Basics of Stress Adaptation 1 and Implications in

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
2003 by CRC Press LLC

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 organisms 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 48C for 120 min before the inoculated
mix was heated at 64C. 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., 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 organisms 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 microorganisms 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 cells 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 (45C 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 48C 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 71.6C 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 145C 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 proteinprotein hydrophobic interactions. According to LeChateliers
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 37C 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 37C for 30 min, cells developed resistance to lethal pressures (at
300 MPa) and heat (57C). Heat shocking the pathogen at 46C 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 (),
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, and UV radiation technologies only will be addressed.
Process
Treatment with 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 cells 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 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) ( 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 cells 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 proteins 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 Krmer, 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 factors Molecular probes to
Transcription Anti 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 (70 in E. coli and A 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 S 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 S-dependent genes in the presence
of high S 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, B, 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 B (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 (32) 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 37C 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 S
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, S, in E. coli and other Gram-negative bacteria and B 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 S and B 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 S. Very little if any S is
detectable in non-stressed E. coli cells. When cells are exposed to stress, S is
induced, activating the s-controlled promoters. Expression of these genes is neces-
sary for survival under stress conditions. S 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 B 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) (Arsne 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, 32. Approximately 50 genes are induced by 32 when denatured proteins are
detected in the cytoplasm (Yura and Nakahigashi, 1999). 32 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 32 also increases dramatically upon
exposure to heat (Arsne et al., 2000; Yura and Nakahigashi, 1999).
Two other alternative sigma factors, E and 54, control other regulons induced
by heat. E, 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 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 E is not understood. 54 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 A-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 B 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 37C, 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 37C; 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 S-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 S, the two-component signaling system PhoPQ, and the iron regulator, Fur
(Foster, 1999, 2000). The S-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 S
resulting in a general stress tolerance and induction of acid stress proteins by OmpA
(Foster, 2000). A deletion in the gene encoding B 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 S-
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
Krmer, 2000). S (E. coli) and B (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 S,
the 32 and E 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 B regulon in B. subtilis, but also induces the extracy-
toplasmic function (ECF) sigma factor W (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 S, 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 microorganisms 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 microorganisms 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
45C for 1 h (Lou and Yousef, 1997). By comparison, injury of L. monocytogenes
is most apparent at 55 to 60C (El-Shenawy et al., 1989) and neither stress response

nor injury can be reliably detected at 70C. Heat shocking E. coli O157:H7 at 45 to
46C 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 microorganisms
response to stress. Proteins synthesized in response to stress include regulatory pro-
teins (e.g., 32 in E. coli and B 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 -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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Basics of Stress Adaptation

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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 microorganisms 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 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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Physiological changes in response to cold include changes in the membrane fatty

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In foods, bacteria may be exposed to increased levels of reactive oxygen species

MONITORING STRESS RESPONSE

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INDUCTION OF STRESS ADAPTIVE RESPONSE: PRACTICAL CONSIDERATIONS

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