsible for induction of the heat shock response and thermotolerance. At low pH
values, autophosphorylation was enhanced by several hundred-fold and ATPase activ-
ity by nearly two orders of magnitude. DnaK needs rapid ATPase functioning to
efficiently repair damaged proteins and co-chaperones function to bring this about;
the enhancement of DnaK activities at high temperatures could clearly also play a
role. These authors also suggest that, since binding of inactive DnaK to σ32 reduces
both its stability and activity, that activation (of the ATPase activity) of DnaK might
lead to its release from σ32 and enhanced stability and activity of this σ-factor, leading
to the heat-shock response. On this basis, DnaK would be a biological thermometer.
It is also possible that DnaK might act indirectly as a thermometer by detecting
thermal changes by their results, i.e., by detecting damage to proteins.

Activation of the Thermotolerance ESC by Other Stresses
As indicated above, thermotolerance is generally induced by temperature shifts, and

induction follows the activation of a thermotolerance ESC to the corresponding EIC,
which switches on the response. There are, however, several cross-tolerance
responses, which switch on thermotolerance, and induction by alkalinity (Humphrey
et al., 1991) or acidity (Leyer and Johnson, 1993) is well-known. It has now been
shown, in my laboratory, that UV irradiation and exposure to metal ions, such as
Cu2+, also induce thermotolerance, and that for all four stresses (exposures to acid,
alkali, UV irradiation and Cu2+), induction follows activation of the thermotolerance
ESC (by the stress) to the EIC.
On the Factors Governing the Heat-Shock Response and Its Possible

Relation to Thermotolerance
The initial aim is to give an account of how the heat-shock response is switched on
in E. coli. This response is not only switched on by thermal stress, but also appears
after exposure of bacteria to ethanol, amino acid analogues and alkali. Some believe
that this response does not relate to inducible thermotolerance, i.e., to responses that
lead to survival in the face of potentially lethal thermal stress. This seems highly
unlikely and I propose to outline how the major heat-shock response is induced,
TABLE 8.7
Induction of Stress Responses and Chaperone Synthesis
Inducible Stress Response Stimulus Chaperones Synthesised
Thermotolerance Temperature up-shock1 DnaJ, DnaK, GrpE, GroEL, GroES,

HtpG
Heat-shock response Temperature up-shock1 DnaJ, DnaK, GrpE, GroEL, GroES,
HtpG
Cold-shock response Temperature down-shock Hsc66, HscB, CsdA, CspA2
Acid shock response Low pH, generally pH 2.0-5.5 GroEL, DnaK, HtpG, HtpM
Weak acid shock Weak acid at pH 6.5 or 8.0 GroEL, GroES, DnaK, GrpE, HtpG
Alkali shock response High pH (pH 8.5-9.0) GroEL, DnaK
Osmotic shock response High osmotic pressure GroEL, GroES, DnaK
Starvation response Starvation for carbon compounds3 GroEL, GroES, GrpE, DnaK, HtpG
Oxidative stress response H2O2, O2– or AHP GroEL, GroES, DnaK
SOS response or other DNA damage, e.g., by irradiation GroEL, DnaK, GroES?
response to DNA damage
1 The heat-shock response involves the de novo synthesis of several novel proteins and the increased
synthesis of other proteins, which is induced by mild heat shocks (e.g., shift to 40°C) and by exposure to
agents such as ethanol and nalidixic acid. The organisms sometimes, but not always, become thermotolerant.
To induce thermotolerance, exposure is to higher temperatures (generally 45 to 50°C) and a novel second
heat-shock response induced by σE generally occurs, in addition to the classical heat-shock response.
2 CspA and CsdA may act as RNA chaperones.
3 Other forms of starvation response can occur.

with the production of a large group of heat-shock proteins (HSPs). The HSPs are
components present at low levels in unstressed cells; amounts increase very
rapidly, but often transiently, after a temperature shift up, maximum rates of synthesis
being achieved within 5 min. Several HSPs are molecular chaperones (Table 8.7),
which are also induced by other stresses (Table 8.8). Chaperones mediate proper
assembly/folding of proteins during synthesis, as well as protecting proteins from
damage and aiding repair during heat-shock. Where heat-shock temperature is poten-
tially lethal, nearly all protein synthesis ceases on transfer, virtually all proteins
synthesized after such transfer being HSPs. This behavior at lethal temperatures
suggests HSP involvement in thermotolerance.
The major response after a temperature shift up involves the initial activity of
RpoH. This gene product is present in very low amounts in non heat-stressing
conditions but on transfer, e.g., from 30 to 42°C, amounts increase within a few
seconds and rise to a maximum within 5 min, followed by a rather sharp fall, i.e.,
the rise in RpoH is transient. The amount of RpoH rises for two reasons. The major
reason lies in its stability/instability properties. Under non-stressed conditions, this
component has a half-life of about 60 sec, whereas it becomes stabilized on transfer
to 42°C, and this allows a rapid rise in its level. In addition, rate of synthesis of
RpoH increases at 42°C and this also relates to a stabilization process. In this case,
m-RNA for RpoH is stabilized at 42°C, allowing increasing RpoH formation, i.e.,
increased synthesis depends on increased m-RNA translation. It is RpoH (σ32) that
induces synthesis of the major HSPs since a nonsense mutation in rpoH leads to
markedly reduced induction of HSPs at, e.g., 42°C, and the strain with this mutation
fails to grow at >20°C. This σ-factor binds to the RNA polymerase core enzyme
and is needed for recognition of heat shock promoters, i.e., the RNA polymerase
only binds to these if associated with σ32 (Grossman et al., 1984). HSPs induced by
RpoH include the DnaJ, DnaK, GrpE, GroEL, GroES, Lon, ClpB, ClpP and HtpG
gene products. Many of these are chaperones, e.g., Dna J, Dna K, GrpE, GroEL,
GroES and HtpG, and are needed for numerous processes because of their role in
protein processing and folding, and in protection against heat inactivation and in
repair. A few minutes after the rise in the concentration of RpoH has occurred, its
level falls off again. This appears to be due to the functioning of chaperones in
regulation of RpoH stability and rpoH m-RNA stability.
As indicated above, some researchers believe that the heat-shock response
induced by σ32 is not involved in appearance of inducible thermotolerance. This
proposal has been made because, if σ32 induction occurs at low temperatures, ther-
motolerance does not appear. One suggestion is that there is a second heat-shock
response, induced at elevated temperatures, which is responsible for the thermotol-
erance response. There is the possibility that this response is σE-induced. This sigma
factor induces transcription from the rpoH P3 promoter and also leads to transcrip-
tion of other genes; this sigma factor appears to be particularly functional at lethal
temperatures, in accord with its being involved in survival at such temperatures,
rather than just being needed for accommodation.
My view is that both regulons (i.e., that induced by σ32 and that induced by σE)
are needed for induction of thermotolerance and resistance to potentially lethal
temperatures, such as may occur during food production and preparation procedures

TABLE 8.8
Components Involved in Induction of Responses, Especially Tolerance Responses
to Heat Stress, Cold Stress, and to Other Selected Stresses and Components
Involved in the Biochemistry of Stress Tolerance
Are Regulatory
Components Enzymes and
Stabilized or Other Proteins
Regulatory Destabilized, and Involved in
Components Mechanism of Biochemistry of
Response Sensor Involved These Changes Stress Tolerance
Thermotolerance1 ESC EIC, RpoE, RpoH destabilized by Chaperones? IbpA,

RpoH? GrpE? GroEL? GroES? IbpB, OtsA,B2
Heat-shock Ribosome? ESC? RpoH, EIC? GrpE, GroEL, GroES Chaperones, Lon,
response DNA, DnaK destabilize RpoH Clp
Cold-shock Ribosome? ESC? CspA, H-NS, 1. m-RNAs are stabilized H-NS, GyrA, CspA,
response Membrane RecA* 2. m-RNA translation is CsdA, RecA*
enhanced by DB
enhancer
Osmotic tolerance KdpD, ProP, ESC?3 KdpE, cAMP, Stress may stabilize RpoS Kdp-ATPase, ProP,
H-NS, RpoS by stopping proteolytic ProU, OtsA,B,
cleavage4 TreA
Salt shock NhaA, NhaR, ESC?5 NhaR, H-NS NhaR plus Na+ may NhaA
responses stabilize NhaA m-RNA6
Irradiation RecA, ESC RecA, LexA LexA becomes stabilized RecA, UvrA, B, C
tolerance due to reversible loss of
protease activity
Starvation stress 1. ESC? RpoS, H-NS, Stress stabilizes RpoS by ClpB, OtsAB, Dps,
for carbon protease deactivation Catalase HPII
2. ESC? cAMP-CAP
AlgAC
1See Table 8.7 footnote for details of thermotolerance induction.
2OtsA and B function in stationary-phase thermotolerance, and may be involved in heat-induced thermotol-
erance, although this has been discounted by some.
3Also see Table 8.9.
4RpoS is needed, e.g., for osmotic induction of OtsA and B; there is evidence that some stresses prevent
proteolytic breakdown of RpoS.

5An EIC functions in salt-induced acid sensitivity (see Table 8.3) and, therefore, an ESC probably acts as
NaCl sensor.
6Suggested as an unlikely possibility by Dover, N. et al., J. Bacteriol., 178, 6508, 1996; for pex mutants and
for cst mutants.

* RecA may have a regulatory function, as well as simply functioning in repair.
and in cooking at low temperatures. This possibility is of particular interest, as is

the possibility that different thermal sensors function to switch on the two heat-
shock responses. In addition, however, there is strong evidence for the involvement
of trehalose in some thermotolerance responses and, accordingly, the heat-induced

thermotolerance response could be governed by the sensor and regulatory compo-
nents which control trehalose accumulation. However, there have been claims that
trehalose is not needed for heat-induced thermotolerance. My view is that, since the
heat-induced thermotolerance response will need repair processes as well as protec-
tion processes (trehalose may, if it functions at all, play a role in the latter), there
is likely to be involvement of σ32 and σE-governed processes in this response.
The role of H-NS in thermotolerance induction ought to be mentioned again
here. There is no doubt that a lesion in hns leads to derepression of the thermotol-
erance response, i.e., to thermotolerance in organisms grown at low temperatures.
It seems highly likely that there are groups of genes needed for thermotolerance and
that some of these are repressed by H-NS binding. Presumably, H-NS is ejected
from these regions at high temperatures, leading to thermotolerance induction, and
absence of H-NS in hns mutants gives the same phenotype at low temperature.
Mutants in himA show a similar phenotype, suggesting that IHF plays a role with
H-NS in repressing the thermotolerance response.
Sigma 32 is not essential for growth at low temperatures, since the rpoH deletion
mutant can grow at below 20°C but not above. Some products controlled by RpoH
are needed for growth at below 20°C, e.g., some chaperones, but there is presumably
some transcription at their promoters, catalyzed by RNA polymerase activated by
another sigma factor.
On the Involvement of Chaperones in Regulation
Chaperones synthesized in the presence of RpoH mainly function in normal protein

assembly, folding and processing and in repair (Tables 8.7 and 8.8). Several, however,
also play a major part in governing synthesis and stability of RpoH and, accordingly,
are important in regulating the heat-shock response, including their own synthesis.
Thus, DnaJ, DnaK and GrpE gene products destabilize RpoH by enhancing activity
of proteases which degrade it, e.g., mutations in these chaperone-encoding genes
lead to RpoH stabilization and, on a temperature shift up, HSP synthesis for an
extended period. These chaperones also appear to inhibit RpoH activity under some
conditions.
RESPONSES TO COLD
Contaminating organisms in food face three types of cold stress. First, at low
temperature they need to acclimate to cold and there are a group of proteins involved
in acclimatization. Secondly, transfer to low temperatures can involve a sudden drop
in temperature, termed a cold shock. Finally, polluting organisms can also be exposed
to freezing conditions and, for survivors, to thawing during recovery. Substantial
studies have been made of cold shock recently, although the work has rarely involved
consideration of the applied importance of findings, and experimental design has
not generally borne in mind questions related to cold shock in food microbiology.
There is evidence that the responses which aid growth and recovery after exposure
to each of the above three processes are related and, in particular, that failure to

form a regulator, CspB, required for induction of cold-shock proteins, leads to
sensitivity to freezing/thawing in Bacillus (Graumann and Marahiel, 1996).
Sensing of Cold in Bacteria
First, one must ask how low temperature is sensed. One consideration is how
organisms accommodate to reduced temperature; there are changes in protein syn-
thesis with reduced translation, and since it is deficiencies in ribosomal function
that reduce growth rate in the cold (Das and Goldstein, 1968), it has been proposed
that ribosomes detect falling temperature. Studies of antibiotic effects on protein
synthesis show that there are C (cold-shock) antibiotics which induce a cold-shock
response (van Bogelen and Neidhardt, 1990). These agents block the ribosomal A-site,
e.g., one C antibiotic, chloramphenicol, inhibits peptidyl transferase and the accu-
mulating charged t-RNA blocks the A-site. It is proposed that a down-shift leads to
reduced translation and associated blockage of the A-site, which induces the cold-
shock response, whereas making the A-site empty leads to the heat-shock response,
so this ribosomal theory explains responses at high and low temperatures. The
blocking of the A-site in the cold plus C antibiotics leads to a fall in (p)ppGpp.
Evidence for the ribosome as cold sensor is better than for its functioning as heat
sensor. First, the proposed sensor is present under stressed (cold-shock) and
unstressed conditions. Second, interaction of cold with ribosomes leads to a fall in
(p)ppGpp (Pao and Dyess, 1981) and third, a fall in (p)ppGpp (the proposed inducing
condition) switches on cold-induced protein (CIP) synthesis; conversely, a rise in
(p)ppGpp leads to reduced synthesis of CIPs following cold shock (Jones et al.,
1992). In summary, the evidence appears quite good for the ribosome as cold sensor.
A second possibility for a cold sensor, responsible for inducing the cold-shock
response, is a membrane component; there is no evidence so far for this, although
the work of Nishiyama et al. (1999) on thermotaxis shows that a CM protein can
act as a cold sensor.
A third possibility, as melting of DNA becomes a problem at low temperatures,
is that an altered DNA configuration could be involved in sensing cold, or that low
temperatures might lead to regulatory components being dislodged from some areas
of DNA, with associated operon derepression. There is no evidence for this so far.
In view of the involvement of ECs in thermotolerance, it seems likely that such
components could function during temperature down-shifts. Although internal and
external temperatures will be the same when there is a temperature down-shift, to
have an extracellular cold sensor could lead to early warning of stress for unstressed
cells. This is because the EIC, which would arise by cold activation of the proposed
cold-sensing ESC, could diffuse away from the cold region and interact with
unstressed cells before they face cold shock. This could be investigated as follows.
The proposal would be to down-shift E. coli, prepare a cell-free filtrate from the
down-shifted culture, expose organisms to the filtrate at 37°C and examine whether
major cold-induced proteins appear at this temperature. Use could be made of
fusions, of cold-induced genes to lacZ, so that induction by the EIC could be followed
by studying β-galactosidase levels. If evidence were obtained for a cold-shock EIC,
a study could be made of whether this EIC arises in the cold from an ESC formed

at normal temperatures, and whether these ECs are involved in protection from
freezing/thawing.
Proteins Synthesized after Triggering of the Cold-Shock Response

by Sensor Activation
On cold shock to 10°C, growth stops in E. coli and synthesis of most cellular proteins
is abolished. Some time after, synthesis of a group of some 20+ proteins begins,
with one of the earliest appearing being the low MW CspA (Goldstein et al., 1990).
In addition, regulatory proteins are induced, the most significant being H-NS and
GyrA. Cold shock is distinct from heat shock, and not only are HSPs not induced
on a transfer from 37 to 10°C, but their levels also fall substantially at the lower
temperature. This applies, for example, to DnaK and GroE gene products, so there
are tiny concentrations of the heat-shock chaperones present at 10°C. Components
involved in protein folding and damage repair are, however, formed at low temper-
atures, since specific cold-shock chaperones probably occur; e.g., Hsc66 appears to
be a classic Hsp70 homologue, induced at low temperatures in E. coli but not formed
during heat shock (Lelivelt and Kawula, 1995). It is probably a chaperone and a
DnaJ homologue, HscB, also appears. In addition, the CspA and CsdA proteins may
function as RNA chaperones under some conditions.
E. coli, on cold shock, shows very great induction of a group of small acidic
proteins. Of these, CspA is the first and most markedly induced (Goldstein et al.,
1990), forming as much as 10% of protein synthesized at 10°C. There may be no
cold-shock sigma factor and for some CSPs, at least, derepression involves stabili-
zation of m-RNA (e.g., for CspA) and increased translation of m-RNA (Etchegaray
and Inouye, 1999) due to presence of a downstream box enhancer (for CspA and B).
Strikingly, neither chloramphenicol nor kanamycin appreciably inhibits the syn-
thesis of CspA, CspB or CspG at low temperatures and the only proteins synthesized
in the presence of these antibiotics are the above three cold-shock proteins (Etche-
garay and Inouye, 1999). All three are of very low molecular weight (70, 71 and 70
amino acids, respectively, for CspA, B and G), and it is possible that ribosomal
translation of m-RNA for such small proteins is less affected by inhibitory antibiotics.
The alternative is that some stress-related proteins are synthesized by a slightly
modified synthetic pathway, since the synthesis process for the ESC for acid toler-
ance induction is also refractory to antibiotics that normally block ribosomal function
(Rowbury and Goodson, 1999a).
RESPONSES INDUCED BY CHANGES IN OSMOTIC PRESSURE OF MEDIA

Whereas there may be multiple thermal sensors, there definitely are multiple osmo-
sensors. As illustration, three responses will be considered. First, E. coli grown at
low osmolality produces high levels of OmpF porin and low levels of OmpC, whereas
growth in richer media leads to the reverse. These Omp proteins function in the OM
to ensure passage across the OM of low (up to ca. 600 Da) molecular weight
uncharged or positively charged hydrophilic molecules.

It has been proposed that shift from high OmpF/low OmpC to low OmpF/high OmpC as the osmotic pressure is raised has evolved
to ensure that the porin with the smaller pore size is predominant in rich medium, e.g., in the body; growth would be supported well by
OmpC because of the high nutrient content, but inhibitory agents would permeate less well across the OM because of the smaller pore
size of OmpC porin. In contrast, in very poor media, e.g., in natural waters, where osmolality is low, derepression of OmpF would ensure
that the poor nutrient supply is used most effectively. One must note that the change from high OmpF/low OmpC to low OmpF/high
OmpC occurs at a low osmotic pressure compared to other responses considered and that the OmpF/OmpC changes have not evolved to
protect from very high osmotic pressure, as is the case for the other responses (Table 8.9). Two systems that have evolved for such a
purpose are that involving the ProP transporter, which takes up proline to protect cells from osmotic damage, and that using the Kdp
products to take up K+ for the same purpose. The ProU system is also used for such protection; it involves the functioning of ProV, W,
X to import, for example, glycine betaine (see Table 8.9).
Do Both Intracellular and Extracellular Osmosensors Occur?
One osmotically controlled system is porin regulation. The intracellular sensor is well established; mutants in the ompB group are aberrant
in control and one of the ompB genes, namely the envZ gene, controls synthesis of EnvZ gene product which is an integral CM protein.
It has been shown in vivo that raising osmotic pressure leads to phosphorylation of EnvZ. The proposal is that EnvZ is the osmosensor
and on sensing a rise in such pressure, the protein changes in conformation, leading to auto-phosphorylation. Sensor activation sends a
signal to shift porin synthesis, by phosphorylating the gene product of the other OmpB gene, OmpR. The striking fact is that both the
sensor and the component altered by the activated sensor are intracellular, by contrast with extracellular stress sensors, that on activation
produce extracellular EICs.
Increased osmolality also leads to autophosphorylation of the sensor kinase KdpD, which then phosphorylates KdpE. This phospho-
rylated component then interacts with the kdp promoter, leading to increased transcription (Wood, 1999) and induction of the kdpFABCDE
operon. The KdpA, B, C and F components form the Kdp-ATPase which catalyses K+ uptake. The activity of this complex is also activated

TX69124 ch08(280) frame55 Page 280 Thursday, October 31, 2002 2:01 PM
TABLE 8.9
Sensing, Induction and Biochemistry of Osmotic Responses
Osmotic Effect of High OP
Shift on Activity
Inducing Function of Gene Products Induced of Induced
Physiological Response Response Response Osmosensor Regulator by Sensor Activation Components
1. Alteration to porin levels Slight upshift To choose most EnvZ OmpR OmpC (OmpF repressed) ?
suitable porin
2. Induced K+ uptake Large upshift Accommodation to KdpD KdpE KdpA, B, C, D, E, F Activation of
high OP Kdp-ATPase
3. Induced glycine-betaine uptake Large upshift Accommodation to Glutamate N.E.1 ProV, W, X (i.e., ProU system) Activation of ProU
high OP dehydrogenase*
4. Induced proline uptake Large upshift Accommodation to Glutamate N.E.2 ProP Activation of ProP
high OP dehydrogenase*
OP = osmotic pressure. N.E. = not established.

1 H-NS inhibits proV,W,X transcription by binding downstream of its promoter; K glutamate reverses.
2 CRP-cAMP binds to P1 promoter of proP and stops transcription; K glutamate reverses.
* Glutamate dehydrogenase would be activated by the increased pHi which may occur at high OP. If so, it is acting as an osmosensor, in the sense that the rise in internal
glutamate produced by its increased activity would induce the response by reversing the effect of H-NS (on proV,W,X transcription) or of CRP-cAMP (on proP transcription).
Alternatively, other osmosensors may indirectly lead to proV,W,X or proP induction, e.g., those osmosensors which induce K+ uptake may act indirectly as transcription
of the above operons may need K+.

by high osmotic pressure (see Table 8.9). The factors governing induction of ProP
and ProV, W, X by osmotic shock are not so clear (see Table 8.9). Transcription of
the respective genetic regions is blocked (Xu and Johnson, 1997) at low osmotic
pressure (OP) by CRP-cAMP (proP) and H-NS (proV, W, X); K glutamate reverses
this and, accordingly, osmosensing may be indirect, with components which sense
high OP and respond by raising internal K+ and/or internal glutamate, being indi-
rectly responsible for inducing the above two genetic regions.
Several other proteins have been proposed as osmosensors. Two — ProP and
MscL — should be mentioned. They have been proposed as sensors because high
OP activates ProP and low OP activates MscL.
The above osmosensors are intracellular; extracellular ones have not been looked
for. The rationale for evolution of intracellular osmosensors would be that they must
detect differences between external and internal OP, and one would expect intrac-
ellular sensors to do this. However, it is likely that extracellular sensors would have
evolved to give early warning of rising solute level in the medium with some
responses being switched on, not by altered difference between internal and external
OP, but by the sensing of a change in the concentration of extracellular solutes. Such
sensing could alter a solute concentration-sensing ESC to an EIC and induce a
response. This would ensure earlier induction than with a sensor which detects an
internal versus external change. In addition, if EIC produced by a rise in external
solute concentration were to diffuse away to unstressed cells, this would give early
warning. Tests will be made for such an ESC, using exposure to increased solute

levels, such as induce OmpF; filtrates from such cultures and those from cultures
not exposed to increased solute concentrations, but then exposed to such stress, will
be compared to filtrates from unstressed cultures with respect to ability to induce
OmpF-LacZ in unstressed ompF-lacZ+ strains.
RESPONSES TO SALT STRESS

Two responses will be considered here; inducible resistance to salt switched on by
high Na+ and acid sensitization switched on by high salt.
Sensing of Salt Stress
There may be sensing of salt by an intracellular sensor, but it is also likely that an
ESC functions to detect high levels of salt, since acid sensitization by salt depends
on functioning of an EIC as inducer of the response (Rowbury, 1999), and all EICs
studied in detail so far arise from ESCs, i.e., for all such responses examined, an
ESC/EIC pair functions, and this is likely to be so for salt stress.
Other enzymes, CM components and regulatory components are also induced
by salt; for example, the NhaA antiporter is induced at high Na+ concentrations. It
is proposed to examine whether ESCs and EICs play any role, e.g., by looking for
NhaA-LacZ induction at low salt concentrations, by medium filtrates from cultures
exposed to high Na+ or to filtrates from low Na+ cultures exposed to high Na+,
followed by dialysis to remove Na+. If ESCs and EICs play any role, suitable filtrates
should induce NhaA-LacZ at low salt levels.
Also, NhaR senses Na+ intracellularly, high salt levels leading to induction of
NhaA, induction being enhanced by the changes in NhaR binding to nhaA DNA
caused by rise in Na+ (Padan et al., 1999).
Regulation of Salt-Induced Responses
Tolerance of high [NaCl] involves induction and activation of NhaA. This component
is an integral CM protein which functions as an Na+/H+ antiporter with stoichiometry
of 2H+/Na+. This component is the major protein determining NaCl tolerance and
is induced by Na+; studies of NhaA-LacZ synthesis show that intracellular levels of
Na+ are the signal for NhaA synthesis and that, at specific concentrations of Na+,
alkaline pH enhances induction (Dover et al., 1996; Rowbury, 1997). NhaR is an
activator required for NhaA synthesis and nhaR deletions are Na+-sensitive because
of the greatly reduced levels of NhaA (Rahav-Manor et al., 1992). NhaR binds
directly to the nhaA gene and Na+ specifically affects the interaction of NhaR with
base –60 of nhaA (Padan et al., 1999). NhaR binds Na+ and such binding causes a
conformational change which alters the footprint of NhaR on the DNA, altering
attachment of NhaR to –60. Such altered binding is pH-dependent, occurring most
favorably at alkaline pH.
Salt induces acid sensitivity and this process is independent of both the NhaA and
NhaR gene products (Rowbury, 1997). As with so many stress responses, ECs are
required. As stated above, an EIC has been implicated in the response but there is no
information on possible involvement of an Na+-sensing ESC, although its involvement

is likely. Studies have been made of which regulatory components are needed for
induction of this response and results (see Table 8.3), show that H-NS, RelA and
cAMP are needed for induction (Rowbury, 1997). It had earlier been established that
acid sensitivity depended on the level of the OM pore protein PhoE (Rowbury and
Goodson, 1993) and it appears that it is increased PhoE synthesis which allows salt-
induced sensitization (see Table 8.5). As stated above, the OM impedes protons from
entering the periplasm and lesions in OM components (Bielecki et al., 1982) or
induction of certain OMPs leads to easier permeation of H+ and acid sensitivity.
As stated above, there is evidence that the PhoE pore is involved directly or
indirectly in passage of protons from the outside across the OM. Salt induces both
PhoE-LacZ and PhoE-PhoA and lesions or agents which block sensitization by salt
usually prevent synthesis of PhoE-LacZ (Lazim et al., 1996), e.g., glucose stops
sensitization by salt and stops induction of PhoE-LacZ while cAMP which reverses
effects of glucose on sensitization induction also allows PhoE-LacZ synthesis in the
presence of glucose.
REGULATION OF RESPONSES TO IRRADIATION
Irradiation damages the DNA, and is lethal at appropriate levels. It is, therefore,
important to consider both regulation of responses to irradiation and the biochemistry
of inducible irradiation tolerance. Here, the regulation of the SOS response will be
considered.
Sensing of Irradiation Stress
Although regulation of the SOS response has been studied in great detail, sensing
is still not fully solved. It is generally considered that the RecA gene product, or a
component associated with it, functions as the DNA damage sensor; DNA damage
switches on expression of numerous SOS genes, because RecA, on activation by
damage, gains protease activity. This activity destroys LexA and allows transcription
for SOS genes to begin. Accordingly, the idea is that RecA interacts directly or
indirectly with a stimulus produced by DNA damage, and this interaction unmasks
the protease activity which allows the SOS response to be derepressed (Walker,
1984).
The finding, in early studies, that oligonucleotides induced ϕ80 (Irbe et al., 1981)
suggested that these nucleotides might arise from a damaged region of DNA and
seemed ideal as an induction stimulus. This now seems unlikely, since some mutants
that show little DNA degradation show a strong SOS response. The likelihood now
is that SS DNA regions arise either directly following DNA damage, e.g., by nalidixic
acid (the damaged regions being unwound to give SS DNA), or result from DNA
replication following DNA damage (Sassanfar and Roberts, 1990). Such replication
leaves gaps and SS regions arise from these. In accord with this idea, SS DNA plus
NTP leads to in vitro activation of RecA gene product. Interestingly, there may be
a second intracellular sensor of DNA damage, since some processes are switched
on by DNA damage in recA mutants.

It could be argued that ESCs would not be involved in sensing, because internal
and external sensors would be equally affected by irradiation, with an internal sensor
being as efficient as an ESC. This, however, overlooks the fact that EICs could
diffuse away to an area not exposed to irradiation, and prepare cells there to resist,
i.e., could give early warning of impending irradiation. One would expect such an
ESC to be converted to an irradiation tolerance-inducing EIC. Such ESCs and EICs
had not been looked for, but it seemed likely that they occurred, since we had shown
that an acidity-sensing ESC is activated by irradiation (Rowbury and Goodson,
1999b). The same applies to a thermotolerance ESC; its irradiation converts it to
the thermotolerance EIC, and to an alkali tolerance ESC, which is activated to the
alkali tolerance EIC, by irradiation at pH 7.0. I have now taken filtrates from
organisms grown with mild irradiation, from those grown without irradiation and
the latter filtrates irradiated in the absence of organisms and have shown that the
activated filtrates on incubation with unirradiated organisms induce UV tolerance,
i.e., a UV tolerance ESC/EIC pair occurs.
Stages in the Switching-On of Responses to Irradiation Following

Sensor Activation
The switching-on of the SOS response is rather well understood apart from sensing.
Once the protease activity of the RecA gene product has been unmasked by inter-
action with SS DNA and NTP, RecA cleaves LexA. This protein normally (i.e., in
the absence of DNA damage) binds to the so-called SOS box of numerous (at
least 25) genes, preventing their expression; there is evidence that LexA dimerizes
onto the operator region of these genes to block expression, with the strength of
binding varying from gene to gene. After RecA activation, initially, when the LexA
gene product level begins to fall appreciably, several genes that have weak binding
of LexA become derepressed. Later, as LexA level falls further, other genes with
stronger LexA binding are derepressed.
STARVATION STRESS
Although other types of starvation occur (e.g., for N or P), this account will be of
carbon starvation. Matin and his group (Jenkins et al., 1988, 1990; Matin, 1991)
described two classes of genes governing the response to starvation stress. The first
class are the cst genes, which are controlled by cAMP-CAP; in these studies, 19 cst
loci were revealed. None of the lesions alter stress tolerance, e.g., there is no loss
of stress tolerance in cya mutants. These genes are involved in aiding organisms to
escape from starvation by inducing pathways that can degrade novel carbon com-
pounds. It would be expected that these genes would show induction by derepression
of cAMP synthesis due to carbon starvation, this being sensed as follows: assuming
that carbon starvation is due to a fall in glucose level, this leads to a rise in the level
of Protein IIIGlc-phos. Increased phosphorylation of this CM component leads to
greater adenyl cyclase activity, increased cAMP synthesis, and cst induction. Accord-
ingly, the fall in carbon, i.e., glucose is sensed by the CM, so there is intracellular
sensing.

The other genes defined by Matin were pex genes, governing tolerance to carbon
starvation and cross tolerances. Matin showed that starvation induced not only
starvation tolerance, but also thermotolerance, osmotic tolerance, oxidative tolerance
and acid tolerance (salt tolerance also appears). Matin revealed that the general
stationary-phase response showed different characteristics than specific tolerance
responses, e.g., H2O2 tolerance induced by H2O2 is regulated by OxyR whereas the
stationary-phase H2O2 tolerance is governed by RpoS.
Many protection/repair systems are controlled by RpoS, although some are not.
Also, during starvation or in stationary phase, organisms become spherical and show
altered RNA polymerase structure, components are stored for protection or as
reserves, and changes in DNA conformation occur (Hengge-Aronis, 1993). RpoS is
the major component controlling starvation responses, with relation to tolerance (but
see “growth phase and stress tolerance” for role of chromosome termination in
tolerance responses), and it functions as a σ-factor, allowing RNA polymerase to
bring about increased transcription of a huge number of genes. Stress enhances the
translation of rpoS-mRNA and increases RpoS stability (see Table 8.8), stopping its
cleavage by protease. The major interest here, namely, how carbon starvation leads
to RpoS induction, is not well understood; the nature of neither the precise stimulus
nor of the sensor is clear.
It is assumed that both cst genes and pex genes are switched on by mechanisms
involving intracellular sensors. However, since cAMP can function via extracellular
components (Rowbury, 1999), it is important that the possibility of extracellular
sensors functioning to detect starvation should be considered.
During growth into stationary phase, the levels of RpoS increase, leading to the
induction of numerous proteins. As indicated above, this increase in RpoS levels
results partly from increased m-RNA translation and partly from increased stability
of this sigma factor. This stabilization occurs due to changes in the ClpXP protease.
Aside from degrading abnormal or damaged proteins, this complex specifically
targets RpoS (and some other proteins) and destroys it. In log phase, this sigma
factor is actively cleaved by the ClpXP complex and, accordingly, shows a very
short half-life. This is because, in log phase, the complete clpP, clpX operon is
transcribed and gives so-called long transcripts, which are translated into complete
ClpXP complexes (Li et al., 2000), which have protease activity. On growth into
stationary phase, however, there is premature termination of transcription, so that
most of the transcripts (short transcripts) arise only from the proximal gene of the
operon, clpP. These transcripts cannot be translated into complete ClpXP (i.e., active
protease) complexes and so degradation of RpoS is greatly reduced in stationary
phase, and the half-life of this σ-factor increases markedly. As the organisms leave
the stationary phase, the longer transcript begins to form, the full ClpXP arises on
its translation, and this enzyme functions proteolytically to cleave both RpoS and
many of the numerous proteins which have been induced by RpoS in stationary
phase (Li et al., 2000).

STRESS TOLERANCE INDUCTION BY KILLED CULTURES
Polluting and contaminating bacteria often face naturally occurring or man-produced
conditions which are lethal to them. This can occur in the natural environment for
organisms that go on to enter foods. It would be expected that killing cultures would
prevent them from growing and further altering the properties of the environment;
also that it would prevent them from going on to cause disease. They would also
not be expected to influence behavior of any organisms which enter the environment
later. Recent findings have established, however, that dead cultures killed by several
means can confer stress tolerance legacies on living organisms appearing in the same
environment later (Rowbury, 2000). As indicated above, organisms in natural waters
or in other environmental situations can eventually enter foods and food materials
and, therefore, the likely effects of killed organisms in a range of locations, including
the natural environment, will be considered here.
ACID TOLERANCE INDUCTION BY KILLED CULTURES

Appropriate cultures killed by many means confer an acid tolerance legacy on other
organisms entering their environment. This applies to cultures killed by acidity,
alkalinity, heat, irradiation, metal ions and antibiotics. For example, exposure of
appropriate cultures to levels of alkali which kill more than 99.9% of organisms
leads to preparations which can induce acid tolerance in living cultures (Table 8.10).
Similarly, cultures killed by heat have essentially no living organisms (<0.001%)
but they induce acid tolerance in living cultures. The highest percentage survival is
for cultures killed by novobiocin, where as many as 2% survive the lethal agent.
This survival rate is too little to explain acid tolerance induced in the living culture,
i.e., acid tolerance is not due to survivors from the antibiotic treatment. Also, use
of genetically marked pairs of strains established that indeed tolerant organisms
arose by induction of tolerance, by the killed culture, in the organisms of the added
living culture; the acid-tolerant organisms are not survivors of the killing process.
The killed preparations do not act by slowing the growth-rate of the living cultures,
neither do they lower the pH of the mixture. Properties of the active agents in the
killed cultures (protease sensitivity and non-dialysability) suggest that it is EICs in
the killed cultures which induce acid tolerance in the living organisms. Appropriate
cultures killed by other means — by exposure to irradiation, to metal ions and to
acidity itself — also induce acid tolerance (Rowbury, 2000).
Other Stress Responses Induced by Killed Cultures
It seems highly likely that numerous other stress responses will be induced by killed
cultures. So far, it has been established that alkali tolerance can be induced by
appropriate killed cultures and alkali sensitivity by others. More recently, killed
cultures have been shown to confer thermotolerance also (Rowbury and Goodson,
2001). It is essential that ability of killed cultures to induce tolerance responses to
irradiation, starvation, oxidative components and metal ions be established.

TABLE 8.10
Acid Tolerance Induction by Cultures Killed by Alkali, Heat, Acidity
and Novobiocin
Culture Induced % Survival ( ± SEM) after
to Acid Tolerance Acid Challenge for Culture
Filtrate or Killed Culture by Added Filtrate Incubated with or without
Used to Induce Acid Tolerance or Killed Culture Filtrate or Killed Culture
None pH 7.0-grown 0.6 ± 0.1

None pH 5.0-grown 45.7 ± 3.1
Filtrate from pH 7.0-grown culture pH 7.0-grown 0.93 ± 0.07
Filtrate from pH 5.0-grown culture pH 7.0-grown 19.0 ± 1.96
Alkali-killed pH 7.0-grown culture pH 7.0-grown 2.5 ± 0.22
Alkali-killed pH 7.0 culture → pH 5.0 pH 7.0-grown 11.9 ± 0.7
Alkali-killed pH 5.0-grown culture pH 7.0-grown 11.3 ± 1.8
Heat-killed pH 7.0-grown culture pH 7.0-grown 6.9 ± 1.1
Heat-killed pH 7.0-grown culture → pH 5.0 pH 7.0-grown 42.0 ± 2.0
Heat-killed pH 5.0-grown culture pH 7.0-grown 11.15 ± 0.6
Acid-killed pH 7.0-grown culture pH 7.0-grown 4.9 ± 0.23
Acid-killed pH 7.0 culture → pH 5.0 pH 7.0-grown 16.8 ± 0.7
Acid-killed pH 5.0-grown culture pH 7.0-grown 16.8 ± 1.1
Novobiocin-killed pH 5.0-grown culture pH 7.0-grown 31.2 ± 1.4
Organisms of E. coli strain 1829 ColV were grown to log phase at the stated pH and cell-free filtrates
were prepared. Some cultures were killed by alkali (pH 11.0 for 15 min), by heat (70°C, 15 min),
by acid (pH 2.0, 15 min) or by novobiocin (5 µgml-1 for 120 min at pH 5.0). After activation at pH
5.0 (if required) and neutralization, the filtrates or killed cultures were incubated for 45 min at pH
7.0 with log-phase strain 1829 ColV cultures grown at pH 7.0. After incubation, mixtures were washed
once with broth before acid challenge (pH 3.0, 7 min). Plating for survivors after challenge was on
NA for 20 h at 37°C.
BIOCHEMICAL CHANGES LEADING

TO STRESS TOLERANCE
ROLE OF CHAPERONES IN THE BIOCHEMISTRY OF STRESS TOLERANCE
Chaperones and chaperonins are tiny intracellular biological “work horses” used for
processing proteins under normal and stressed conditions, and function as essential
components in many inducible tolerance responses (see Table 8.7). Under normal
conditions, they function to fold, assemble and process newly synthesized proteins,
aid secretion of proteins through membranes, de-oligomerize proteins and play a
role in degradation of seriously abnormal proteins. On exposure to stress, these
molecules play some role in protection of proteins from damage, but they work
mainly on damaged proteins, converting them back to a native state.
HSP70 chaperones like DnaK are proteins folded to give a structure with a lidded
“pocket” into which polypeptides with non-native exposed hydrophobic regions are
introduced. Entry is allowed by opening and closing of the “lid,” so the structure

can occur in an open state which has ATP bound. In this state, the polypeptide to
be folded or repaired enters the pocket, associates by hydrogen bonding with the
chaperone and the short hydrophobic region is refolded; following refolding, the lid
opens, releasing folded polypeptide and the ADP and inorganic phosphate resulting
from ATP cleavage. Association of non-native polypeptide and DnaK involves very
short hydrophobic (leucine-rich) regions on the polypeptide, spanned by basic
regions. Each entry into the pocket folds only a short region of the polypeptide, and
so there must be rapid cycles, i.e., following release there must be rapid reattachment,
entry into the pocket, folding and release with ATP being bound and cleaved at the
appropriate time. This cycle may then have to be repeated many times for the same
non-native or damaged protein. For this reason, each cycle must be rapid and this
is ensured by the binding of co-chaperones; DnaJ binds both to the substrate and to
DnaK, aiding their interaction, stimulating the ATPase activity and allowing more
rapid functioning. GrpE also enhances DnaK functioning, probably by enhancing
release of ADP and inorganic phosphate. DnaK functioning is increased in another
way also. McCarty and Walker (1991) showed that ATPase of DnaK is greatly
enhanced at high temperatures, allowing more rapid protein repair.
The 60 kDA chaperones are also known as chaperonins (Ellis, 1996). They are
used for the folding of some proteins during synthesis, with the further function of
repairing stress-damaged polypeptides. In bacteria, the GroEL HSP60 forms cylin-
ders with a channel through them. The bacterial situation is complicated, however,
in that these tiny GroE machines also contain the ca. 10 kDA GroES protein (co-
chaperonin) which binds to the GroEL cylinder, forming a lid. It has other functions,
however, in that on GroES and ATP binding, the interior of the channel is profoundly
altered, so that the compartment becomes larger, its hydrophobicity is modified and,
in the open form, the polypeptide can enter for folding or repair.
Small cytoplasmic chaperones also play a role in repair of damaged components,
often after oligomerization to form large complexes. The small HSPs IbpA and IbpB,
for example, are believed to be chaperones, which function to confer resistance to
heat and oxidative stresses (Kitagawa et al., 2000), presumably repairing damage
caused by such challenges.
Chaperones occur not only in the cytoplasm of the Gram-negative bacterium,
but also in the periplasm. Chaperones in this compartment differ from cytoplasmic
ones, especially in being ATP-independent. In this environment, these proteins
function, first, to aid the maturation (e.g., by appropriate folding) of newly synthe-
sized periplasmic and outer membrane proteins and, secondly, to refold abnormal
proteins arising on exposure to stress. Accordingly, a role as a chaperone can be
most readily established by implicating components in normal folding or by showing
that they can stop aggregation following stress damage. On this basis, several proteins
seem to function as chaperones in the periplasm, amongst them the SurA, Skp and
DegP proteins. Skp, which is a small protein, can bring about the maturation of
unfolded OmpF protein, while the larger (448 residue) DegP protein aids the folding
of the periplasmic MalS protein (Speiss et al., 1999). The third protein of this group,
the 46 kDA SurA protein, shows peptidyl prolyl isomerase activity, its functioning
as a chaperone being revealed both by its ability to aid LamB folding and by its
inhibiting thermal aggregation of proteins (Behrens et al., 2001). After considering

mutants with lesions in one or more of these three components, Rizzitello et al.
(2001) proposed that the Skp and DegP proteins function as chaperones in one
pathway, and SurA in another. Strains altered in one pathway can survive, albeit
with poor growth in some cases, whereas those altered in both cannot.
One exciting recent finding in the chaperone field has been that some proteins
or protein complexes can function both as proteases and chaperones. This dual
functioning is of particular relevance when considering abnormal or damaged pro-
teins. If these show major abnormalities or damage, then proteases degrade them, so
that their components can be reused. In contrast, proteins with less damage or fewer
abnormalities can be refolded by chaperones, to reconstitute the active molecule. In
addition, it may be that growth conditions influence which pathway is chosen, namely,
whether a damaged or abnormal protein is repaired or not. Such dual functioning
applies to the cytoplasmic ClpXP and ClpAP complexes. These were originally
believed to be proteases, with the P sub-unit having the enzyme activity, and the
ClpX or A moieties simply determining the specificity of the proteins degraded. It
is now known, however, that the X or A components can, as well as determining
the above specificity, switch the complex to a chaperone mode. Such duality also
applies to periplasmic components, with the DegP protein aiding folding of, for
example, the periplasmic MalS at low temperatures, while degrading abnormal
proteins at high temperatures (Spiess et al., 1999).
BIOCHEMICAL CHANGES IN HABITUATED ORGANISMS MAKING THEM

ACID-TOLERANT
It has not been possible to establish the relevance of most ASPs to the biochemical
changes that lead to acid tolerance but there appear to be three main biochemical
mechanisms by which tolerance appears. First, many tolerant organisms, but not all,
gain novel pH homeostasis. Enteric bacteria growing at neutral external pH (pHo)
maintain their internal pH (pHi) slightly alkaline at ca. 7.8 and at neutral pHo there
is, therefore, a significant ∆pH, and the inside is alkaline. As the medium is acidified
with inorganic acid, pHi is maintained at or near neutrality down to a pHo value of
ca. 4.5; in this situation ∆pH is ca. 2.0 to 2.5; thus a neutral internal milieu is
maintained with pHi only falling ca. 0.8 units compared to a fall of 2.5 units in pHo.
Once pHo falls appreciably below 4.5, however, the non-habituated organism cannot
maintain pHi at close to neutrality, and by the time pHo 3.0 is reached, death ensues
unless tolerance has been induced during challenge. For tolerant organisms, one
process preventing death is the new pH homeostatic system which acts at lower pHo
values and still maintains pHi at near to neutral. Early studies suggested atp involve-
ment in this novel system of homeostasis (Foster and Hall, 1990) but atp involvement
has not been mentioned recently. It has also been claimed that there is a lysine
decarboxylase-dependent homeostasis system, which maintains pHi near neutrality
at acidic pHo.
The second biochemical change that occurs in inducibly acid-tolerant cells is
that, independent of the pH homeostasis changes, tolerant cells gain ability to better
resist acid damage, i.e., there is a damage limitation system induced, in habituated
organisms. It is likely that DNA is the critical macromolecule, with respect to acid

damage. Certainly, DNA is readily damaged by acid, as evidenced by the finding
(Sinha, 1986) that mutants altered in DNA repair are acid-sensitive. Assuming the
DNA damage is the major lethal change, then it is highly significant that acid-tolerant
cells of E. coli are far more resistant to such damage (Raja et al., 1991); perhaps
tolerant cells contain SS DNA-binding proteins which can protect DNA replication
forks from low pH.
Third, acid-tolerant cells are better able than non-habituated ones to repair any
acid damage to DNA which occurs. Acid-habituated organisms repair acid-damaged
plasmid better than non-habituated ones do; more plasmid-containing (p+) transfor-
mants are produced when acid-damaged plasmid is transformed into an habituated
culture (Raja et al., 1991). The occurrence of novel DNA repair mechanisms in
tolerant organisms is also established by the finding that acid-habituated organisms
are better able than non-habituated ones to repair UV-damaged phage (Goodson and
Rowbury, 1991). Foster has proposed that Ada plays a role in repair of acid damage
to DNA, since ada mutants are acid-sensitive (Foster and Moreno, 1999). It should
be noted that acid sensitivity is shown by polA, recA and uvrA mutants (Sinha, 1986;
Goodson and Rowbury, 1991), suggesting that acid damage repair involves several
repair systems. The new repair system induced at acidic pH (Goodson and Rowbury,
1991) was able to function in mutants altered in any of the above genes (polA, recA
or uvrA), suggesting that it involves novel DNA repair processes, independent of
the PolA, RecA and UvrA gene products.
BIOCHEMICAL AND PHYSIOLOGICAL CHANGES MAKING ORGANISMS

INDUCIBLY ALKALI-TOLERANT
Organisms transferred from pH 7.0 to pH 9.0 become alkali-tolerant (habituate to
alkali; Goodson and Rowbury, 1990; Rowbury, 1997). There appear to be at least
two biochemical components in the tolerance response. First, there is less alkali
damage to DNA in tolerant organisms: NhaA, which is induced at high pHo, is
needed for tolerance induction (Rowbury, 1997); it seems likely that high levels of
this gene product are responsible for the ability of tolerant organisms to resist alkali
damage. A second component of tolerance involves alkali-habituated organisms
showing better repair of alkali damage to DNA (Rowbury, 1997); NhaA appears not
to be involved. The new repair system seems independent of RecA and PolA.
BIOCHEMICAL CHANGES LEADING TO HEAT TOLERANCE IN PHENOTYPICALLY

THERMOTOLERANT ORGANISMS
Kusukawa and Yura (1988) established a key role of GroEL/GroES proteins of E. coli
in governing normal maximum growth temperature. These workers took a strain
deleted in rpoH which was unable to grow at higher than 20°C and isolated revertants
able to grow at higher temperatures. They showed that a series of strains able to grow
at temperatures above 20°C and up to 40°C produced increasing amounts of the HSPs
GroEL and GroES. Inability of the deletion strain to grow at >20°C was due to low
levels of GroE proteins being produced in the rpoH deletion strain, and derepression
of these gene products allowed growth at increased temperatures. These strains with

derepressed GroE were unable even with the highest levels to grow at 42°C but
increased synthesis of DnaK protein allowed growth; similar changes might be needed
as some component of inducible thermotolerance.
It seems likely that both σE-induced responses (with DegP protease being one
critical component induced) and σ32-induced responses are involved in thermotol-
erance induction by heat, although little is known of the biochemical changes, which
are important. It is also possible, however, that trehalose is involved in heat-induced
thermotolerance, since the ability to accumulate this sugar is linked to starvation-
induced thermotolerance (Hengge-Aronis, 1993). It should also be reiterated that
the apparent expulsion of H-NS and IHF from appropriate regions of the DNA also
appears to be needed for the induction process.
PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN ORGANISMS INDUCIBLY

TOLERANT TO COLD
One important example of how the cold-shock response aids tolerance to cold relates
to the CsdA protein. At low temperatures, stable secondary structure forms in RNAs,
especially in m-RNAs, and this acts to reduce m-RNA translation. The CsdA protein
is a major cold-shock protein (Jones et al., 1996) associated with the ribosomes. It
acts to destabilize the DS RNA helix, i.e., it functions to unwind stable secondary
structure and, accordingly, allows increased translation at low temperatures. Strik-
ingly, another major cold-shock protein, namely, CspA, may function with CsdA.
CspA binds to SS RNA (or SS DNA) and hence may prevent re-annealing of
unwound m-RNA.
Accordingly, this mechanism allows a third means of increasing synthesis of
Csp proteins at low temperatures, without increasing m-RNA synthesis, the other
two being stabilization of Csp m-RNA at low temperatures and increased Csp
m-RNA translation, due to the presence of the downstream box translational
enhancer.
OSMOTIC STRESS TOLERANCE: PHYSIOLOGICAL AND BIOCHEMICAL CHANGES

INVOLVED IN INDUCIBLE RESPONSES
Increased osmotic pressure (OP), leads to numerous inducible responses, and some
are shown in Table 8.9. On an increase in external OP, organisms are protected from
growth inhibition, damage and death by increased synthesis or accumulation of
compatible solutes such as K+, glutamate, proline, glycine betaine and trehalose.
The responses that occur function to bring about such changes in synthesis and/or
accumulation; for example, the changes in synthesis and activity of the ProP and
ProU systems function to take up increased amounts of proline, glycine betaine and
related compounds, and similar changes in Kdp-ATPase synthesis and activity aid
K+ uptake, while changes in the activity of glutamate dehydrogenase (and possibly
glutamate synthase) raise internal glutamate concentration.

BIOCHEMISTRY OF TOLERANCE TO SALT
Factors governing the protection of organisms have been considered earlier, but one
should note that a major factor influencing salt tolerance is the level of the NhaA
sodium/hydrogen antiporter. Organisms which induce this protein (rises in intra-
cellular Na+ appear to induce NhaA synthesis, NhaR regulating induction) can expel
Na+ more effectively, but it is the massive increase in activity of the antiporter at
alkaline pH which has the greatest influence on Na+ expulsion.
BIOCHEMICAL CHANGES FOLLOWING STARVATION

Firstly, RpoS controls synthesis of many proteins involved in tolerance (Hengge-
Aronis, 1993; but see “growth-phase and stress tolerance” in this chapter for role
of chromosome termination in stress tolerance, induced by starvation). Among these
are: 1) enzymes for trehalose synthesis and uptake; trehalose is needed for starvation-
induced thermotolerance and for other tolerances, and has membrane and protein
protection properties; 2) enzymes for H2O2 degradation, e.g., catalase HPII; 3) enzymes
for DNA protection or repair, e.g., the Dps DNA-binding protein and exonuclease III;
and 4) enzymes involved in glycogen synthesis (not all are RpoS-controlled). Many
other proteins are induced by starvation but are not rpoS-controlled, e.g., GlgA and
C glycogen synthetase proteins are controlled by cAMP. Starvation-induced ther-
motolerance needs RpoS-controlled OtsAB, implicating trehalose accumulation in
thermotolerance, but starvation also induces HSPs like GroEL, GroES, DnaK, GrpE
and HtpG. (see Table 8.7). In addition, of critical importance during starvation are
proteases which break down dispensible proteins to give amino acids for synthesis
of essential proteins. Mutants altered in the genes which encode such proteases show
poor starvation survival.
Another physiological change, which occurs in organisms as they enter station-
ary phase, may play a major role in inducing the stress tolerances that appear in this
growth phase; it has been established that the phospholipids of the CM become
enriched in cyclopropane fatty acids (CFAs), a change which may itself lead to some
stress tolerances. For example, such a fatty acid change has been implicated in the
acid tolerance that appears at acidic pH in E. coli (Brown et al., 1997), and may
lead to other stress tolerances. Initially, it was believed that such CFA synthesis was
controlled by RelA, since mutations in the gene which encodes this product lead to
CFA deficiencies in stationary phase. In fact, ppGpp is needed for RpoS synthesis,
and, accordingly, it is RpoS which controls CFA synthesis, one synthase promoter
needing this stationary-phase sigma factor for functioning (Eichel et al., 1999).
COUNTERACTING THE INDUCTION OF STRESS

TOLERANCE RESPONSES
Very few efforts have been made to inhibit the induction of stress responses, although
if a common metabolite were able to prevent or counteract a stress response of applied
or medical importance, this could provide a means of preventing the response.

SUBVERTING STRESS RESPONSES USING METABOLITES
Efforts have been made to prevent induction of acid tolerance in E. coli. As
Table 8.11 shows, many metabolites and other agents prevent tolerance induction at
TABLE 8.11
Counteracting Acid Tolerance Responses by Adding Metabolites
During Induction
Agent Used to Effect of Agent on Acid Tolerance Induced by:

Subvert Response Acidic pH Glucose L-glutamate L-aspartate FeCl3
cAMP Tolerance Marked Tolerance Tolerance Tolerance

abolished inhibition abolished abolished reduced
NaCl Tolerance Tolerance Tolerance Tolerance Tolerance
abolished abolished abolished abolished abolished
Sucrose Tolerance Tolerance Tolerance Tolerance Tolerance
abolished abolished abolished abolished abolished
Ethanol Tolerance Slight Slight No effect Slight
abolished inhibition inhibition inhibition
Urea Very marked No effect No effect Marked Slight
inhibition inhibition inhibition
Phosphate Tolerance Marked Marked Slight Slight
abolished inhibition inhibition inhibition inhibition
SDS Tolerance Tolerance Tolerance Marked Tolerance
abolished abolished abolished inhibition abolished
DOC Tolerance Tolerance Tolerance No effect Slight
abolished abolished abolished inhibition
N-acetyl serine Tolerance N.T. N.T. N.T. N.T.
abolished
L-leucine Tolerance N.T. N.T. N.T. N.T.
abolished
N.T. = not tested.
pH 5.0, or by specific inducers at neutral pH. In the case of a few of the agents that
act at pH 5.0, the possibility that they act on synthesis or action of the ECs has been
examined. For example, phosphate inhibits synthesis of the ESC and cAMP acts
both on ESC synthesis and on interaction of EIC with pH 7.0-grown organisms. The
results also indicate that HCO3– can interfere with tolerance induction; HCO3– has
not been tested on acid habituation, because it is decomposed at pH 5.0. Because
both main biological stages in the functioning of ECs in acid habituation can be
examined at pH 7.0 (ESC → EIC, which needs pH 5.0, is probably simply a chemical
reaction), the potential role of bicarbonate can be studied; it is able to inhibit both
ESC synthesis and interaction of EIC with organisms.

The effects of urea and N-acetyl-L-serine on acid habituation at pH 5.0 are of
interest, since CysB-dependent responses (acid habituation at pH 5.0 is CysB-
dependent) are often inhibited by these agents. The striking additional finding was
that urea markedly inhibited L-aspartate-induced acid tolerance and this may indicate
CysB-dependence of this response.
SUBVERTING OTHER STRESS RESPONSES

Several of the pH responses described above can be abolished or strongly inhibited
by metabolites and other agents, and a few effects are discussed next.
Alkali Tolerance Induction
First, induction is strongly inhibited by glucose, and of interest here is the finding
that cyclic AMP does not reverse this inhibition; this nucleotide usually reverses
inhibitory effects of glucose on inducible responses. Second, phosphate also inhibits
alkali tolerance induction and the same is true for NaCl.
Acid-Induced Alkali Sensitization
Induction of this response is greatly reduced by phosphate, NaCl and FeSO4.
Cu2+-Induced Thermotolerance
Several tested metabolites reduced such tolerance induction by copper. The most
significant effects were with glutathione (GSH), L-cysteine and urea, which all
substantially reduced thermotolerance induction, and ethanol which virtually abol-
ished the response. It is now known (Rowbury and Goodson, 2001) that copper-
induced thermotolerance involves an ESC/EIC pair; accordingly, it would be of
interest to know whether GSH, L-cysteine and urea act on ESC synthesis, on ESC →
EIC, on interaction of EIC with sensitive organisms or on some other process.
EXTRACELLULAR ALARMONES AND

CELL-TO-CELL COMMUNICATION
In the early 1980s, Bruce Ames and his group (Bochner et al., 1984) proposed that
cellular damage, by several chemical stresses, could lead to the production of
nucleotide “alarmones,” the sensing of which could switch on damage limitation
and damage repair processes. These alarmones were polyphosphates and, although
they were able to leak poorly from the producing organisms, they would be virtually
unable to permeate into other cells and, accordingly, only influence the producing
organisms. For this reason, the success of these alarmones, in protecting organisms,
depended on their being synthesized rapidly, and following low levels of damage.
It was also essential that the sensing system was able to detect tiny concentrations
of the alarmones.
In contrast, the discovery of the ESC/EIC pairs demonstrated that chemical and
physical stresses could produce extracellular alarmones, the stress activating the ESC

to the EIC alarmone, with activation occurring in the medium. Since organisms have
receptors which allow the uptake of the EICs, these alarmones are able to warn other
organisms (including unstressed ones, and those which cannot produce the ESC),
and trigger favorable responses. Table 8.12 lists the properties of the ESC/EIC pairs;
TABLE 8.12
Characteristics of the ESC/EIC Pairs Allowing Them to Provide Early Warning
Systems against Chemical and Physical Stresses
1. The ESCs are extracellular stress sensors and are secreted into the media of stressed or unstressed
cultures; accordingly, the stress is sensed in the medium with no delay, producing EIC and, therefore,
inducing the response with no delay.
2. The ESCs are extremely sensitive to activation by chemical stress, so that a response can occur at
very low levels of toxic agents.
3. The ESC occurs in several forms; the form synthesized under particular conditions is that which can
most rapidly respond (Rowbury, R.J. and Goodson, M., FEMS Microbiol. Lett., 174, 49, 1999a).
Sometimes, the ESC can be activated even before the level of the agent (e.g., protons) becomes stressing.
4. Formation of the EIC from the ESC is essential for response induction in the presence of the stress,
but the EIC can also induce the response in unstressed cells.
5. The EIC can also induce its response in non-producers (cells which fail to form ESC and, therefore, EIC).
6. The EICs are small molecules and can, therefore, diffuse away to other regions, including those not
subject to stress; this behavior of EICs makes them alarmones, and the system constitutes an early
warning against stress, warning organisms of impending stress and preparing them to resist it.
7. The characteristics listed in 4, 5 and 6 allow cell-to-cell communication, with the EICs acting
pheromonally.
8. The EIC receptors on the cell surface can occur in different forms, the form synthesized being that
which can most favorably bind the EIC present in the medium.
9. The ESCs and EICs are highly resistant to irreversible inactivation by lethal conditions, making the
ESC/EIC pair system highly robust, and allowing stress-killed cultures to induce responses.
these properties, especially three of them, namely, the diffusibility of these agents,
their ability to act on unstressed organisms, and their cross-feeding characteristics,
allow these EC pairs to influence other organisms and so these EC pairs are acting
as pheromones. Accordingly, the functioning of the ESC/EIC pairs allows other
organisms to be warned of impending stresses, with damage limitation and damage
repair processes being triggered.
FUTURE EXPERIMENTS AND CONCLUSIONS

The stresses relevant to the survival of organisms in food have been outlined here,
and the responses affecting tolerance to such stresses listed. The regulation of such
responses has then been considered in detail; in particular, the switching-on of such
responses by both intracellular and extracellular stresses has been discussed.
Although internal levels of some stresses are detected by classical means, i.e., by
intracellular sensors with all other reactions and components related to induction

being intracellular, many extracellular stresses are sensed by extracellular compo-
nents (ECs), and interaction of the stress with an extracellular sensing component,
ESC (which is synthesized in the absence of stress), activates the ESC to an extra-
cellular induction component (EIC). The EICs are usually small diffusible proteins
and can diffuse to and warn unstressed organisms, i.e., they are alarmones which
allow cross-talk between stressed and unstressed organisms. Such EICs associate
with receptors on organisms in the medium, leading to the stress response. Such
ESCs and EICs occur for many stress responses and it appears that each ESC exists
in several forms, depending on the culture conditions (see Table 8.12); the receptors
on the cell surface which interact with the EIC can also be modified according to
the culture conditions. Such ESC/EIC pairs are needed for numerous pH responses
and for salt-stress responses.
Most recently, it has been shown in my laboratory that UV tolerance induction
involves a specific ESC/EIC pair, with increased temperature leading to increased
UV tolerance, following the ESC to EIC conversion. The ribosome has been pro-
posed as the thermal sensor involved in triggering the heat-shock and cold-shock
responses, and DnaK is another proposed thermometer, but the evidence for these
is mostly indirect. The finding that a heat-activated EIC is involved in thermotoler-
ance induction suggests that the heat-shock and cold-shock responses may be
switched-on by ESC/EIC pairs, and that these ECs are involved in induction of most
other stress responses. Further support for a role for ESCs as biological thermometers
comes from the demonstration that the triggering of acid tolerance, alkali tolerance
and UV tolerance by temperature rises involves activation of the appropriate ESCs
by the temperature shift. Further work on the role of ECs in the switching-on of
responses to cold, irradiation, osmotic shock and to starvation should be urgently
undertaken. Although the nature of the sensor(s) for induction of heat shock and
cold shock is controversial, there is good information on how the later stages of
these two responses are regulated and, for heat shock, on which biochemical changes
in the organisms allow protection against heat and ability to repair heat damage.
Substantial studies have also been made on the molecular biology of the regu-
lation for several other stress responses, e.g., relating to killing and growth inhibition
by irradiation, starvation, osmotic stress and oxidative stress (although not on the
role of ECs) and this work has been reviewed. Additionally, a brief account has been
given of some important biochemical changes which lead to tolerance, although only
a few examples have been given of the changes occurring on induction of each stress
response. It was pointed out that little has been done to subvert stress tolerance
induction, except that many agents are known to prevent acid tolerance induction
and, in a few cases, agents that subvert responses have been shown to inhibit ESC
synthesis or EIC interaction with organisms or both processes. Recent work reveals
that appropriate killed cultures induce stress tolerance in living organisms (Rowbury,
2000) and these findings are of public health significance and important in food
microbiology; it is critical that the basis for these responses is established, although
all the evidence so far suggests that it is stress response ECs in the killed cultures
which induce the tolerance responses.

GLOSSARY
AdiA arginine decarboxylase
ahpC gene encoding the smaller subunit of alkyl hydroperoxide reductase
ASI acid sensitivity induction at pH 9.0
cAMP adenosine-3′-5′-cyclic phosphate
CAP catabolite activator protein
CFA cyclopropane fatty acid
clpA, B, P, X structural and regulatory genes governing synthesis of heat-shock
protease/chaperones
CM cytoplasmic membrane
CsdA cold-shock protein
CspA, B cold-shock proteins
cysB/CysB a gene and its gene product implicated in regulation of cysteine
biosynthesis and other processes
cyclic AMP adenosine-3′-5′-cyclic phosphate
⌬pH the difference between internal pH of a cell and the external milieu
DOC deoxycholic acid
DnaJ a chaperone protein which is induced by heat-shock and certain other
stresses
DnaK a chaperone protein which is induced by heat-shock and certain other
stresses
DNase deoxyribonuclease
EC extracellular component
EIC extracellular induction component
EnvZ cytoplasmic membrane component involved in regulation of OmpC/F bio-
synthesis
ESC extracellular sensing component
fur/Fur ferric uptake regulator, gene and gene product
GABA γ-amino-butyric acid
gadA, B genes which encode two forms of glutamate decarboxylase
GlgA/C glycogen synthetase components
GroEL a heat-shock chaperone
GroES a heat-shock chaperone
GrpE a heat-shock chaperone
GSH glutathione
GyrA a DNA helicase
himA/HimA the gene which encodes one subunit of integration host factor, and
its gene product
H-NS a histone-like regulatory protein, which affects numerous genetic regions
after binding to the DNA
HSP heat-shock protein
hsp-lacZ a fusion of a heat-shock protein gene to the lacZ gene
hsp-phoA a fusion of a heat-shock protein gene to the alkaline phosphatase gene
HtpG a heat-shock protein
HtpM a heat-shock protein

Hyd components proteins needed for formic hydrogenlyase activity
IHF integration host factor
kdp genes a group of genes encoding or regulating synthesis of Kdp components
Kdp components proteins involved in governing K+ uptake and osmotic pressure
LexA regulator of the SOS response
Lon a heat-shock protein with protease activity
NhaA the major sodium/hydrogen antiporter of E. coli
NhaB a secondary sodium/hydrogen antiporter of E. coli
NhaR the regulator of NhaA induction
NMW nominal molecular weight
OM outer membrane
ompA/OmpA a gene and its gene product involved in outer membrane stability
and permeability
OmpC/F porins for uptake of uncharged molecules and cations
OmpR protein involved in regulation of OmpC/F biosynthesis
O.P. osmotic pressure
OtsA, B proteins involved in osmotic tolerance and thermotolerance induction
oxyR/OxyR a gene and its gene product involved in regulation of H2O2 tolerance
pHi internal pH
pHo external pH
PhoE porin for anion uptake
PhoP/Q regulatory gene products which control some acid tolerance responses
and other processes
PhoS periplasmic phosphate-binding protein
PolA the Kornberg polymerase
ppGpp a guanosine tetraphosphate involved in controlling the stringent response
pppGpp a guanosine pentaphosphate involved in controlling the stringent response
(p)ppGpp refers to enzymes or processes using ppGpp or pppGpp
pro genes genes involved in governing proline biosynthesis and some responses
to high osmotic pressure
RecA a protein which functions in recombination and in the regulation of the
SOS response
RelA (p)ppGpp synthetase
RNase ribonuclease
RpoH the major heat-shock sigma factor
rpoS gene encoding the RpoS stationary-phase sigma factor
␴E a heat-shock sigma factor
␴32 the major heat-shock sigma factor
SDS sodium dodecyl sulfate
TonB a cytoplasmic membrane protein, which aids entry of components across
the OM and into the periplasm
TreA protein involved in trehalose synthesis and in osmotic tolerance induction
ts temperature-sensitive, referring to mutants unable to grow at the wild-type
growth temperature
UvrA, B and C gene products needed for repair of DNA damage

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Strategies to Control Stress-
9 Adapted Pathogens
John Samelis and John N. Sofos
CONTENTS
Introduction
Foods Involved in Bacterial Foodborne Outbreaks
Potential Reasons for Pathogen Emergence
Effects of Stress on Bacteria
Effects of Food-Related Stresses on Bacteria
Research Needed to Control Stressed Pathogens in Foods
Research Approaches for Control of Stressed Pathogens
Novel Pathogen Control Strategies
Practical Application of Pathogen Control Strategies
Conclusions
References
INTRODUCTION
Despite the extensive scientific progress and technological developments achieved
in recent years, food safety problems continue to exist and may actually increase in
the future. It is estimated that foodborne diseases cause approximately 76 million
illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year,
most due to unknown causative agents (Mead et al., 1999). Among the known
pathogens associated with foodborne illness, there is an increasing involvement of
environmentally resistant and host-adapted species or strains, which may be difficult
to inactivate or control with traditional food preservation methods (Alterkruse et al.,
1997; Foster, 1997; Tauxe, 1997).
Intensified research in recent years indicates continuous adaptation and devel-
opment of resistance by pathogenic microorganisms to antibiotics (Threlfall et al.,
2000) and to various food-related stresses, such as low pH or acidity, heat, cold
temperature, dry or low water activity environments, and chemical preservatives
(Abee and Wouters, 1999; Bower and Daeschel, 1999; Brul and Coote, 1999;
Sheridan and McDowell, 1998). Prolonged exposure of adapted pathogens to anti-
biotics and other stresses may lead to the rise of new genotypes, as a result of
bacterial evolution resulting in adaptive mutations (Lederberg, 1997, 1998). These

mutants are capable of surviving and potentially multiplying under adverse condi-
tions, while they may also be of enhanced virulence (Archer, 1996). Indeed, in vivo
studies with animals indicate that stressed bacterial pathogens may increase their
virulence and, accordingly, decrease their oral infectious dose (Bearson et al., 1997;
Foster, 1995; Gahan and Hill, 1999; O’Driscoll et al., 1996; Robertson and Roop,
1999; Wong et al., 1998). Examples are the multidrug-resistant Salmonella Typh-
imurium DT104 (Davis et al., 1999; Glynn et al., 1998) and the acid tolerant Escher-
ichia coli O157:H7 (Armstrong et al., 1996; Park et al., 1999), both associated with
recent foodborne illness outbreaks (Alterkruse et al., 1997; Mead et al., 1999). There-
fore, it seems that, as a response to exposure of bacteria to sublethal stresses, the
microbial ecology of our food supply is undergoing changes toward an increasing
occurrence of resistant pathogens of enhanced virulence (Archer, 1996; Lederberg
et al., 1992; Lederberg, 1997, 1998; Sheridan and McDowell, 1998; Sofos, 2001).
The genetic and associated biochemical mechanisms that bacteria possess or
develop, and then express or activate, to enhance survival in stressful environments
and during food processing, are discussed in previous chapters. The scope of this
chapter is to present a brief overview of existing knowledge on pathogen stress
responses in correlation with known or potential consequences in foods, and to
discuss potential strategies for control of stress-adapted pathogens with the objective
of enhancing the safety of our food supply.
FOODS INVOLVED IN BACTERIAL

FOODBORNE OUTBREAKS
Occurrence of foodborne disease outbreaks has increased in the past 15 to
25 years (Bean et al., 1997; Mead et al., 1999; Tauxe, 1997) and foodborne illness
remains a global problem despite major scientific and technological developments
in food science and technology. Foodborne illness episodes are still frequent even
in countries or regions with advanced food chains, such as the U.S., Canada and
Europe. In addition to the increasing number of pathogenic agents involved (Alterk-
ruse et al., 1999; Beuchat, 1996b; Bryan and Doyle, 1995; CDC, 1999; Davis et al.,
1999; Doores, 1999; Park et al., 1999; Sterling and Ortega, 1999), the number of
the types of foods associated with foodborne illness has also increased (Alterkruse
et al., 1997; Beuchat, 1996a, Doores, 1999; Keene et al., 1997; Tauxe et al., 1997;
Tilden et al., 1996).
Examples of food vehicles and associated pathogens involved in foodborne
illness episodes include E. coli O157:H7 and other hemorrhagic E. coli serotypes
from ground beef, apple juice and cider, other fruit juices, alfalfa, radish and other
types of sprouts, jerky, mayonnaise, watermelon, other produce and dry fermented
meats; Salmonella from ice cream, cantaloupes, watermelon, potatoes, alfalfa
sprouts, tomatoes, and other produce; Salmonella Enteritidis from eggs and ice
cream; Shigella from produce; Camplylobacter from poultry and garlic butter;
Yersinia enterocolitica from chitterlings and tofu; Yersinia pseudotuberculosis from
milk, pork and possibly fruit juice; Vibrio vulnificus from oysters; Clostridium
botulinum from potato salad, garlic sauce, sauteed onions, eggplant, bean dip, clam

chowder, olives, summer sausage, and canned bamboo shoots; Listeria monocyto-
genes from milk, cheeses, coleslaw, hot dogs and luncheon meats; Cryptosporidium
parvum from water and fresh-pressed apple juice; Cyclospora cayetanensis from
raspberries and basil; Hepatitis A virus from frozen strawberries; and Norwalk-like
virus from oysters, salads and frostings (www.cdc.org).
Considering the increasing number of pathogenic agents transmitted by an also
increasing number of foods, including products traditionally considered as low-risk,
an important question is why and how specific pathogens are transferred to, and
established in, specific foods to cause foodborne illness. Several reviews (Armstrong
et al., 1996; Beuchat, 1996a; Gill, 1998; Sofos, 1994, 2001) and recent studies (Elder
et al., 2000; Keene et al., 1997; Samelis and Metaxopoulos, 1999; Sofos et al.,
1999a) have dealt with the factors leading to cross contamination of a specific food
with a specific pathogen. Knowledge of the sources and routes of contamination of
bacterial pathogens from the environment to the food, and their mechanisms for
transfer, attachment, distribution and survival in food processing environments is
essential for pathogen control (Buchanan, 1997; Tauxe, 1997).
In most cases, existing knowledge on the microbial ecology of different types
of foods was adequate to explain the incidence and potential establishment of a
pathogen in a specific food. For example, the prevalence of Salmonella and Campy-
lobacter on live birds, their eggs and fresh poultry (Bryan and Doyle, 1995), or that
of L. monocytogenes on fresh meat (Farber and Peterkin, 1999; Sofos, 1994) are
well established. Therefore, safety problems caused by these pathogens, following
their survival or post-processing contamination in such products, could be expected.
For example, the survival of Salmonella (Mertens et al., 1999) and L. monocytogenes
(Samelis and Metaxopoulos, 1999) in stuffed ham and ham-like products due to
undercooking could be recognized. Also, the growth of L. monocytogenes that
potentially occurred on post-processing contaminated frankfurters and luncheon
meats to result in the fatal 1998–1999 multistate outbreak in the United States (CDC,
1999) is a known safety risk associated with such products (Farber and Peterkin,
1999). Likewise, the cross contamination of ground beef with E. coli O157:H7 due
to bovine fecal contamination, and the hemorrhagic colitis outbreaks caused by
consumption of undercooked hamburgers (Bell et al., 1994; Riley et al., 1983) could
be expected considering the habitat of the pathogen (Armstrong et al., 1996). In all
these situations, the occurrence of the pathogens in the final products and the
associated outbreaks were due to faulty processes that allowed survival of the food
poisoning agent.
In several recent instances of food products serving as vehicles of bacterial
foodborne outbreaks the route of contamination and the potential for survival of the
pathogenic agent were unexpected, but not surprising. For example, contamination
of apple juice and cider with E. coli O157:H7 may be due to its survival in wounds
of fallen apples after contact with manure (Dingman, 2000), while experimental
transmission in apples by fruit flies has also been demonstrated (Janisiewicz et al.,
1999a). Also, E. coli O157:H7 and Salmonella may have caused foodborne illness
(Ackers et al., 1998; Besser et al., 1993; CDC, 1995a,b,c; Keene et al., 1997; Sauer
et al., 1997; Tauxe et al., 1997; Tilden et al., 1996; Wall et al., 1994; Wood et al.,
1991) because of their ability to attach firmly and survive on fresh produce (Beuchat,

1996a), in unprocessed fruit juices (Miller and Kaspar, 1994; Roering et al., 1999;
Ryu and Beuchat, 1999b; Zhao et al., 1993) and vegetable salads (Abdul-Raouf
et al., 1993), or on dried or fermented meats and fruits (Burnham et al., 2001;
Calicioglu et al., 1997; Clavero and Beuchat, 1996; Glass et al., 1992; Harrison and
Harrison, 1996; Hinkens et al., 1996; Ihnot et al., 1998; Nissen and Holck, 1998;
Riordan et al., 1998), with their survival being potentially enhanced by refrigeration
of stored products (Clavero and Beuchat, 1996; Faith et al., 1997; Tsai and Ingham,
1997; Zhao et al., 1993; Zhao and Doyle, 1994). Water contaminated with human
waste, subsequently used to apply fungicide, was reported as the potential route of
contamination of raspberries with Cyclospora (Sterling and Ortega, 1999).
Based on new routes of transmission of emerging foodborne diseases, Tauxe
(1997) stated that, while in the past prevention involved avoidance of contamination
of human food with sewage or animal manure, in the future prevention will increas-
ingly depend on controlling contamination of feed and water by the animals them-
selves. In other words, pre-harvest control measures to prevent or minimize transfer
of contamination with pathogens from the field or the stable to the plant need to be
established. Use of water of good microbiological quality for spraying or rinsing is
an essential good agricultural or processing practice, while poor hygiene and sani-
tation practices and inadequate chilling or cooking should be avoided throughout
food processing.
POTENTIAL REASONS FOR PATHOGEN EMERGENCE

The reasons for the increasing numbers of foodborne disease outbreaks in recent years
seem to be multiple. They are associated with changes in consumer lifestyles and food
preferences, food production and distribution practices, consumer lack of proper food
handling knowledge and habits, advances in microbiological detection methods, and,
more importantly, the adaptive responses of microorganisms in the environment
(Alterkruse et al., 1997; Lederberg et al., 1992). Thus, the factors that contribute to
pathogen emergence, either singly or through their interactions, may be classified as
biological, environmental, food-related, societal and consumer-associated.
As indicated, the types of pathogens that may, expectedly or unexpectedly, be
found in a food niche depend on natural selection of species, bacterial evolution
(Lederberg, 1997, 1998) or environmental cross contamination. The responses of
bacteria to stressful environments may also lead or contribute to the emergence of
pathogenic strains or species. The recognition and documentation of E. coli
O157:H7, S. Typhimurium DT104 and several other previously unknown or unrec-
ognized pathogenic bacteria, as the causative agents of diseases transmitted by foods,
have been based on advanced microbiological detection methods (de Boer and
Beumer, 1999; Meng et al., 1994; Swaminathan and Feng, 1994; Vernozy-Rozand,
1997). Thus, advances in molecular microbiology, immunology and detection meth-
ods have contributed significantly to the recognition of agents classified as emerging
foodborne pathogens.
Environmental factors associated with variations in geographic location and
climate, as well as natural stresses, may also induce biological changes and lead to
new pathogens or enhanced virulence. This probability is supported by differences

found in the microbial ecology of similar foods harvested or processed in different
geographical zones or countries with diverse climates and food preferences (Mossel
et al., 1995). However, the increasing international food trade and centralized food
production and processing in large volumes have the potential to transfer and dis-
tribute foodborne bacteria and other food-related pathogenic agents between distant
continents or countries (Kaferstein et al., 1997).
Food-related factors that may lead to pathogen emergence, increased resistance
or enhanced virulence include changes in food production and harvesting, processing
modifications, marketing developments, preparation practices and development of
new food products to meet consumer demands (Lammerding and Paoli, 1997; Zink,
1997). As mentioned, establishment and transmission of pathogenic agents is
enhanced in large-scale production of agricultural products or feeding of animals.
Since animal and plant diseases may cause high economical loses in such large
production units, the demand for and the use of new and more effective antibiotics
and pesticides in agriculture is under continuous consideration. However, by releas-
ing large amounts of chemicals in nature and altering or increasing the level of
traditional food preservation methods, humans play a major role in the development
of resistance and the adaptive, cross-protective responses of bacterial pathogens to
stresses (Bower and Daeschel, 1999; McManus, 2000; Tollefson and Miller, 2000).
The increasing use of decontamination interventions to reduce microbial con-
tamination on harvested produce (Beuchat and Ryu, 1997), slaughtered animals
(Siragusa, 1995; Smulders and Greer, 1998; Sofos and Smith, 1998) and during
other food processing steps (Sofos, 1993) may lead to new or evolving bacterial
pathogens. There is a potential risk for decontamination to alter the microbial ecology
of a food by substantially reducing the numbers of the food-specific natural flora,
and allowing the underlying, potentially new or more resistant, pathogenic species
to grow (Jay, 1996, 1997). Also, food manufacturers may adopt processing modifi-
cations which are inadequately validated for their microbiological safety, while
attempting to meet consumer demands for more convenient and “healthy” preser-
vative-free foods (Zink, 1997). These changes may lead to new sublethal stresses
exerted on microorganisms or reduction of existing food preservation hurdle inten-
sities, leading to increased pathogen resistance or virulence, as well as failure of
traditional hurdles to assure food safety (Sofos, 1993). For example, product for-
mulations with reduced fat or salt may result in increased food safety risks. This is
because reduced fat levels are usually replaced with added water in the product,
which further dilutes the low level of salt and other preservatives in the water phase
of the product; thus, microbial growth may become more prolific (Sofos, 1993,
2001).
Marketing efforts undertaken to make food products more attractive to consum-
ers may also have an impact on pathogen emergence if factors such as the packaging
conditions (e.g., the type of container, film permeability, packaging atmospheres) of
the product are altered without previous testing. Such alterations may change the
hurdle effect and the microbial ecology of foods and may enhance outgrowth of
certain pathogens, which otherwise would be inhibited. Therefore, processes for new
food products need to be validated for their ability to lead to microbiologically safe
products prior to their commercial application (Lammerding and Paoli, 1997).

Societal factors may also contribute to pathogen emergence. Following urban-
ization in most countries, there is a need for transportation of large amounts of food
products from centralized production and processing locations to distant markets at
urban centers. In developed countries, large quantities of foods are manufactured in
big factories at centralized locations (Tewari et al., 1999). Seasonal food products
are now available throughout the year, while consumers may travel throughout the
world with no need to change their dietary preferences. Thus, food products need
to be of adequate shelf life for distribution, marketing and consumption in distant
areas. Also, food product transportation from exporting to importing countries is
steadily increasing. Accordingly, transmission of foodborne disease and infection of
large populations is much easier than in the past (Kaferstein et al., 1997; Majkowski,
1997). Improper processing, handling and storage of foods during transportation and
distribution may result in foodborne illness outbreaks affecting large numbers of
consumers (Hall, 1997; McMeekin et al., 1997). Furthermore, it may be more dif-
ficult to recall faulty food prior to infecting large populations of consumers
(Majkowski, 1997).
The globalization of the food industry and the increased travelling of people
enhance the transfer of pathogenic agents between countries (Kaferstein et al., 1997),
and this may also lead to new pathogens following their adaptation to diverse
environmental conditions. Nowadays, patients undergo more sophisticated medical
treatments, which may prolong the life of immunocompromised individuals who are
more susceptible to foodborne illness from pathogens at lower infectious doses
(Morris and Potter, 1997). Outbreaks are more thoroughly investigated, and detailed
surveillance data are available in developed countries (Bean et al., 1997; Mead et al.,
1999), while news media undertake extensive, but sometimes uninformative, reports
to address an outbreak and increase public awareness. The internet and telecommu-
nications have dramatically increased the exchange of information, and have helped
consumers to express increased interest in food safety issues (Bruhn, 1997).
Consumer-associated factors include changing demographics and human life-
styles, increased life expectancy, modified eating habits and, most importantly, lack
of adequate food handling education (Alterkruse et al., 1997; Collins, 1997; Hall,
1997; Lederberg, 1997). Human lifestyles have undergone major changes in the last
15 to 20 years, and so have consumer demands for all goods, particularly foods
(Collins, 1997). People living in big cities are interested in maintaining fitness and
having a healthy diet. Therefore, it is not surprising that most consumers and public
media characterize foods as “healthy” or “unhealthy” based more on a compositional,
rather than a microbiological safety, basis. Consumers demand foods that have
reduced levels of calories, fat and additives, while being “natural” or “organic,” and
potentially having properties that do not enhance incidence of cancer, heart disease
and other illnesses. New generation foods are less processed, enriched in nutrients,
pre-prepared and convenient for use to reduce the time required for preparation of
meals (Zink, 1997). Moreover, following the major changes in demographics and
consumer lifestyles (e.g., urbanization and reduction of human populations directly
involved in agriculture), domestic or small-scale production and consumption of
foods has shrunk. Increasing numbers of consumers who do not prepare their own
meals at home but purchase ready-to-eat foods or meals from commercial suppliers

or food service outlets, increase the potential for spread of foodborne illness out-
breaks. Moreover, it is disappointing that in urban societies most consumers lack
basic knowledge on how to prepare, handle and keep food safely to protect them-
selves from food poisoning; this was not the case in the past, especially with people
living in the countryside and involved in agriculture. Since improper food handling
in the kitchen appears to be increasing (Collins, 1997), educational material through
public news media and detailed food labeling (specific warnings, expiry dates and/or
directions for use) have been increasingly used or legislated to enhance food safety
(Bruhn, 1997). Educating consumers on proper food handling practices should be a
major priority for food scientists, regulators, industry and trade associations, and
public health providers (www.foodsafety.gov).
EFFECTS OF STRESS ON BACTERIA

Bacteria may encounter several environmental stresses or less than optimal condi-
tions affecting their survival and growth, and bacteria that do not find ways to cope
with stress may die (Foster, 1995; Hengge-Aronis, 1993). Stresses on bacteria may
include starvation, cold, heat, acid, osmolarity, low moisture, high atmospheric
pressure, low oxygen or anaerobic conditions, bactericidal gases (i.e., carbon dioxide,
carbon monoxide, etc.), other antimicrobials occurring naturally or applied by
humans (e.g., antibiotics, sanitizers, preservatives), and competing bacteria. Expo-
sure of bacteria to environmental stresses may be continuous and of varying intensity,
starting in the soil and water, and continuing in environments where bacteria establish
niches, including animal hides, harvested plant products, slaughtered animals, or
plant and food service surfaces (Abee and Wouters, 1999; Bower and Daeschel,
1999).
Passage of bacteria through the host may be long and tortuous (Foster and
Spector, 1995; Gahan and Hill, 1999). Exposure to acid excretion in the stomach
and other multiple or sequential stresses in the small intestine, such as volatile fatty
acids, bile, low oxygen and competition with the intestinal flora are among the
primary defensive mechanisms of the host to inactivate pathogens and prevent
infection. Following invasion through the intestinal epithelial cells, bacteria may be
taken up by macrophages and internalized within phagosomes, where specialized
organelles prevent their multiplication by means of acidic pH and/or production of
defensins, hydrogen peroxide and superoxide radicals (Foster and Spector, 1995;
Gahan and Hill, 1999). Consequently, a bacterial pathogen either finds ways to
survive these multiple stresses, or it dies. According to Dorman (1994), “bacterial
cells have a remarkable capacity to reinvent themselves as they endeavor to adapt
to changing environmental conditions. The host-pathogen interaction represents a
form of biological politics in which two contending parties seek to reconcile their
competing interests. If the host prevails, the infection fails; if the bacterium wins an
outright victory, the host suffers disease and the bacterium may find itself without
a host.” Thus, the primary effect of stress on bacteria is the triggering of mechanisms,
modulated by specific genes or gene groups, to adapt, develop resistance, survive
and potentially multiply under stressful conditions (Archer, 1996; Hengge-Aronis,
1993, 1996). These responses may lead to unpredictable difficulties in controlling

bacterial pathogens in human and animal infections, and foods (Abee and Wouters,
1999; Bower and Daeschel, 1999).
Overall, bacteria attempt to cope with environmental stress by two major types
of responses, namely temporary changes and mutations (Archer, 1996; Sheridan and
McDowell, 1998). Detailed information on these aspects in relation to food research
is given in other chapters, while the fundamentals and the latest knowledge on
bacterial stress responses can also be found in a publication of the American Society
for Microbiology (Storz and Hengge-Aronis, 2000). Therefore, only some principles
of bacterial stress responses are summarized below.
• Bacteria appear to possess complex sensory systems that alert them to the
presence of one or more stresses. Such systems are always activated, irre-
spective of stress, when a bacterium enters into its stationary phase of growth.
• Trusting their sensors, bacteria develop adaptive stress tolerance
responses, and activate various defensive mechanisms, to prevent irrevers-
ible injuries, develop resistance and eventually survive the stress.
• Appropriate genes are induced to activate essential defensive mechanisms,
in most cases temporarily. This gene expression does not result in perma-
nent genomic changes (mutations), and when response to a stress is not
required, the genes involved are switched off.
• Although stresses and their target cell sites might be different, in several
cases the same or related genes, such as rpoS, are involved in the adaptive
processes to regulate the cell defense. As mentioned, this often results in
cross-protection effects (e.g., a bacterium successfully adapted to one
stress may develop resistance to other stresses).
• Cells are more resistant to stress in their stationary phase compared to
the exponential phase, as they develop/possess a generalized stress
response (GSR) system, which is regulated mainly by RpoS to face
upcoming starvation; the GSR is independent of specific-stress.
• Upon an extended exposure to one or more stresses, mutant strains may
arise to enhance bacterial survival, and some of these mutants may possess
increased virulence. Mutations are of two major types: spontaneous and
adaptive or directed. Spontaneous mutations occur mostly in exponentially
growing cells, when all intracellular activities and the replication of the
genome are at high speed to respond to sudden stress. Such mutations
may yield large numbers of cells, which temporarily become resistant to
the stress encountered, but the mutation is not permanent to benefit the
bacterial population at later times. In contrast, mutations that occur in
stationary phase cells, in the absence of growth (e.g., no genomic repli-
cation), are more stable than spontaneous mutations and may be advan-
tageous to the bacterial population as they provide permanent increased
resistance to one or more stresses. When a specific selective agent is
present in the environment to induce bacterial adaptation and achieve its
utilization or prevention from its lethal effects, these stationary-phase
mutations are termed adaptive or directed. Overall, adaptive mutations of
stationary-phase cells are more frequent and, accordingly, of greater sci-

entific concern than spontaneous mutations of growing cells. This is
because mutants of bacterial pathogens triggered by exposure to specific
stresses may be significantly more stress-resistant and virulent than their
parental strains. Escherichia coli O157:H7, which is reported to have
originated from an O55:H7 ancestor through horizontal transfer and
recombination (as cited by Park et al., 1999) and to have evolved in a way
that is more a Shigella with a little cloak of E. coli antigens (Lederberg,
1998), appears to be a classic example. This E. coli O157:H7 evolution
may have occurred in the bovine gastrointestinal tract, where it attained
an exceptional acid resistance (Armstrong et al., 1996; Park et al., 1999).
In several strains of E. coli O157:H7, acid resistance is permanent, while
in other strains it may be increased by a pH-inducible adaptive acid
tolerance (Benjamin and Datta, 1995; Buchanan and Edelson, 1999a;
Conner and Kotrola, 1995; Lin et al., 1996).
To survive and grow, bacterial cells must maintain their integrity and homeostatic
balance within their surrounding environment. However, environmental stresses may
cause disruption of cell homeostasis, while the cell attempts to prevent or minimize
such disruption (Gould, 1995; Leistner, 2000). The membrane is the cell component
that primarily protects the cell from external factors and, therefore, it is the first
component that suffers damage and it is where most cellular changes occur to prevent
or repair damage. Exposure of most bacteria to cold temperatures induces phospho-
lipid and fatty acid alterations (e.g., increases in the proportion of unsaturated fatty
acids in the cell membrane), resulting in increased membrane fluidity (Berry and
Foegeding, 1997; Sofos, 1989). Also, in most bacteria, specific sets of cold shock
proteins are induced upon abrupt shifts to cold temperatures, and functional enzymes
become simpler in structure and more flexible. These changes enhance survival and
potential growth in cold environments, but at much lower than optimal reaction rates
(Berry and Foegeding, 1997). Opposite phenomena occur when cells are exposed
to elevated temperatures. The concentration of saturated fatty acids in the membrane
increases, and there is a heat shock response expressed by the synthesis of specific
proteins, which results in increased thermotolerance.
Changes in membrane lipid composition may also confer increased resistance
to certain antimicrobials, which may attack the cell by binding on, creating pores
and disrupting the proton motive force of the membrane (Berry and Foegeding,
1997; Sofos and Busta, 1999). Weak organic acids penetrate the cell membrane in
their undissociated form and thereafter dissociate and acidify the cytoplasm, leading
to cell death (Alakomi et al., 2000; Gould, 1995; Young and Foegeding, 1993).
Bacteria respond to the lowering of the intracellular pH by expelling protons and
by regulating the pH membrane gradients (Diez-Gonzalez and Russell, 1997; Dil-
worth and Glenn, 1999; Gould, 1995; Slonczewski and Foster, 1996; Sofos and
Busta, 1999). Also the membrane cyclopropane fatty acid content is a major factor
in acid resistance of E. coli (Brown et al., 1997; Chang and Cronan, 1999).
Another example of bacterial response to stress is that associated with osmotic
pressure. When the osmotic pressure in the surrounding environment increases, water
efflux occurs from the cell; to prevent shrinkage and eventually plasmolysis, cells

activate osmoregulation systems (i.e., mechanisms that provide equilibration of the
intracellular with the environmental pressure) (Gould, 1995; Pichereau et al., 2000).
Osmoregulation is achieved either by active passage of charged (e.g., K+ or glutamate)
solutes, followed by passage of compatible solutes (e.g., trehalose, proline, glycine
betaine, carnitine), or by de novo biosynthesis and accumulation of such osmopro-
tectants, when they are not available in the substrate (Pichereau et al., 2000).
Bacterial stress responses become more complex when cross induction of pro-
tective mechanisms occurs, which confers cross-protection to several stresses regu-
lated by the same gene or group of genes, such as rpoS (Bearson et al., 1997; Hengge-
Aronis, 1993, 1996). As mentioned, significant progress has been made in recent
years in elucidating the underlying genetic and biochemical mechanisms that protect
bacteria from stress, but still these mechanisms need further elucidation (Storz and
Hengge-Aronis, 2000).
EFFECTS OF FOOD-RELATED STRESSES ON BACTERIA

Food-related stresses may occur naturally or may be applied purposefully or inad-
vertently during processing and storage of foods to inactivate or prevent growth of
spoilage and/or pathogenic microorganisms (Archer, 1996; Bower and Daeschel,
1999). It is important to consider that there is a high degree of similarity between
the stresses bacteria encounter in the host and those in foods and food processing
environments (Sheridan and McDowell, 1998). For example, major stresses present
in the human body, such as acid, osmolarity, anaerobiosis, and temperature shifts
from the host to the environment at excretion, are also present in acidic, dried,
cooked/refrigerated, and canned or frozen foods, respectively (Table 9.1). Thus, there
TABLE 9.1
Food-Related Stresses against Pathogenic
Bacteria in Food Environments, Processes
and Products
Stress Food Products or Processes
Acid Mayonnaise, juices, fermented foods

Heat Cooking, minimum thermal processing
Cold Refrigeration
Osmolarity Brine, marinades, fish
Oxidation Hydrogen peroxide treatment
Anaerobiosis Vacuum packaging, sous vide
Starvation Food contact surfaces
(Modified from Sheridan, J.J. and McDowell, D.A., Meat

Sci., 49, 5151, 1998.)
is a need to consider foods as potential stressful environments for bacteria, because

“stresses introduced in foods by preservation or naturally present in foods have a

profound effect on gene expression in bacterial pathogens” (Archer, 1996). This
indicates that the intensity of total stress that may be encountered by bacteria in a
food is a function of the nature of the food and its “hurdle effect,” as affected by
the type, number and severity of technologies applied to preserve it (Leistner, 2000).
When the intrinsic, extrinsic, processing or implicit factors affecting the dynam-
ics of microbial growth in foods (Mossel and Ingram, 1955; Mossel et al., 1995)
move toward their upper or lower limits during food processing or storage, they act
as hurdles which stress the bacteria (Gould, 1995; Leistner, 2000; Sofos, 1993).
Hurdles in foods are dynamic as they continuously change during processing and
storage (Sofos, 1993), while in most instances they are combined to exert a syner-
gistic antimicrobial effect as multiple barriers (Leistner, 2000). As indicated, hurdle
factors that may stress foodborne microorganisms include, among others, tempera-
ture (Palumbo, 1986; Berry and Foegeding, 1997), pH (Dilworth and Glenn, 1999;
Rowbury, 1997), water activity (Gailani and Fung, 1986; Pichereau et al., 2000),
redox potential and anaerobiosis (Potter et al., 2000), carbon dioxide (Genigeorgis,
1985; Hotchkiss and Banco, 1992), sodium chloride (Sofos, 1984), other chemical
preservatives (Brul and Coote, 1999; Sofos, 1989; Sofos and Busta, 1999), natural
antimicrobials (Sofos et al., 1998), nutrient limitation or starvation (Gill, 1976; Rees
et al., 1995), and microbial competition (Fredrickson and Stephanopoulos, 1981;
Hugas, 1998). How, and to what extent, these factors shift from optimal to stressing
and, thus, cause variable effects on bacterial survival or growth depends on the
compositional, processing and storage conditions of the food, as well as on the
physiological and biochemical properties of the bacterial populations present in the
food (Mossel et al., 1995; Sofos, 1993).
Bacteria may respond to stressful food environments in the same manner they
respond to any stress in nature (i.e., by developing respective tolerances, such as
acid tolerance, osmotolerance, thermotolerance, cryotolerance and tolerance to oxi-
dative stress). Expression of the induced tolerances, which may potentially shift to
longer term resistances, depends on stress intensity and whether acid, heat, cold and
other stresses are applied instantaneously or increase gradually over longer periods
of time in the food and, more importantly, in the food plant environment. In most
cases, the bacterial responses to stress involve complex mechanisms, which may be
regulated by groups of genes (Archer, 1996; Sheridan and McDowell, 1998). Expres-
sion of these genes may be variable with bacterial species, and may be affected by
the physiological state of the cells, the rate and severity of the stress applied and
other environmental factors. For example, one of the most important and intensively
investigated food-related resistances of bacterial pathogens is the acid tolerance
response (ATR) (Davis et al., 1996; Foster, 1995; Rowbury, 1997; Slonczewski and
Foster, 1996). The development of acid tolerance can be pH-dependent, pH-inde-
pendent or a combination of both types, depending on the growth phase of the
bacteria undergoing the acid stress (Lee et al., 1994; Lin et al., 1995). There are at
least 11 different ATRs induced under various conditions during growth (Rowbury,
1997), which differ from the acid resistance (AR) responses that only occur in
complex culture media during stationary phase (Lin et al., 1995, 1996).
Overall, bacterial pathogens acquire increased resistance to pH upon exposure
to sublethal pH while they are growing exponentially (Davis et al., 1996; Jordan

TABLE 9.2
Effect of the Type of Acid on the Inactivation Rate of Acid-
Adapted (TSB+G) or Nonadapted (TSB-G) Enterohemorrhagic
Escherichia coli Strains at pH 3.0 and 37ºC
Cells Grown In
Strain TSB+G TSB-G
S2 HCl = malic<citric<acetic<lactic HCl<malic<citric<acetic = lactic

S4 HCl = citric = malic<acetic<lactic HCl<citric<malic<acetic<lactic
S8 HCl<citric<acetic<malic<lactic HCl<citric = malic = acetic = lactic
TSB+G: Tryptic soy broth with 1% added glucose

TSB-G: Tryptic soy broth without glucose
(Modified from Buchanan, R.L. and Edelson, S.G., J. Food Prot., 62, 211, 1999a.)
et al., 1999; Lin et al., 1996; O’Driscoll et al., 1996). In addition, they possess a
GSR that is expressed upon entry into stationary phase, results in a pH-independent
acid tolerance and is regulated by RpoS (Arnold and Kaspar, 1995; Cheville et al.,
1996; Hengge-Aronis, 1996). The complex mechanisms and regulation of expression
of acid resistance, the description of which is out of the scope of this chapter, may
overlap in a way in which more than one ATR mechanisms are simultaneously
activated to protect the cells in concert (Dilworth and Glenn, 1999; Jordan et al.,
1999; Lin et al., 1995, 1996). To what extent each of the concerted ATR contributes
to acid resistance expressed under a given set of environmental conditions is difficult
to determine. For example, stationary-phase cells may exhibit an induced pH-depen-
dent acid resistance, which further increases their GSR (Buchanan and Edelson,
1996, 1999a; Foster, 1995; Lee et al., 1994), but this response varies greatly between
different strains, and in relation to the type of acidulant (Table 9.2). Also, different
bacterial genera or species, such as Salmonella, Shigella, and E. coli, possess dif-
ferent combinations of ATR (Lin et al., 1995).
The complexity of various bacterial responses to acid has resulted in variation
in the techniques and the terminology used to describe these phenomena in food
studies. In general, shifting an exponentially growing culture from neutral to suble-
thally acid conditions (pH ≤ 5.5) may confer protection of pathogens to lethally acid
conditions. When acid shocked cells are subsequently exposed to pH values below
4.0, they may exhibit an increased ATR and cross-protection to other stresses com-
pared to non-shocked cells (Davis et al., 1996; Leyer and Johnson, 1993; Leyer
et al., 1995; Lou and Yousef, 1996, 1997; O’Driscoll et al., 1996). This approach
would be better described as acid shock (Ryu et al., 1999a), rather than acid adap-
tation (Leyer and Johnson, 1993; Leyer et al., 1995; Lou and Yousef, 1997;
O’Driscoll et al., 1996). Another approach is to produce stationary-phase cells under
conditions that result in acid adaptation, (e.g., culturing E. coli O157:H7 in glucose
containing media to induce higher levels of ATR compared to cultures grown without
glucose) (Buchanan and Edelson, 1996). When acid-adapted cells are subsequently

TABLE 9.3
D-Values (min) at Different Heating Temperatures of Previously
Non-Heat-Shocked and Heat-Shocked Escherichia coli O157:H7
in Correlation with the Nature of the Heated Substrate
D-Value at Non-Heat Heat Shocked
Substrate Temperature Shocked (45ºC, 30 min)
Tryptic soy broth 54 12.1 16.6

58 2.2 3.7
62 0.6 0.9
Ground beef slurry 58 4.2 4.1
(Modified from Williams, N.C. and Ingham, S.C., J. Food Prot., 60, 1128, 1997.)
transferred to real or simulated food environments with low pH, they demonstrate
an enhanced survival compared to the non-adapted cells (Berry and Cutter, 2000;
Buchanan and Edelson, 1999a; Gahan et al., 1996; Leyer and Johnson, 1992; Ryu
et al., 1999a). Also, acid adapted cells may be cross-protected against heat or other
stresses (Buchanan and Edelson, 1999b; Ryu and Beuchat, 1999a,b). Acid adaptation
may be considered more realistic than acid shock in food microbiology studies
because microorganisms in foods are more often likely to occur in stationary phase
under nutrient deprivation (Rees et al., 1995), thus, with an activated GSR. In
addition, acid conditions in many foods are built up slowly by fermentation (Caplice
and Fitzgerald, 1999; Lucke, 2000; Samelis et al., 1998), enabling bacterial acid
adaptation by exposure to a gradual decrease in pH, rather than an immediate
exposure to low pH. There are, however, food processes or interventions, which
cause acid shock to bacteria as, for example, processing of acidified foods and
decontamination of meat or fresh produce with organic acids (Beuchat and Ryu,
1997; Smulders and Greer, 1998).
In addition to acid, other stresses (e.g., heat, cold) may also be applied either
instantaneously (shock) or gradually (adaptation) during food processing. Heat shock
has been shown to increase the D values of E. coli O157:H7 (Table 9.3) and Salmo-
nella Enteritidis (Table 9.4), especially in culture broth and under anaerobic condi-
tions of growth. On the other hand, the tempering or slow heating rates of bulk
foods, such as cooked ham and other cured meat products permit gradual bacterial
exposure and adaptation to heat and may result in survivors of increased thermal
resistance in the finished product (Carlier et al., 1996; Mackey et al., 1994; Samelis
and Metaxopoulos, 1999). Likewise, slow cooling rates after cooking and prolonged
periods of drying or refrigerated storage of foods enhance adaptation to cold envi-
ronments or low water activity (Mackey et al., 1994). In contrast, the spraying of
carcasses with hot water (Sofos and Smith, 1998), the rinsing of fresh produce with
nonacid disinfectants (Beuchat and Ryu, 1997), the spray-drying of liquids to form
powders, the blast freezing of small food pieces (e.g., patties, sticks), or the cleaning
of food equipment surfaces with sanitizers (Kumar and Anand, 1998; Mah and
O’Toole, 2001) are short-term shocking treatments. Some of these treatments, how-

TABLE 9.4
D-Values (min) of Heat-Shocked and Non-Heat-Shocked
Salmonella Enteritidis in Culture Broth
Heat Shock D-Value at Temperature (ºC)

(42ºC, 60 min) Incubation Atmosphere 52 58
Control Aerobic 5.3 0.9

Heat shocked Aerobic 16.9 1.3
Heat shocked Anaerobic 20.0 1.8
(Modified from Xavier, I.J. and Ingham, S.C., J. Food Prot., 60, 181, 1997.)
ever, may be followed by extended times of exposure to the residual activity of the
stress applied, resulting in adaptation also.
Starvation is probably the most common stress on bacteria in foods (Rees et al.,
1995). Although starvation does not induce a specific stress resistance per se or
trigger mutations (Archer, 1996) it induces multiresistances as it accelerates bacterial
entry into the stationary phase to enhance survival (Hengge-Aronis, 1993; Pichereau
et al., 2000). As a consequence, starvation has been associated with cross-protection
to several stresses (Arnold and Kaspar, 1995; Jenkins et al., 1990; Nystrom et al.,
1992), while, as mentioned, any stress can confer cross-protection to another stress
sharing regulatory systems (Rowe and Kirk, 1999). For example, starvation and acid
adaptation (pH 4 to 7) increased thermotolerance (56°C) of L. monocytogenes (Lou
and Yousef, 1996), whereas starvation induced cross protection against osmotic
challenge in E. coli (Jenkins et al., 1990). Acid adaptation (pH 5.5, 1 h) protected
L. monocytogenes against thermal (54°C) and osmotic (2.5 M NaCl) stresses
(O’Driscoll et al., 1996). An increased thermotolerance at 50°C was induced by high
osmolarity (0.15 to 0.3 M NaCl) in Salmonella (Fletcher and Csonka, 1998). Acid
adaptation (pH 4.5 to 5.0) protected L. monocytogenes against lethal doses of ethanol
(17.5% vol/vol) and hydrogen peroxide (0.1% wt/vol) (Lou and Yousef, 1997), while
it cross-protected (pH 5.8) S. Typhimurium against heat (50 to 57.5°C), salt (2.5 M
NaCl) and an active lactoperoxidase system (Leyer and Johnson, 1993), and provided
(pH 5.5) cross-protection to sodium lactate (10 to 30%) and sodium chloride (5 to
15%) in E. coli O157:H7 (Garren et al., 1998). Adaptation to ethanol (5%, vol/vol)
increased resistance of L. monocytogenes to 25% sodium chloride (Lou and Yousef,
1997). Heat shock (45°C for 1 h) increased resistance of L. monocytogenes to 1%
hydrogen peroxide (Lou and Yousef, 1997), while it enhanced (48°C, 10 min) acid
tolerance (pH 2.5, minimum glucose medium) of E. coli O157:H7 (Wang and Doyle,
1998). Habituation of Salmonella spp. at reduced water activity (0.95) in media with
various solutes increased its heat tolerance (Mattick et al., 2000b). Listeria mono-
cytogenes cultivated under low nutrient conditions showed increased tolerance to
chlorine sanitizers (Lee and Frank, 1991).
In general, research and experience have shown that foodborne bacteria display
a broad spectrum of resistance responses to common food preservation techniques,
and resistant bacterial pathogens, such as E. coli O157:H7 (Armstrong et al., 1996;

Park et al., 1999), S. Typhimurium DT104 (Davis et al., 1999; Glynn et al., 1998),
C. jejuni (Alterkruse et al., 1999; Humphrey, 1995), and L. monocytogenes, have
emerged and are a global problem (Foster, 1997; Mead et al., 1999; Tauxe, 1997).
These pathogens and other causative agents of foodborne illness outbreaks may
possess unusual growth characteristics, may be multi-stress and multi-drug resistant
or may be virulent at low infective doses (Archer, 1996; Gahan and Hill, 1999;
Threlfall et al., 2000). The potential existence, development or establishment of such
multi-stress-resistant pathogenic strains in food environments may explain why
traditionally low risk foods, such as fruit juices (Besser et al., 1993), fermented
meats (CDC, 1995a,b,c), fresh produce (Ackers et al., 1998), and dried products
(Keene et al., 1997) have become vehicles of foodborne illnesses. This suggests that
traditional preservation barriers are becoming ineffective against multi-stress resis-
tant pathogenic bacteria developing in our food supply. Also, the intentional or
inadvertent exposure of bacteria to sublethal food-related stresses may enhance their
stress adaptation and survival under unfavorable conditions. Thus, development of
bacterial resistances and cross-protection effects, which are well documented with
pure pathogenic cultures grown under laboratory conditions, may also be a reality
in foods. Since adaptive mutations of pathogens may also be associated with
increased virulence (Sheridan and McDowell, 1998), stress-adapted, multi-resistant
mutants that may develop in food environments may be difficult to control and may
dramatically increase food safety risks. This concern is particularly important for
at-risk populations, such as the elderly, infants, children, pregnant women, patients
with chronic diseases and other groups of immunosuppressed individuals (Morris
and Potter, 1997).
In summary, any food processing, decontamination or storage method has the
potential to cause shock, adaptation, or shock/adaptation, and lead to cross-protection
to environmental food-related stresses. This necessitates the reconsideration of tra-
ditional food preservation barriers by including pathogen stress responses in food
validation studies to avoid underestimation of potential risks and to enhance food
safety (Knochel and Gould, 1995; Sofos, 1993, 2001).
RESEARCH NEEDED TO CONTROL STRESSED

PATHOGENS IN FOODS
The complexity of, and our developing but still limited knowledge on, the microbial
responses to environmental stresses makes the control of stress-adapted pathogens
in foods a difficult task. Achieving this objective, however, is a necessity that will
challenge food microbiologists in the coming years. According to Lederberg (1997),
“we have barely started to study the responses of microorganisms under stress,
although we have examples where root mechanisms of adaptive mutability are
themselves responses to stress.”
Development of strategies for pathogen control in foods should aim at:
(1) controlling resistant and stress-adapted pathogens; (2) minimizing resistance
development; and (3) avoiding virulence changes. Thus, the overall strategic plan
for pathogen control should have two major targets: (1) trying to maintain control of

the existing situation and (2) preventing the existing situation from becoming worse.
Bower and Daeschel (1999) proposed five approaches to control stress resistant bac-
terial pathogens in foods and, overall, in nature: (1) change antibiotic usage practices;
(2) develop new antibiotics; (3) apply hurdle preservation approaches; (4) prevent
bacterial adhesion; and (5) utilize competitive exclusion. These approaches are among
the many useful ones that can be applied, and indicate the need to consider foods as
a part of the environment, which is the main reservoir of stress-resistant microorgan-
isms. On this basis, it is important to consider that, while antibiotic-resistant bacteria
are an increasing problem for patients at home or in hospitals, current information
on the potential of bacteria to overcome food preservation hurdles solely because
of their drug resistance is scanty. Indeed, multidrug-resistant pathogens, such as
S. Typhimurium DT104, have been isolated from animal feeding and food processing
environments (Davis et al., 1999), but the impact of their antibiotic resistance on
survival under stressful conditions in foods is unclear. Thus, more research is needed
to address the problem by a better understanding of potential cross effects between
antibiotics and other stress factors on bacteria.
Research should be multidisciplinary and proactive to determine mechanisms
and controls rather than only describe unpredicted bacterial responses (Buchanan,
1997; Sofos, 2001). In addition, basic research should investigate stress responses
at the cellular or subcellular level. Factors involved in global stress response need
to be identified, while accurate determination and correlation of the physiological
reactions of bacteria responding to stress with the respective biochemical and
genomic alterations occurring inside the bacterial cell are required. Continuous
updating of our knowledge on microbial genetics and physiology is a prerequisite
for a better understanding of bacterial responses to stress. Stress-related mutations
and gene transfer, growth requirements, survival characteristics and pathogenicity
of bacteria under stress and their mechanisms to cope with the stress by maintaining
cell homeostasis and developing resistance or enhanced virulence need further elu-
cidation (Buchanan, 1997; Lederberg, 1997; Sofos, 2001; Tauxe, 1997).
Research should also examine whether responses similar to those triggered
in vitro or in vivo in the laboratory possibly occur in situ in multifactor ecosystems,
including foods. This is because, unlike pure cultures in laboratory media, bacterial
pathogens in foods are usually a minor component of a complex microbial associ-
ation dominated by food-specific, environmentally adapted microbial species (Agge-
lis et al., 1998; Dainty and Mackey, 1992; Mossel et al., 1995; Samelis et al., 1998;
Sofos, 1994). While the food itself may represent an oligotrophic and stressful
environment, the different microorganisms present compete strongly for uptake of
limiting nutrients to initiate growth, and eventually predominate. Thus, survival and
development of pathogens in foods frequently takes place under limited nutrient
availability and adverse conditions of pH, osmolarity, oxidation, temperature, chem-
ical residues and competition by other microorganisms. Therefore, to accurately
determine or predict the response of a pathogen to stress, we need to consider all
micro-ecological factors involved in the processing of a specific food.
Intensified research is needed to clarify the behavior of foodborne pathogens in
their natural environment. Despite progress in epidemiology, the origins and main
reservoirs of contaminating pathogenic strains, and the sources and routes of con-

tamination (e.g., pathways of transmission from non-food to food environments or
throughout the food processing line) are largely unknown. This is because, until
recently, characterization of foodborne microorganisms, including pathogens, was
primarily based on physiological, biochemical or immunological criteria determined
by classical methods (Farber, 1996). These methods are valuable, but they are time-
consuming and cannot follow up bacterial transmission and cross contamination
involved in the chain of food processing steps. However, the recent developments
in polyphasic taxonomy, based on genotypic molecular characterization and finger-
printing methods (Vandamme et al., 1996), should enable food microbiologists to
compare bacterial strains originating from different natural environments, food
niches or food processing steps, and thereby permit the establishment of their mode
of transmission (Farber, 1996).
Another limitation has been the difficulty in detecting small numbers of unpre-
dictably distributed target pathogens among high numbers of a diverse, non-patho-
genic, spoilage flora in complex and nonhomogeneous foods. As sampling and
detection methods improve (Brown et al., 2000; de Boer and Beumer, 1999; Manafi,
2000; Swaminathan and Feng, 1994; Vernozy-Rozand, 1997), their application will
enhance pathogen surveillance during food processing. For example, advanced
molecular techniques, such as denaturating gradient gel electrophoresis (DGGE),
are currently in progress and may allow rapid monitoring of microbial population
dynamics directly in a food, without the need of obtaining bacterial isolates (Ben
Omar and Ampe, 2000; Cocolin et al., 2001). Also, on line pathogen detection in
the field and in the food industry with biosensors is receiving increasing attention.
There is also a need to improve virulence testing and toxin detection methods
(Pimbley and Patel, 1998) and to investigate virulence changes in relation to resis-
tance mechanisms and the behavior and stress response of pathogenic bacteria in
foods. At present, there is evidence that stress enhances virulence under laboratory
conditions (Archer, 1996; Buncic and Avery, 1998; Foster, 1995; Gahan and Hill,
1999; O’Driscoll et al., 1996). However, whether this response may also be inducible
to stressed pathogens present in foods, and the magnitude of increased virulence
these mutants may attain compared to their non-stressed parental strains, are
unknown. It is, therefore, necessary to investigate potential strain variations in stress
resistance and virulence in relation to the sources and routes of food contamination
and the sensitivity of at-risk populations (Takumi et al., 2000). Further areas to be
investigated for more effective control of resistant pathogens in foods include the
mechanisms through which bacteria examine and sense their surrounding environ-
ment (Dorman, 1994; Lazim and Rowbury, 2000; Rowbury, 1997, 2001), how they
process and transmit this information to their genome, and how the mechanisms of
changes in gene expression are activated (Storz and Hengge-Aronis, 2000). These
are challenging research issues that require copious studies.
In addition to the elucidation of the genetics and physiology of resistant patho-
gens and determination of mechanisms of survival and resistance development in
restrictive environments, advanced food-related research approaches are required.
As indicated, the ideal approach is to evaluate food systems by considering them as
stressful environments. On this basis, farm to fork evaluations and measures are
needed to increase the effectiveness and practical significance of the research under-

taken. Animal and plant production practices may be decisive for induction of
pathogen resistance. For example, the potential role of diet shifts on the acid-
resistance of E. coli in cattle has been an important, but controversial, research issue
in the United States in recent years (Diez-Gonzalez et al., 1998; Russell et al., 2000).
This underscores the difficulty in understanding complex responses of foodborne
pathogens in complex environments. Also, pathogens resistant to antibiotics, pesti-
cides and other chemical residues are increasingly being isolated from animal feeding
(Davis et al., 1999; Tollefson and Miller, 2000) and plant cultivation (McManus,
2000) environments, which otherwise would have been exposed to normal-range
magnitudes of natural environmental stresses. Exposure of multidrug-resistant iso-
lates from these habitats to common stresses encountered in food systems may reveal
potential correlation between those two types of resistances.
An additional area to be examined by reasearch is bacterial stress resistance
development in response to changing food preparation practices at home. As indi-
cated, increasing numbers of consumers lack the knowledge necessary to handle and
prepare foods safely, while preparation of mildly cooked or undercooked meals and
application of non-validated cooking procedures in microwave ovens or other home
appliances is increasing. Although these consumer-related changes in food prepara-
tion may induce bacterial resistance, their impact on food safety has been largely
overlooked. During home-drying of foods, for example, multiple stresses are applied
(e.g., marination, gradual heating to enhance vaporization, decrease in water activity
and increase in osmolarity due to water removal and salting of cured meats, low pH
and acidity due to concentration of solids, etc.). However, the magnitude of stresses
and the drying times may be low and long enough, respectively, to allow stress
adaptation, resistance development and eventually survival of bacterial pathogens in
the final, ready-to-eat product. Following that, some bacterial pathogens may survive
during storage of dried foods, temperature and atmosphere permitting, while others
become inactivated. Recent research data support these concerns. For example, acid-
sensitive enteric pathogens were protected from killing under acidic conditions when
attached onto solid foods, such as ground beef (Waterman and Small, 1998), while
an increased thermotolerance may be induced by high osmolarity in Salmonella
(Fletcher and Csonka, 1998; Mattick et al., 2000b). Salmonella Enteritidis and S.
Typhimurium DT104 could survive at a water activity of 0.92 for long periods by
increasing their biomass via filament formation, and their survival was enhanced as
the temperature was lowered (Mattick et al., 2000a). As expected, increasing the
osmotic pressure by addition of sodium chloride increased the lag phase and
decreased the growth rate of L. monocytogenes (Vasseur et al., 1999); however, the
osmotically stressed pathogenic cells survived on processed meat surfaces by accu-
mulating osmolytes (Smith, 1996). The inactivation of E. coli exposed to osmotic
stress was biphasic; the initial high death rate was followed by a slower second
phase decline, or “tailing” effect (Shadbolt et al., 1999). Osmotic resistance of E. coli
was greater upon starvation (Jenkins et al., 1990), while exposure of E. coli O157:H7
to acidic environments increased its heat tolerance (Buchanan and Edelson, 1999b;
Ryu and Beuchat, 1999a); a major concern when drying fruits at home after acid
soaking.

Escherichia coli O157:H7 could survive in dried beef powder and other foods,
but its survival was suppressed as the water activity and the pH of the product were
decreased and the storage temperature was increased (Deng et al., 1998; Ryu et al.,
1999b). Also, previous storage or pre-incubation of ground beef at higher temper-
atures resulted in increased sensitivity and decreased survival of E. coli O157:H7
following a decrease in its acid tolerance (Cheng and Kaspar, 1998). Importantly,
E. coli O157:H7, as well as Salmonella and L. monocytogenes, increases its ability
to recover heat damage in foods when stored under anaerobic conditions or under
a low redox potential; thus, the anticipated level of safety in foods dried by heating
might not be achieved (George et al., 1998; Murano and Pierson, 1993).
In summary, plant cultivation, animal feeding, and food production, harvesting,
transportation, decontamination, sanitation, processing, distribution and storage
technologies, as well as food preparation methods in restaurants, catering or at home,
need to be evaluated for contribution to bacterial resistance. This evaluation is
important to perform for both the traditional and novel technologies and procedures
used in food processing and handling, by considering all variables that may contrib-
ute to resistance.
RESEARCH APPROACHES FOR CONTROL

OF STRESSED PATHOGENS
In order to meet current research needs associated with control of stress-adapted
pathogenic bacteria in foods, we also need to advance our research approaches by
introducing more complex and challenging objectives and multifactorial experimen-
tal designs, and by using advanced microbiological detection and typing methods.
Considering the recent salmonellosis and hemorrhagic colitis outbreaks implicating
acidic, fermented, or home-dried foods, it appears necessary to reconsider critical
limits at critical control points for traditional food manufacturing practices.
Advanced research approaches are required to explain complex mechanisms of
pathogen emergence, resistance and virulence in foods and to avoid false estimations
of potential food safety risks.
Significant modifications in current food production, processing and handling
systems may be necessary to avoid or minimize resistance and virulence changes in
bacteria and to assure food safety. Modifications should be based on results of well-
designed pathogen challenge and validation studies (e.g., finding combinations of
interventions or processes that guarantee the delivery of pre-specified reductions of
target pathogens) to achieve established food safety objectives (van Schothorst,
1998). These studies should be based on worst case scenarios (e.g., high levels of
food contamination with very resistant pathogenic strains; pathogen protection and
survival enhancement due to the food microenvironment; food storage under con-
ditions that prevent destruction of survivors, etc.). For example, in validation studies,
it is important to use multistrain and stress-resistant inocula, which originate from
similar niches and, better yet, have been exposed to conditions as stressful as they
are expected to encounter in real foods. Thus, inocula can be previously grown in
synthetic media of minimal composition or food extracts, in the presence of chemical

TABLE 9.5
D-Values (min) of Escherichia coli O157:H7 at Different Heating
Temperatures or Concentrations of Lactic Acid as Affected
by the Growth Temperature of the Pathogen Prior to Exposure
to Heat or Acid and the Origin of the Strain
Culture Growth Origin Heating Temperature Lactic Acid Concentration
Temperature of Strain (ºC) (%)
(ºC) 52 56 0.1 0.5
10 Beef 11.2 2.5 3.7 0.35

Cider 40.7 5.1 5.3 0.44
Beef 17.7 9.3 6.4 1.7
Cider 89.4 14.1 4.8 0.47
(Data from Semanchek, J.J. and Golden, D.A., J. Food Prot., 61, 395, 1998.)
or natural preservatives, or competitive flora, or harvested following prior habitua-

tion/growth in liquid foods or on solid food surfaces.
Strains that have previously been isolated from outbreaks caused by related foods
and are of increased resistance or virulence are the best candidates for inclusion in
multistrain inocula. For example, an outbreak E. coli O157:H7 strain associated with
apple cider demonstrated higher D values than a strain from beef (Table 9.5), poten-
tially due to its origin from a more stressful, acidic food environment. For this reason,
it is essential to deposit in culture collections more strains and their respective
epidemiological and biochemical data, isolated from foodborne outbreaks and indi-
cating unusual stress resistance.
It is very important to understand the significance of testing resistance of stressed
bacterial populations isolated directly from foods, rather than to monitor the survival
of purposefully stress-adapted laboratory cultures inoculated in challenge media or
foods. On this basis, research should investigate pathogen responses to sequential
applications of stresses of varying intensity in situ in foods. Research with mixed
microbial cultures in real foods, or in media that are simulatory to foods, should be
intensified. In other words, we need to determine survival and development of
resistance of surviving pathogens to initial or secondary food-related stresses, with
the natural flora present, and to follow or simulate their passage from diverse
environments in the field to the food processing chain and the host.
Succession of stresses, as affected by their type, order of application, magnitude
and duration, may allow survival and resistance development. The responses of
surviving pathogen populations in the presence of food-specific natural flora should
be examined. This is necessary because the metabolic activity and competition by
this natural flora may induce additional stresses, may alter the intensity of existing
stresses, or may affect the sensing ability and the timing of pathogen responses to
these stresses. Indeed, recent data from basic microbiological studies have revealed
that early sensing of environmental stresses by bacteria may be due to extracellular

sensors to alarmones and cross-talking between cells rather than due to intracellular
compounds (Lazim and Rowbury, 2000; Rowbury et al., 1998; Rowbury 2001). Thus,
compounds found or added to a food or formed by natural flora may act as extra-
cellular stress sensors to alarmones to pathogenic cells, or may suppress the formation
of such alarmones to eventually sensitize pathogens. Alarmones and their precursors
or inhibitors may be absent in cultivation or challenge media, which are sterile or
defined in chemical composition. It appears, therefore, that the natural competitive
flora may do more than simply affect growth, which is well documented in the
literature (Breidt and Fleming, 1998; Buchanan and Bagi, 1999; Drosinos and Board,
1994; Duffy et al., 1999; Farrag and Marth, 1989; Janisiewicz et al., 1999b). Com-
petitive or synergistic interactions of mixed microbial associations may alter or
monitor stress resistance responses of bacterial pathogens under conditions encoun-
tered in food environments, which may have a significant impact on food safety
(Samelis et al., 2001b).
Advanced research approaches are needed to investigate long-term effects of
food processing, decontamination and storage technologies on development of bac-
terial stress resistance and to examine subsequent responses of surviving bacteria
exposed to a set of stressors. The benefits from the commercial application of novel
technologies may be attractive enough to adopt them without much concern or
skepticism as to potential negative impacts on the environment or humans and
without evaluation of potential undesirable effects. For example, modified atmo-
sphere packaging (MAP) of foods had been introduced commercially for at least 20
years before major concerns were expressed regarding the microbiological safety of
this technology (Genigeorgis, 1985; Hotchkiss and Banco, 1992; Sofos, 1993). It
was suggested that low-oxygen packaging methods may enhance survival and growth
of facultative or anaerobic pathogens, such as L. monocytogenes and clostridia. The
concerns may have been reasonable, as indicated by the early botulism outbreaks
associated with consumption of vacuum packaged smoked fish in the 1960s and the
recent 1998–1999 listeriosis outbreak from the consumption of ready-to-eat vacuum
packaged processed meats in the United States (CDC, 1999). The latter outbreak
renewed interest on hurdles for control of post-processing contamination of
L. monocytogenes in processed meat products. Potential hurdles under investigation
are the inclusion of chemical antimicrobials in the formulation (Bedie et al., 2001;
Blom et al., 1997; Harmayani et al., 1993; Wederquist et al., 1994), immersion in
post-processing antimicrobial solutions (Samelis et al., 2001e), post-packaging thermal
pasteurization (Roering et al., 1998), inoculation with protective cultures (Bredholt
et al., 1999; Degnan et al., 1992; Hugas, 1998; McMullen and Stiles, 1996) and
potential application of emerging technologies such as high pressure and irradiation
(Farber and Peterkin, 1999). Most of these hurdles have shown promise in controlling
L. monocytogenes, but their potential long-term effects in inducing stress resistances
to pathogen survivors in the packaged food or in the host and the environment are
largely unknown.
Another recent case of using a new technology without really knowing its
potentially long-term negative impact on bacterial stress resistance is decontamina-
tion of fresh foods. As mentioned, the commercial use of antimicrobial interventions
to reduce contamination of meat and fresh produce with pathogenic bacteria is

steadily increasing, particularly in North America (Beuchat and Ryu, 1997; Siragusa,
1995; Smulders and Greer, 1998; Sofos and Smith, 1998). Also, following several
outbreaks from fresh produce in recent years, consumers have adopted recommen-
dations to increase washing of raw foods with disinfectants at home. Research has
clearly demonstrated that decontamination technologies or treatments with disinfec-
tants deliver significant reductions (1 to 3 logs) in populations of total and pathogenic
bacteria, particularly in those of enteric pathogens such as E. coli O157:H7 and
Salmonella (Cutter and Siragusa, 1994; Dorsa et al., 1998a; Hardin et al., 1995;
Smulders and Greer, 1998; Sofos and Smith, 1998). However, the impact of heat,
organic acids, sanitizers and other types of chemicals used as decontaminants or
disinfectants in inducing resistance to pathogens surviving the process or contami-
nating the food post-decontamination has not been addressed adequately. Most
studies are limited at determining the immediate antimicrobial effect of decontam-
ination on the target pathogens or the total bacterial populations (Bacon et al., 2000;
Beuchat, 1996a; Delmore et al., 2000; Hardin et al., 1995; Siragusa, 1995; Smulders
and Greer, 1998; Sofos and Smith, 1998). In recent years, however, there is an
increasing scientific interest in evaluating the consequences of decontamination on
the microbial ecology of fresh meat and produce and the impact of possible impli-
cations of flora changes on product safety and shelf life (Jay, 1996, 1997; Van Netten
et al., 1994).
In response, the residual effect of several intervention treatments on bacterial
growth during subsequent refrigeration storage of products that were intact after
decontamination or originated from decontaminated primal units was evaluated
(Dorsa et al., 1998b; Kenney et al., 1995; Prasai et al., 1997; Van Netten et al., 1994,
1998). Results were conflicting as to whether growth of natural flora and inoculated
pathogens was affected in stored product, while experimental conditions varied
among studies to allow for clear conclusions. However, an overall conclusion has
been that pathogens that may survive or contaminate meat after decontamination do
not seem to represent an additional health risk (Dorsa et al., 1998a,b; van Netten
et al., 1998). Additional studies, however, have shown that bacterial pathogens on
decontaminated meat may recover and multiply to be equal to or even higher than
levels of respective pathogens surviving on or contaminating the untreated product.
Specifically, high levels of natural flora inhibited growth of E. coli O157:H7 in
ground beef (Vold et al., 2000), while the numbers of the pathogen on decontami-
nated beef (e.g., by steam vacuuming combined with spraying with 0.2 M lactic
acid) increased by nearly 3 logs compared to 1 log and virtually no increase on the
untreated beef stored (10°C) in air and vacuum, respectively (Nissen et al., 2001).
It should be stressed though that inhibition of pathogens by the background flora
on nondecontaminated meat stored at temperatures ≤10°C appears to be more due
to retardation of growth because of the high population increases of the competitive
flora, rather than due to an antimicrobial effect. For example, pathogens more
psychrotrophic than E. coli O157:H7, such as S. Enteritidis and Y. enterocolitica,
increased to similarly high levels (7 to 9 logs) on decontaminated and untreated
chicken or pork skin, respectively, indicating increased competitiveness with the
natural flora at 10°C (Nissen et al., 2001). It should be emphasized, however, that
there is a significant percentage reduction of Salmonella or E. coli biotype 1- or

O157:H7-positive beef carcasses in United States plants that use decontamination
(Bacon et al., 2000; Elder et al., 2000; Sofos et al., 1999a, b, c).
Decontamination of fresh foods packaged under vacuum or MAP may also
significantly reduce the numbers of indigenous lactic acid bacteria that potentially
inhibit L. monocytogenes and other pathogens during anaerobic storage (Babic et al.,
1997; Carlin et al., 1996; Nissen et al., 2001; Vold et al., 2000). For this reason, the
use of salts of organic acids, natural antimicrobials or protective cultures as additional
microbial barriers is also under investigation for fresh vacuum packaged meats or
produce (Ajjarapu and Shelef, 1999; Bennik et al., 1999; McMullen and Stiles, 1996;
Smulders and Greer, 1998). However, potential effects of chemical antimicrobials,
competitive natural flora or protective cultures on stress resistance of the coexisting
pathogens, and on their secondary stress responses, are largely unknown.
There is a need for research to examine the long-term benefits of risks of
decontamination of fresh meat or other foods as they relate to pathogen reduction
and potential development of long-term bacterial resistances. Such research should
determine benefits and recognize risks, which may be eliminated or minimized by
proper application of technologies. Numerous studies have shown that healthy E. coli
O157:H7 inocula may survive for extended times in fresh fruit juices or other acidic
foods, especially if previously acid-shocked or acid-adapted and the food is stored
at refrigeration temperatures (Glass et al., 1992; Leyer et al., 1995; Miller and
Kaspar, 1994; Tsai and Ingham, 1997; Zhao and Doyle, 1994). In contrast, few
studies have evaluated whether acid-injured survivors that may be generated in such
foods maintain high resistance upon subsequent exposure to acid or other stresses
(Ingham and Uljas, 1998; Riordan et al., 2000; Uljas and Ingham, 1998). One study
found that E. coli O157:H7 cells that had previously been exposed to acidic (pH 3.5)
apple juice were of equal or lower acid tolerance in a simulated gastric fluid,
compared to their parental strains habituated in nonacidic juice (pH 6.5) or broth
acidified with various acids (Uljas and Ingham, 1998). Also, E. coli O157:H7 cells
that had previously survived in pepperoni fermented to final pH 4.5 were of decreased
thermal tolerance in heated pepperoni, compared to their parental strains that sur-
vived in pepperoni of higher (4.8) final pH (Riordan et al. 2000). Likewise, Salmo-
nella strains previously adapted for growth in acidic conditions by sequential transfer
in broth (pH 5.0; final growth 107 CFU/ml) had either equal or greater sensitivity
to organic acid rinses of beef tissue and significantly lower D55 values than their
parental strains (Dickson and Kunduru, 1995). It appears, therefore, that based on
the results of studies with laboratory cultures, pathogen survivors from acidic foods
or fresh foods decontaminated with organic acids, or survivors in the processing
environments of such foods, may acquire resistance to acid or other stress to a level
different from that anticipated.
Recent and ongoing research in our laboratory has been undertaken to examine
potential safety risks and to determine critical control points and limits associated
with stress-adapted pathogens in foods (Lakkakula et al., 2001; Samelis et al.,
2001a,b,c,d, 2002a,b; Stopforth et al., 2001). Based on previous data (Brackett et al.,
1994; Cutter and Siragusa, 1994), it was assumed that meat decontamination may
be incomplete or that poor sanitation may allow pathogenic bacteria to establish
niches in the plant (Sofos and Smith, 1998). Therefore, we used decontamination

washing residual waste fluids (washings) from fresh meat, either acidic or plain
water washings, as a natural stressful and oligotrophic environment to monitor
survival and growth, as well as the initial and subsequent ATR of acid-adapted,
partially acid-adapted or nonadapted stationary-phase inocula of L. monocytogenes,
E. coli O157:H7 and S. Typhimurium DT104 in the presence or absence of natural
flora at incubation temperatures ranging from 4 to 15°C (plant conditions) to 35°C
(abusive plant conditions or optimum temperature range of the pathogens in model
studies). Initially, it was shown that E. coli O157:H7 has greater potential compared
to L. monocytogenes or S. Typhimurium DT104 for survival (up to 7 days) in 2%
organic acid meat washings, even with moderate previous adaptation to acid, and
mainly when acetic rather than lactic acid is sprayed and the washings are kept at
4°C compared to 10°C (Samelis et al., 2001a). As expected, acid adaptation of E. coli
O157:H7 enhanced survival in acidic washings under the same storage conditions;
acid-adapted populations of the pathogen survived with minimal reductions for 14
days in 2% acetate washings or in mixtures of both 2% acetate or lactate washings
with water washings at ratios of 1:1, 1:9 or 1:99 (Samelis et al., 2002b).
Mixing of acidic with water washings was purposefully done to obtain decon-
tamination waste fluids of sublethal pH and lowered acid concentration, as may be
the case in meat plants. Declines of nonadapted populations followed a similar
pattern, but they were much faster compared to those of acid-adapted populations
(Samelis et al., 2002b). On the other hand, in water (control) washings at 4 and
10°C, E. coli O157:H7 survived but the low storage temperatures, the predominant
growth (>108 CFU/ml at 2 to 4 days) of the natural meat flora and an apparently
limited nutrient availability synergistically inhibited its growth (Samelis et al.,
2001a,b). Importantly, nonadapted E. coli O157:H7 populations showed greater
potential for survival and a tendency to initiate growth in plain water washings
compared to acid-adapted populations at 10°C, suggesting that acid adaptation
negatively influenced the pathogen ability to readapt upon a sudden shift to high
pH conditions (e.g., pH 6.5 to 7.5) prevailing in water washings (Samelis et al.,
2002b).
Based on these findings, additional experiments were done at 15°C and indicated
that, while the behavior of E. coli O157:H7 within treatments was unchanged, the
higher incubation temperature accelerated pathogen death in acidic washings, while
in nonacid (water) washings, it enhanced pathogen growth by approximately 2 logs,
irrespective of type of inoculum (unpublished data). It should be also stressed that
acid-containing washings diluted with water washings, at ratios up to 1:9, suppressed
growth of the predominant Pseudomonas-like natural meat flora while they were
simultaneously selective for growth of lactic acid bacteria and yeasts. This natural
species selection was not observed in very dilute acid:water (1:99) washings where
the normal Gram-negative meat flora could overcome the inhibitory effect of acid
and eventually grow to high (8 logs) levels similar to those in plain water washings
(Samelis et al., 2002b).
These findings are in agreement with earlier studies indicating that the acid
tolerance of E. coli O157:H7 may be pH-inducible, and underscored potential meat
safety risks associated with this induction (Buchanan and Edelson, 1999a; Berry
and Cutter, 2000). In fact, results were consistent with survival and growth of E. coli

O157:H7 on acid-decontaminated vs. water-decontaminated or untreated meat
(Berry and Cutter, 2000; Brackett et al., 1994; Dorsa et al., 1998a,b; Nissen et al.,
2001; Siragusa, 1995; van Netten et al., 1998). Furthermore, it was clearly demon-
strated that acid decontamination interventions do have the potential to alter the
microbial ecology of meat plant environments (Samelis et al., 2002b) in addition to
that of the acid-treated meat (Van Netten et al., 1994).
Interesting and potentially important results were obtained by evaluating the
ATR attained by E. coli O157:H7 following exposure to meat washings (unpublished
data). Acid-adapted or nonadapted survivors (3.0 to 5.0 logs) of the pathogen in 1:9
or 1:99 mixtures (pH 3.1 to 4.7) of 2% lactate or 2% acetate washings with plain
water (pH 6.8 to 7.1) meat washings after 2 or 7 days of storage at 10°C were re-
exposed to broth acidified to pH 3.5 with lactic acid. Survivors (4.2 to 5.2 logs)
from plain water washings were also acid-challenged to serve as control. Day-2
survivors from acid-containing washings survived at pH 3.5 much better than sur-
vivors from water washings, especially when the mixtures contained acetic acid and
the original inoculum was acid-adapted. High ATR was maintained in day-7 survi-
vors from acetate washings, which were more numerous, as suffering less acid injury,
but not in day-7 survivors from lactate washings. It may be postulated that the latter
survivors were either energetically exhausted (1:9 dilution) to be able to survive the
secondary acid stress, or that they shifted to acid sensitive soon after the metabolic
activity of the Gram-negative natural flora eliminated the acid stress (1:99 dilution)
in the washings. It should be indicated that acid-adapted populations from plain
water washings continued to exhibit very low ATR at day 7, while nonadapted
populations showed a clear reverse trend (Samelis and Sofos, 2002).
The above findings indicate that acid stress may lead to development of acid-
resistant strains of E. coli O157:H7 in fresh meat environments, when its magnitude
(i.e., pH and type of acid) and duration (i.e., time of exposure) are not adequate to
energetically exhaust the pathogen to death, or the natural flora cannot eliminate
long-term sublethal pH effects. This may lead to potential risks because, as men-
tioned, acid decontamination of fresh meat may be ineffective per se to completely
inactivate pathogens (Brackett et al., 1994; Cutter and Siragusa, 1994; Dorsa et al.,
1998a). Moreover, acid decontamination is not followed by other lethal stresses in
immediate sequence, as in the case in fermenting meat products with a gradually
decreasing water activity. Therefore, acid-injured pathogen survivors may have time
and enough energy remaining to survive (Berry and Cutter, 2000; Samelis et al.,
2001a) and grow (Nissen et al., 2001; Van Netten et al., 1998) as potentially adaptive
mutants on the meat or in the plant. Similar phenomena may occur in any other type
of food where the intensity of total stress is insufficient to eliminate or exhaust and
irreversibly injure surviving bacterial pathogens. This postulation, which requires
experimental verification, leads to a proposed novel hypothesis for development of
strategies to enhance food safety (unpublished data), as discussed later.
In our studies, an important case of an unexpected secondary bacterial stress
response has been a dramatic acid sensitization of E. coli O157:H7 following expo-
sure to plain water washings for 2 or 7 days at 10°C, particularly when the pathogen
was previously acid-adapted (Samelis et al., 2002a). A similar trend was also noted
for L. monocytogenes and S. Typhimurium DT104 in water washings under the same

incubation conditions (Samelis et al., 2001c,d). Indeed, all three pathogens exposed
to water washings at 10°C for 2 to 8 days became more acid-sensitive compared to
their acid-adapted, partially acid-adapted or nonadapted inocula (day 0) previously
grown at 30°C, or pure cultures previously grown in broth at 10°C. It needs to be
stressed though that acid sensitization of L. monocytogenes and S. Typhimurium
DT104 at 10°C was not so prominent because both pathogens were of lower acid
resistance compared to E. coli O157:H7 (Samelis et al., 2001a). Also, both pathogens
were acid-sensitized while they were growing exponentially (e.g., ca. 2-log increase)
in water washings at 10°C (Samelis et al., 2001c,d), which was not the case for the
less psychrotrophic E. coli O157:H7 (Samelis et al. 2002a). Accordingly, the log-
phase populations of L. monocytogenes and S. Typhimurium DT104 during the first
days in water washings were of expectedly greater acid sensitivity compared to
1-week-old stationary-phase populations, which were also acid-sensitized. Thus,
despite these differences between pathogens, their acid sensitization in nonacid meat
decontamination waste fluids was shared, suggesting a role of the dominant natural
flora, its catabolic products and potentially other compounds transferred to the
washings from the meat in enhancing this response, which may be of major practical
importance.
As discussed, the potential effects of natural flora on pathogen responses to
stresses have not been examined adequately, despite the coexistence and competition
of spoilage and pathogenic bacteria in foods. To examine these effects, a logical
research approach is to determine the initial and secondary stress responses of
bacterial pathogens in real or model food systems under identical conditions, in the
presence of natural flora or after its removal prior to processing or at the beginning
of storage. Very few studies have systematically investigated this issue, mainly using
laboratory media rather than foods. In culture broth incubated at 37°C, Duffy et al.
(1995) reported that the addition of 108 CFU/ml viable competitors (i.e., E. coli,
Citrobacter freundii and Pseudomonas fluorescens) protected an underlying, expo-
nentially growing population (105 CFU/ml) of S. Typhimurium against thermal
inactivation, as the pathogen D values at 55°C increased from 0.43 to 2.09 min. In
a more recent study, Aldsworth et al. (1998) showed that the presence of 108, but
not 105-107 CFU/ml, mixed viable competitors also increased the viability of
S. Typhimurium (105 CFU/ml) under freezing, but addition of equal populations of
heat-killed competitors did not provide this protective effect. Thus, the observed
protective effect to food-related stresses, such as heating and freezing, was associated
with the metabolic activity of the competitive flora, while it could not be correlated
with RpoS (Aldsworth et al., 1998). It was found that by rapidly reducing levels of
dissolved oxygen through active respiration, high levels of competitive flora reduced
oxidative damage to exponential-phase cells of S. Typhimurium and decreased the
RpoS induction time, thus arresting pathogen growth and conferring protection from
stress (Aldsworth et al., 1998). Accordingly, a novel hypothesis was advanced to
explain the increased sensitivity to stress of exponential compared to stationary phase
bacterial cultures: the “suicide” response. Pure cultures of rapidly growing and
respiring bacterial cells exposed to mild stresses will suffer growth arrest but their
metabolism will continue to result in a burst of free-radical production that is lethal
to the cells, rather than the stress itself (Aldsworth et al., 1999). Consistent with this

hypothesis, E. coli O157:H7 was inhibited to a lesser extent in co-culture with a
competitive flora than it was inhibited in pure culture in a simulated fermentation
broth (from pH 5.8 to 4.8) at 37°C, suggesting that the increased numbers of
competitive flora protected the pathogen from acid (Duffy et al., 1999). Thus, pub-
lished data suggest that high numbers of natural flora may increase resistance of
exponentially growing bacterial pathogens to a sudden stress by previous uptake of
available oxygen to prevent oxidative damage (e.g., the burst of free radicals) and
avoid “suicide” through stress.
The “suicide” response of pure cultures and the protection of pathogens from
sudden stress provided by a competitive flora (Aldsworth et al., 1998, 1999) seem
to be in conflict with acid sensitization of E. coli O157:H7, S. Typhimurium DT104
and L. monocytogenes in nonacid meat washings (Samelis et al., 2001c,d, 2002a).
No previous study, however, has investigated whether this protective effect continued
to exist upon extended periods of coexistence of growth-arrested pathogenic cells
lacking an active metabolism with high competitor populations under starvation, as
it happened in water meat washings. Starvation is also known to increase acid
tolerance of pathogens (Arnold and Kaspar, 1995; Buncic and Avery, 1998) and
generally induces multiresistances in bacteria (Hengge-Aronis, 1993, 1996; Jenkins
et al., 1990; Nystrom et al., 1992; Pichereau et al., 2000). Starvation of pure cultures
exposed to synthetic liquid media of minimal composition, as above, or in natural
but previously heat-sterilized water (Gauthier and Clement, 1994), however, may
result in different pathogen responses compared to starvation in limited nutrient food
environments in the presence of high numbers of natural flora. Growth-arrested and
starving pathogens are most likely to occur in complex microbial associations in meat
and other foods (Dainty and Mackey 1992; Labadie, 1999; Mossel et al., 1995;
Samelis et al., 1998; Sofos, 1994), where stress signals, sensors and alarmones may
be crucially altered by the food-specific natural flora.
Considering these differences, we used original (unfiltered) and filter-sterilized
water washings from fresh meat to monitor growth and ATR of inoculated (5 logs)
L. monocytogenes at 35°C, an incubation temperature purposefully selected at the
optimum range of the pathogen to maximize its responses (Samelis et al., 2001b).
Results confirmed an acid-protective effect by the natural flora (>8 log CFU/ml)
that grew rapidly in unfiltered washings, within 24 h. However, this early protective
effect to acid by the natural flora on L. monocytogenes shifted to a dramatic acid
sensitization of the pathogen after incubation at 35°C for 8 days. In contrast, in
filter-sterilized washings the pathogen significantly increased its ATR from days 1 to
8 (Samelis et al., 2001b). As discussed previously, sensitization of E. coli O157:H7,
Salmonella and L. monocytogenes to acid also occurred in unfiltered water washings
at 10°C, where the protective effect of the natural flora was delayed due to the
decrease in microbial growth rate (Samelis et al., 2001c,d, 2002a). However, partic-
ularly for E. coli O157:H7, additional data have shown that acid sensitization occurs
only when this pathogen, at least the acid-resistant strain ATCC 43895 used, com-
pletely fails to grow in the water washings (unpublished data). This growth primarily
depends on the storage temperature, rather than the competition by the natural flora
present. Indeed, growth-arrested acid-adapted cultures of E. coli O157:H7 in unfil-
tered washings at 10°C, and any pathogen culture at 4°C, were very sensitive at pH

3.5, while the respective stationary-phase cultures following growth (ca. 2 logs) in
the same washings at 15 or 25°C were very resistant at pH 3.5, irrespective of acid
adaptation of the inoculum (unpublished data). Similar behavior at each respective
incubation temperature was observed in parallel cultures of acid-adapted or non-
adapted E. coli O157:H7 exposed to heat-sterilized water washings, which, of course,
were free of natural flora but contained organic material from meat.
In contrast, pure cultures of acid-adapted and nonadapted E. coli O157:H7 in
sterile water (controls), where no growth occurred, became more acid-sensitive at
pH 3.5 with time when the incubation temperature increased from 4 to 10 to 15 and
25°C (unpublished data). These findings suggest that strains of E. coli O157:H7
characterized by a permanent acid resistance, such as strain ATCC 43895, may
express this resistance upon their transfer from niche to niche, provided that envi-
ronmental conditions are favorable for the pathogen to multiply. Additional studies
are required to investigate the behavior of E. coli O157:H7 strains that are acid-
sensitive or their high ATR is primarily pH-inducible.
From a practical point of view, these findings suggest that in order to acid-sensitize
E. coli O157:H7 in meat plants, its growth potential should be minimized by either
keeping nonacid conditions under environmentally low (≤10°C) temperatures or estab-
lishing acidic conditions at ambient temperatures. On this basis, E. coli O157:H7
populations that may have multiplied on slaughtered animals or the meat plant to
subsequently survive on an acid-decontaminated carcass transferred to chill storage,
or acid-adapted survivors that may establish niches in refrigerated plant areas may
pose serious safety risks. Such survivors, if not exhausted due to prevalence of sublethal
acid conditions, may maintain high ATR and potentially mutate, while if they have
chances to multiply at later stages of meat processing due to abusive (>12°C) temper-
ature conditions, risks may be maximized. In contrast, even high E. coli O157:H7
populations still attached on water-decontaminated carcasses stored under refrigeration
temperatures inhibitory to their growth, would be eventually acid-sensitized. It appears
that the same scenario is not the case with Salmonella and L. monocytogenes due to
their lower ATR and because acid sensitization occurred following growth in a fresh
meat environment, such as the washings (Samelis et al., 2001c,d).
In conclusion, acid sensitization of bacterial pathogens after exposure to nonacid,
but otherwise stressful, environments is a complex response which may depend on
the species, temperature, microbial competition, naturally present or microbially
catabolized extracellular compounds that may act as stress sensors and alarmones,
limiting nutrients, etc. Overall, this response seems logical because E. coli O157:H7,
Salmonella and other pathogens are neutrophiles. Alterations in ATR of starving
pathogens in the presence of high numbers of competitors increases in alkali toler-
ance at neutral pH (Lazim and Rowbury, 2000) that may switch off genes expressing
or maintaining ATR, or production of antagonistic compounds by the natural flora
(Cheng et al., 1995) may contribute to acid sensitization. Intensified research is
required in this field to indicate whether the natural flora as affected by food type,
composition, processing and storage conditions may synergistically regulate stress
responses of various bacterial pathogens or strains with varying biochemical capa-
bilities and natural tolerances. Such a correlation may exist and may lead to different
responses because food-related processing and storage factors by themselves have

a strong impact, for example, on acid resistance of E. coli O157:H7 (Brudzinski and
Harrison, 1998; Diez-Gonzalez and Russell, 1999; Ingham and Uljas, 1998; Kauppi
and Tatini, 1996; Semancheck and Golden, 1998).
In summary, improved understanding of the effects of decontamination and other
processes on the antagonistic or symbiotic interactions among pathogenic and spoil-
age microorganisms in foods during subsequent processing and storage may prevent
overlooked or underestimated (or indicate overestimated) risks associated with their
use. In addition to physical or chemical antimicrobial factors (Cheroutre-Vialette
and Lebert, 2000; Fernandez et al., 1997; George et al., 1996; McClure et al., 1994),
specific parameters referring to microbial competition should be incorporated in
mathematical models to enhance accuracy of their predictions regarding food safety
and shelf life (Aggelis et al., 1998; Lebert et al., 2000; Vereecken et al., 2000). Based
on the behavior (e.g., growth, survival, inhibition, resistance in stress sequences and
death) of mixed pathogenic strains in validation studies, food processes and products
can be modified to build in extra safety margins. Then, the data obtained should be
used to re-validate and potentially revise or expand existing food safety models to
increase accuracy of predictions (McMeekin et al., 1997), with emphasis given on
the nonlinear inactivation kinetics of bacterial pathogens exposed to lethal agents
(Peleg and Penchina, 2000).
Quantitative predictions of pathogen responses from model experiments with
real or simulated foods should be the basis for the development of reliable quanti-
tative risk assessment schemes (Lammerding and Paoli, 1997; Walls and Scott,
1996). These schemes should enhance transfer of microbiologically validated tech-
nological developments in the food industry (Roberts, 1997), and should be the basis
for developing approaches to meet food safety objectives (van Schothorst, 1998).
Moreover, extension service bulletins based on realistic and product-specific quan-
titative risk assessment of foods should be used in public education to enhance
pathogen control at the plant or kitchen level. As mentioned, a major deficiency of
our modern society, with respect to food production and processing, is the lack of
education of food industry personnel and consumers based on brief, but practical
and straight forward, recommendations (Bruhn, 1997).
NOVEL PATHOGEN CONTROL STRATEGIES

Control strategies for stressed pathogens may need to be based on novel approaches.
According to Lederberg (1997), “we could imaginably adapt in a Darwinian fashion,
but the odds are stacked against us. We cannot compete with microorganisms whose
populations are measured in exponents of 1012, 1014, 1016, over periods of days…
In the case against microbial genes, our weapon is our wits, not natural selection of
our genes. New mechanisms of genetic plasticity of one microbe species or another
are uncovered almost daily.”
Archer (1996) postulated, “I wonder if a reduction in preservation might not in
fact lead to a reduction in the immediate virulence of certain pathogens, and addition-
ally, to a lowering of the rate of emergence of new or better host-adapted pathogens.”
These statements make us pose some important questions: How can we use our
wits as weapons? Should we eliminate use of common antimicrobial inhibitory

hurdles? How can we balance between short-term antimicrobial activity of hurdles
and potential long-term negative effects? No doubt, it is difficult to decide the best
way to control the evolution of resistant bacterial pathogens in our food supply and,
overall, in the environment. Principally, there are two types of control strategies,
which could be seen as complementary rather than as counteractive: the classical
and the novel.
Classical strategies suggest a continuous increase in the application of safety
barriers in foods by developing new antimicrobials and incorporating more hurdle
technologies or decontamination interventions, without necessarily knowing any
potential secondary effects prior to application. A major concern associated with this
strategy is that multihurdles with “milder” doses of stresses, although detrimental to
many microorganisms in the short term, may enhance generation or evolution of
pathogenic strains of increased resistance and virulence in the future (Archer, 1996).
Based on recent data, however, this disadvantage may be eliminated if we assure that
multihurdles are applied in the right order and to sufficient levels to cause exhaustion
of pathogenic cells due to expenditure of large amounts of cellular energy to maintain
viability during processing of foods. If such conditions are carefully set with either
classical or novel methods, exhausted survivors from an initial stress may have
insufficient energy reserves to cope successfully with secondary stresses or the final
acid stress in the stomach, irrespective of their numbers (which, in any case, would
expectedly be very low). In other words, the principal aim is for stationary-phase
pathogen survivors in foods to become so energetically exhausted while attempting
to maintain viability that their potential adaptation to the initial stress applied, or
cross-protection against secondary stresses, is counteracted by exhaustion.
The above hypothesis was first made following exposures to food-related
stresses, such as acid, high or low temperatures and low water activity, of substrate-
limited pure cultures of E. coli in broth (Krist et al., 1998; Shadbolt et al., 1999,
2001). Initially it was observed that acid stress reduced the efficiency of substrate
conversion to biomass by E. coli compared to osmolarity or cold and heat, potentially
due to its higher demand for energy (Krist et al., 1998). In contrast, E. coli could
survive low levels of water activity (e.g., 0.75 to 0.90) for long times by developing
osmotic-resistant subpopulations that declined very slowly, e.g., “tailing” effect
(Shadbolt et al., 1999). More recently, Shadbolt et al. (2001) demonstrated that when
a non-pathogenic E. coli strain sharing high acid tolerance with E. coli O157:H7
was initially exposed to suboptimal water activity of 0.90, followed by exposure to
a secondary lethal acid stress at pH 3.5 with HCl, survivors from the osmotic stress
displayed a second rapid inactivation period due to acid, but eventually a resistant
subpopulation emerged to maintain viability for nearly 50 h after acid addition to
the broth culture.
In contrast, when these experiments were performed under identical conditions,
but by applying the acid stress first followed by the osmotic stress, acid survivors
were inactivated very rapidly and declined below the detection limit within a few
hours (Shadbolt et al. 2001). Thus, these data indicate that acid should be the initial
stress in food processing, whenever used, in order to sensitize foodborne pathogens
to secondary stresses due to its ability to impose large energetic burdens to bacterial
cells (Krist et al., 1998; Shadbolt et al., 2001). In fact, this approach has been wisely

applied in traditional meat fermentations since antiquity. Acid stress due to pH
lowering/lactate formation by the antagonistic lactic flora is followed by a long-term
lethal osmotic stress ranging from 0.92 to 0.85, during drying; conditions that
ultimately exhaust pathogens to death in the ripened product (Lucke, 2000; Samelis
et al., 1998).
Differences in survival of E. coli O157:H7 to initial and secondary acid stress
may potentially indicate different levels of pathogen exhaustion, as affected by the
type (lactate or acetate) and concentration of acid (dilution factor) and the exposure
times to acid-containing meat washings (Samelis et al., 2002b). These results may
be directly extrapolated to meat plant conditions because pathogen survivors from
the initial acid stress originated from a natural, meat-associated environment rather
than from laboratory cultures.
Additional studies in our laboratory (Burnham et al., 2001; Lakakkula et al.,
2001; unpublished data) involving dried foods have also shown that carefully selected
hurdles may enhance death of bacterial pathogens to very low or undetectable levels,
due to potential depletion of cellular energy and eventual exhaustion of cells. Burn-
ham et al. (2001) found that dipping E. coli O157:H7 inoculated apple slices in an
ascorbic acid solution enhanced destruction of the pathogen during drying of the
product. This synergism has also been observed in sliced dried apples of different
varieties previously treated with citric acid (Lakakkula et al., 2001), and in beef jerky.
Inoculated (7 logs) E. coli O157:H7, Salmonella or L. monocytogenes on beef jerky
strips decreased more rapidly and extensively during drying and ambient storage of
product treated before drying with marinade containing acetic acid or combinations
of lactic acid with ethanol, compared to traditional or no marination (unpublished
data). Notably, subsequent drying of jerky was more detrimental for previously acid-
adapted than nonadapted inocula, which may indicate that the amount of cellular
energy expensed by E. coli O157:H7 for acid adaptation could not be replaced before
and during drying to eventually reduce survival in the dried product.
Another complementary rather than counteractive approach for controlling
stress-adapted pathogens in foods may be to make them misinterpret the environ-
mental signals and, thus, activate wrong genes at the right (for food safety), time
(Sheridan and McDowell, 1998). This strategy was originally termed as “microbial
psychosis” (Dorman, 1994). Practically, it requires avoidance of intentional or inad-
vertent exposure of pathogens to food-related stresses in order to drive them to
become unprepared to cope with sudden stress during food processing, in the kitchen
or in the host. As an inappropriate bacterial response, “microbial psychosis” may
be induced effectively, provided that we have exact knowledge of the microbial
regulatory processes under different sets of environmental conditions. For example,
we believe that acid sensitization of E. coli O157:H7 and L. monocytogenes in
nonacid food environments, such as water meat washings, indicates a “psychotic”
behavior in response to absence of acid stress. However, it is difficult to elucidate
the exact factors and their interactions that induce this behavior, which may differ
among pathogens and be altered by different sets of environmental conditions.
“Microbial psychosis” may be induced on pathogens by changing the food
environment via the competitive natural flora. In other words, we can use microbial
competition in foods not just to inhibit pathogen growth, which is the traditional

approach in food preservation, but to potentially reduce stress resistance of bacterial
pathogens (Samelis et al., 2001b). As more knowledge on pathogen interactions with
other bacteria is being accumulated, new approaches for pathogen inhibition by a
competitive flora in foods will be indicated. For example, we have recently demon-
strated that Pseudomonas may completely inhibit growth of E. coli O157:H7 at
10°C, and may retard its growth at 15°C, if glucose at 1% is present in culture broth
to be converted to gluconate. With no or low concentrations (0.25%) of glucose in
the broth, inhibitory effects are low to zero (Samelis and Sofos, 2002). Thus,
supplementation of fresh foods with higher levels of glucose than those they contain
may increase control of E. coli O157:H7 via competitive exclusion (Bower and
Daeschel, 1999). Also, a competitive flora, which can be purposefully added or
allowed to develop in a food product or plant to exert a targeted metabolic activity,
may significantly contribute to a weakening of pathogen resistance to stress factors
not prevailing during the process (Samelis et al., 2001b). Intensified research is
required in this field. In particular, such competitive effects should be studied on
adherent pathogens with natural flora in mixed biofilms where prevention of adhe-
sion or increased sensitization of pathogens to sanitizers may be induced by the
predominant spoilage flora (Stopforth et al., 2001). Sensitization may be due to
excreting of microbial metabolites, which act as “misleading” signals to pathogenic
cells, or chemical compounds that inactivate alarmones excreted by the cells in order
to cope with a specific type of stress. Directed subsequent exposure of the sensitized
pathogen to the target stress may result in rapid cell inactivation.
Another example of a novel approach may be to create a food environment that
causes bacterial cell growth to cease, without application of severe stress and without
adversely affecting food quality (McMeekin et al., 1997). For this purpose, compat-
ible solutes or osmolytes widely found in foods, such as glycine betaine or carnitine,
may be added to make the pathogens activate specific transport systems to equilibrate
the external increase in osmotic pressure. Depending on the magnitude of this
increase, osmotic conditions may either inactivate pathogenic cells, or inhibit their
growth while the compatible solutes transported inside the cell allow continuation
of enzymatic reactions. Thus, energy reserves of pathogen cells are once again
depleted, but by other means than acid stress, and upcoming starvation may drasti-
cally extend the pathogen lag phase, or lead to death (Krist et al., 1998; McMeekin
et al., 1997; Shadbolt et al., 1999).
PRACTICAL APPLICATION OF PATHOGEN

CONTROL STRATEGIES
The establishment of multistep barriers throughout the food chain is the recom-
mended common approach to effectively control microbial pathogens in foods. Steps
in the food chain that require barrier application include animal feeding, plant
harvesting, cleaning of animals and plants, decontamination of animal carcasses and
produce, plant sanitation and hygiene, food production, processing, storage and
distribution, food retailing and meal preparation, food service facilities, and food
handling at home by consumers. It is important not to simply establish, but to

continuously update, antimicrobial barriers, mainly in consideration of their potential
impact on bacterial stress resistance and virulence development.
Advanced knowledge on bacterial responses to food-related stresses should be
used to benefit the food industry and consumers. Effective application of this knowl-
edge can only be achieved by collaborative action between all components of the
food sector, including the industry, regulators, public health authorities and academic
institutions (Fischhoff and Downs, 1997; Majkowski, 1997; Roberts, 1997). Thus,
an integrated microbial control strategy needs to be elaborated (Sofos, 2001). Food-
specific research data should be used to increase the accuracy of predictive models
(McMeekin et al., 1997; Walls and Scott, 1996), and the results from model valida-
tion studies with resistant pathogens in real food systems can lead to the development
of reliable quantitative risk assessment schemes (Lammerding and Paoli, 1997).
These schemes can be used to establish food safety objectives (van Schothorst, 1998)
and be implemented in hazard analysis critical control point (HACCP) systems to
enhance food safety (FSIS, 1996; NACMCF, 1998). Meanwhile, academic institu-
tions need to coordinate education and provide updated, educational or extension
material to food scientists, producers, handlers, processors and consumers. In fact,
the entire food safety network requires continuous updating.
Although research, development and application of novel food preservation
approaches may help us control stress-adapted and emerging pathogens, better appli-
cation of already known, common pathogen control interventions by food handlers
and consumers may be adequate to greatly reduce foodborne illness episodes. Today,
most outbreaks of foodborne disease are due to mishandling food in ways that we
already know how to avoid; approximately 85% of all outbreaks occur as a result
of food mishandling in food service establishments or homes (Hall, 1997).
Lederberg (1997) stated: “It is important to prevent foodborne disease through
sensible monitoring, standard of cleanliness and consumer and foodhandler educa-
tion and not just care of its victims… Education, however, is a universally accepted
countermeasure, especially important in foodborne disease. Food safety programs
should more specifically target food handlers, examining their hands to determine,
if they are carriers, to ensure they are complying with basic sanitation.” Thus,
education of food handlers and consumers in basic hygiene and the consequences
of temperature abuse are urgently needed, as is a greater depth of understanding of
those in technical positions in the food industry or those with regulatory responsi-
bilities (McMeekin et al., 1997).
Food microbiologists must realize their responsibility to provide industry per-
sonnel and consumers with simplified knowledge on food safety aspects, while all
sectors of the continuum from farm to table should share in the responsibilities
(Bruhn, 1997). Governmental agencies have the responsibility to coordinate collab-
orative actions toward the goal of providing our society with safe foods, although
the globalization of the entire food sector requires effective collaborative action and
international harmonization of food safety objectives and regulations (Kaferstein
et al., 1997).

CONCLUSIONS
When exposed to sublethal doses of a stress, microorganisms may adapt and pro-
gressively develop resistance to stronger doses of the stress, while microbial adap-
tation to one stress can confer cross-protective resistance to other stresses. Bacteria
respond to stresses primarily by temporary intracellular changes, or may undergo
mutations which introduce permanent genomic changes that favor their survival.
Accordingly, stressed pathogens have greater potential for survival and/or prolifer-
ation in foods and under stressing conditions of temperature, acidity, water activity,
chemical or biological preservatives and limited nutrients; highly resistant and vir-
ulent mutants may arise in situ. Traditional food processing safety barriers may be
inadequate, and foodborne diseases may be caused even by pathogenic strains at
very low infective doses. Several recent uncommon outbreaks associated with foods
traditionally considered low-risk support current food safety concerns.
To control the emergence and evolution of stress-resistant pathogens in foods,
we need to undertake effective basic and applied research. The overall goal is to
develop control strategies based on optimized, efficient, economical and integrated
approaches that rely on basic knowledge to prevent resistance development and
virulence enhancement, and to assure product quality, production efficiency, and
control of resistant pathogens. A good approach is to work with real food systems,
considering them as stressful environments, and under experimental conditions that
simulate microbial associations in these environments as much as possible. Work
with pure cultures under laboratory conditions may be useful in verifying mecha-
nisms of microbial resistance development and control.
Except for food processes achieving food safety by extensive microbial inacti-
vation (e.g., canning), other approaches employed for control of pathogens in foods
achieve reduction of prevalence or inhibition of proliferation. Selection of such
treatments, interventions or processes is difficult and requires extensive evaluation.
This is because most nondestructive food processing, decontamination or preserva-
tion technologies, although useful in reducing pathogen prevalence and controlling
growth, may not deliver complete removal, inactivation or control of contamination,
and may allow bacterial survivors to become stress resistant and virulent at very low
infectious doses. Such doses may not affect consumers with a strong immune system,
but may cause infection to immunosuppressed individuals. Thus, pathogen control
strategies should be decided by considering the resistance of stressed pathogens,
and by predicting the virulence of survivors in relation to the susceptibility of at-
risk populations.
Effective commercial application of multihurdle pathogen control strategies in
foods requires multivariable research approaches and, indeed, there are many issues
associated with bacterial stress resistance in complex food ecosystems that require
elucidation. Recent data suggest that the energetic status of pathogenic cells, rather
than their actual numbers, may be more important in deciding whether survivors from
primary stresses can cope successfully with secondary stresses in foods, and the
ultimate acid stress in the stomach. On this basis, food-related sequential stresses
should be carefully applied to assure pathogen exhaustion or should be intelligently
avoided to induce “microbial psychosis” (Dorman, 1994) to pathogen survivors, but

should never have an intermediate effect. In practice, when during food processing
and storage application or exposure to a preservation barrier is unavoidable, the barrier
considered the most effective in reducing and exhausting bacterial survivors capable
of recovering in the product should be selected. When it is wiser to avoid challenging
of bacteria, technologies that may sensitize pathogens to types of stresses applied
subsequently during food processing should be selected. Since multihurdles disrupt
cell homeostasis in multiple ways, they should be selected and applied at the right
magnitude and succession to minimize development of multi-stress resistant strains,
and to lead to as much pathogen inactivation as possible.
Monitoring and validating food processes on the basis of novel pathogen control
strategies, such as “microbial psychosis,” and elucidating whether foodborne patho-
gens can be virulent while being “psychotic,” or still viable but energetically
exhausted, are major future challenges in food microbiology. Potential discrepancies
between what we believe and what may really happen in the food environment have
to be hypothesized and, if possible, predicted and verified. Scientific knowledge and
consumer awareness may reduce common or new routes of transmission for known
or newly emerging pathogens from foods. The objective is to provide consumers
with food products for preparation at home, or ready-to-eat, which are free of active
pathogens, especially those of low infectious doses and high virulence for at-risk
populations.
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Microbial Stress Adaptation and Food Safety

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Microbial

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No claim to original U.S. Government works

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Vijay K. Juneja is supervisory microbiologist and lead scientist in the Microbial

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Stress Adaptive Response

1. Physical treatments such as heat, pressure, electric pulses, ultrasonic

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1. Production of proteins that repair damage, maintain the cell, or eliminate

When microorganisms are stressed, an adaptive or protective response may follow.

Tolerance to a deleterious factor (e.g., low pH) refers to a microorganism’s ability

Damage to cellular components by stresses may impair the ability of microorganisms

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STRESS, ADAPTATION AND FOOD SAFETY

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Heat shock Weather-related Mild processing Temperature control Cooking Fever

Cold shock Weather-related Refrigeration Refrigeration Refrigeration fluctuation —

Starvation Non-nutritious environment — — — Iron starvation in

Metal ions Irrigation water Equipment — Equipment —

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Raw food Pre-processing Mildly-processed Processing Fully-processed

Stressed Response Stress-adapted

response and osmotic shock response in pathogenic bacteria. Pathogens, adapted to

EMERGING PROCESSING TECHNOLOGIES

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HIGH PRESSURE PROCESSING

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FIGURE 1.3 High pressure processing equipment: basic components.

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Potential Stress Adaptation

Treatment with γ radiation involves placing the food in proximity of a radiation

The short wavelengths of UV light inactivate microorganisms through alteration of

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Potential Stress Adaptation

PULSED ELECTRIC FIELD

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Electricity Stepped-up Electric

Power Supply Pulser

potential difference across the membrane is 10 mV. The application of an electric

Potential Stress Adaptation

Russell et al. (2000) treated L. monocytogenes and Salmonella typhimurium with

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Stress- related • 2-D gel

FIGURE 1.5 A simplified representation of general cellular processes involved in stress

REGULATION OF STRESS-RELATED PROTEIN SYNTHESIS

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GENERAL STRESS RESPONSE

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SPECIFIC STRESS RESPONSES

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