Effects of Nutritional and Environmental

Conditions on Salmonella sp. Biofilm Formation

Barbara Speranza, Maria Rosaria Corbo, and Milena Sinigaglia

Abstract: Biofilm formation on food industry surfaces has important health and economic consequences, since they can
serve as a potential source of contamination for food products, which may lead to food spoilage or transmission of diseases.
Salmonella sp. is one of the most important foodborne pathogens and several studies have led to the discovery that these
bacteria are capable of adhering and forming biofilms on different surfaces. The attachment of bacterial cells is affected by
several factors, including the medium in which they are grown, motility, growth phase of the cells, type and properties of
the inert material, presence of organic material, temperature, pH, contact time, and so on. This investigation focused on
the study and quantification of the effects of temperature (20 to 40 ◦ C), pH (4.5 to 7.5), and medium composition (0.5 to
2.5 g/L of peptone) on biofilm formation by Salmonella sp. on stainless steel through surface response modeling. Results
highlighted that the target strain was able to adhere on stainless steel, under all the conditions tested. To assess potential
differences, the aptitude to biofilm formation (ABF), defined as the time necessary to start adhesion on the surface, was
calculated by using the Gompertz equation. This parameter was modeled through a stepwise regression procedure and
experimental conditions resulting in the greater ABF were growth in poor media (1.0 to 1.5 g/L of peptone), incubation
temperature of about 30 ◦ C, pH close to 6.0.
Keywords: biofilm formation, central composite design, growth medium, Salmonella sp.
M: Food Microbiology
& Safety

Practical Application: The importance of this work lies in its extension of our knowledge about the effect of different
environmental conditions on Salmonella adherence to stainless steel food-processing equipment, as a better understanding
of biofilms may provide valuable pathways for the prevention of biofilm formation.

Introduction
Biofilms are currently identified as an assemblage of surface-
associated microbial cells that are enclosed in hydrated extracellular
polymeric substances (EPS) (Sauer and others 2007). Polysaccha-
rides, proteins, phospholipids, teichoic, and even nucleic acids are
the main components of EPS. A biofilm community may comprise
single and/or multiple species of bacteria and form a single layer
or 3-dimensional (3D) structures. Mature biofilms are highly or-
ganized ecosystems in which water channels are dispersed and can
provide passages for the exchange of nutrients, metabolites, and
waste products (Sauer and others 2007). These microbial assem-
blages can be developed on food-processing surfaces, if they are
not properly cleaned, and it has been demonstrated that bacteria
in biofilms exhibit enhanced resistance to cleaning and sanitation
(Joseph and others 2001; Shi and Zhu 2009). Once formed on
food industry surfaces, biofilms have important health and eco-
nomic consequences, since they can serve as a potential source of
contamination for food products, which may lead to food spoilage,
reducing shelf life of products, or transmission of diseases (Jessen
and Lammert 2003). Biofilms not only present a considerable hy-
MS 20100474 Submitted 4/30/2010, Accepted 10/8/2010. Authors are with
Dept. of Food Science, Faculty of Agricultural Science, Food Quality and Health
Research Center, Univ. of Foggia, Via Napoli 25, Foggia 71100, Italy. Direct inquiries
to author Sinigaglia (E-mail: m.sinigaglia@unifg.it).
giene risk in the food industry, but also cause economical losses
by technical failures in water systems, cooling towers, heat ex-
changers, and so on (Shi and Zhu 2009). As a matter of fact, the
phenomenon has serious implications in industrial, environmen-
tal, public health, and medical situations (Gilbert and others 2003;
Hall-Stoodley and others 2004).
It is well recognized that pathogenic microorganisms can also
attach to and grow on food surfaces, equipment, and processing
environments to form biofilms (Shi and Zhu 2009). For exam-
ple, Listeria monocytogenes forms biofilms on floor drains, storage
tanks, hand trucks, conveyor belts, and other food-contact materi-
als (Sandasi and others 2008). Different studies have been focused
on the attachment of bacterial pathogens to food surfaces such as
L. monocytogenes to beef surfaces and Salmonella sp. to chicken skin
(Shi and Zhu 2009).
Salmonellae are food-poisoning organisms, which are of consid-
erable significance for the food industry, since they are one of the
most important foodborne pathogens (Hohmann 2001). Non-
typhoidal salmonellosis is estimated to affect 1.4 million people
each year in the United States, and more than 95% of cases of
infections caused by these bacteria are foodborne. These infec-
tions account for about 30% of deaths resulting from foodborne
illnesses (Hohmann 2001). According to the Annual Report on
Epidemiology of foodborne infectious diseases in the European
Union, published by the European Food Safety Authority (EFSA)
in collaboration with the European Center for Disease Control
(ECDC), Salmonella appears to be the 2nd cause of food poisoning

C 2010 Institute of Food Technologists

 R

M12 Journal of Food Science r Vol. 76, Nr. 1, 2011 doi: 10.1111/j.1750-3841.2010.01936.x
Further reproduction without permission is prohibited
Salmonella sp. biofilm formation . . .

in Europe, after Campylobacter (ECDC 2010). Outbreaks of
salmonellosis have been linked to a wide variety of fresh produce
including alfalfa sprouts, lettuce, fennel, cilantro, cantaloupes, un-
pasteurized orange juice, tomatoes, melons, mango, celery, and
parsley (ECDC 2010). Studies performed so far have led to the
discovery that these bacteria are strongly capable of adhering and
forming biofilms on different surfaces (Hood and Zottola 1997;
Römling and Rohde 1999; Römling and others 2000; Sinde
and Carballo 2000; Joseph and others 2001). Although molec-
ular mechanisms mediating attachment and biofilm formation are
not completely understood, it is recognized that agfD promoter is
involved in Salmonella sp. biofilm formation (Römling and others
2000; Gerstel and Römling 2001). Even if information on regu-
latory mechanisms controlling expression of agfD is limited, it is
known that bacteria regulate gene expression in response to dif-
ferent environmental signals such as temperature, osmolarity, O2 ,
CO2 , pH, nitrogen compounds, nutrient availability, inorganic
ion concentrations (Römling and others 1998, 2000; Gerstel and
Römling 2001). Therefore, attachment of bacterial cells is dif-
ferently affected by numerous factors, including the medium in
which they are grown, motility, growth phase of the cells, type
and properties of the inert material, presence of organic material
(‘‘conditioning film’’), temperature, pH, length of contact time,
production of extracellular polysaccharides, and cell-to-cell com-
biofilm formation on stainless steel were studied and quantified by
a response surface model.
Materials and Methods
Test culture and surface
A strain of Salmonella sp. ATCC 35664 (serotype Infantis, anti-
genic formula 6,7: r: 1,5) was used as test organism. The strain
was stored at −20 ◦ C in vials containing Tryptone Soya Broth
(TSB; Oxoid, Milan, Italy) plus 33% glycerol. Prior to use, a mas-
ter culture was prepared by adding a vial content to 100 mL of
TSB in a conical flask with incubation at 37 ◦ C for 18 h, un-
til late exponential phase was attained. Then, a working culture
was prepared by adding 100-μL aliquot of the master culture to
100 mL of TSB and incubating at 37 ◦ C for 18 h.
Stainless steel was the surface chosen (AISI-316, finish#2B,
ARVEL, Naples, Italy) for the adhesion experiments. This mate-
rial has been reported as the ideal one for food processing, since it
is chemically and physiologically stable at various food-processing
temperatures, easy to clean, and has a high resistance to corrosion
(Verran and others 2001). Before each experiment, the stainless
steel chips (2.5 × 5.0 cm) were prepared by washing in acetone
for a minimum of 30 min, rinsing in distilled water, and then
soaking in 1 N NaOH for 1 h. After a final rinse in distilled water,
the chips were allowed to air dry. This cleansing procedure was

M: Food Microbiology
munication (Chandy and Angles 2001; Chmielewski and Frank
2003; Shi and Zhu 2009; Simões and others 2010). The abil- required to remove fingerprints, oils grease, and other soils that
may have been on the stainless steel (Hood and Zottola 1997).

ity to recognize how foodborne pathogens attach on surfaces is
an important area of focus, as a better understanding of biofilms
may provide valuable pathways in the prevention of their for-
mation. This study, using model systems capable of mimicking
potential industrial food-processing conditions (that is, poor or
rich growth media, different pH, various temperatures), under
controlled established laboratory conditions, was born as an effort
to better understand the physiological requirements of Salmonella
sp. biofilm formation on stainless steel. In particular, the goal of
the present research was to analyze the capacity of Salmonella sp.
to form biofilms during growth under different environmental
conditions. The effects of temperature (20 to 40◦ C), pH (4.5 to
7.5), and medium composition (0.5 to 2.5 g/L of peptone) on
& Safety
Cleaned steel chips were finally autoclaved at 121 ◦ C for 15 min
prior to use.
Sample preparation and biofilm formation
The combined effects of temperature, pH, and medium compo-
sition on biofilm formation by Salmonella sp. were studied through
a 5 levels–3 variables central composite design (CCD) (Box and
others 2005). A total of 17 conditions were tested, as can be seen
in Table 1.
To allow biofilm formation, sterile steel chips were placed ver-
tically into jars containing 20 mL of sterile medium (Bacterio-
logical Peptone, BP, Oxoid) and aliquots of the working culture

Table 1–Combinations of the central composite design and fitting parameters of the modified Gompertz equation.

BP Fitting parameters
Temperature concentration μmax
Combinations (◦ C) pH (g/L) A (log CFU/cm2 )  log (CFU/cm2 )/h ABF (h) R2
1 25 5.2 1.0 5.95 ± 0.25 0.82 ± 0.05 3.96 ± 0.05 0.958
2 35 5.2 1.0 5.81 ± 0.05 1.30 ± 0.01 4.43 ± 0.05 0.998
3 25 6.6 1.0 6.57 ± 0.01 1.12 ± 0.03 4.65 ± 0.08 0.996
4 35 6.6 1.0 5.93 ± 0.05 2.82 ± 0.01 6.62 ± 0.19 0.988
5 25 5.2 2.0 6.15 ± 0.01 0.99 ± 0.02 7.68 ± 0.02 0.998
6 35 5.2 2.0 6.13 ± 0.01 4.75 ± 0.02 7.21 ± 0.02 0.998
7 25 6.6 2.0 6.17 ± 0.19 1.51 ± 0.01 4.68 ± 0.16 0.990
8 35 6.6 2.0 6.10 ± 0.01 1.61 ± 0.05 4.47 ± 0.05 0.994
9 30 5.9 1.5 6.39 ± 0.06 2.97 ± 0.03 6.34 ± 0.15 0.972
10 20 5.9 1.5 5.67 ± 0.26 0.16 ± 0.05 11.54 ± 0.20 0.978
11 40 5.9 1.5 5.99 ± 0.05 3.67 ± 0.05 6.91 ± 0.04 0.996
12 30 4.5 1.5 6.08 ± 0.02 4.45 ± 0.04 7.16 ± 0.10 0.998
13 30 7.5 1.5 6.08 ± 0.01 4.74 ± 0.01 7.26 ± 0.04 0.992
14 30 5.9 0.5 6.55 ± 0.03 3.18 ± 0.01 6.32 ± 0.05 0.984
15 30 5.9 2.5 6.45 ± 0.14 3.29 ± 0.04 6.88 ± 0.05 0.998
16 30 5.9 1.5 6.39 ± 0.06 2.97 ± 0.03 6.34 ± 0.15 0.972
17 30 5.9 1.5 6.39 ± 0.06 2.97 ± 0.03 6.34 ± 0.15 0.972
A = maximum biofilm bacteria load attained at the stationary phase; μmax = maximal adhesion rate; ABF = aptitude to biofilm formation, R2 = coefficient of determination.
Data are presented as average ± standard deviation.

Vol. 76, Nr. 1, 2011 r Journal of Food Science M13

 Salmonella sp. biofilm formation . . .

were inoculated to obtain an initial bacterial population of about Results and Discussion
103 CFU/mL. Incubation temperature, pH, and composition of Several studies indicate that a nutritionally limited environment
the media were modulated according to Table 1. For all samples, increases the transition from planktonic to a sessile mode of life
incubation was done without agitation. (Jefferson 2004). This observation suggested that biofilm forma-
tion may represent a survival strategy in a nutritionally limited
Cell enumeration environment because surface colonization would provide a num-
Biofilm cells were enumerated at 8, 24, 48, 72, 96, and 120 h ber of advantages such as increased capture of nutrients that may be
after inoculum. At these times, chips were aseptically removed absorbed to surfaces. Because the nutrient content of the growth
and rinsed with sterile distilled water by using a Pasteur pipette, in medium has been found to regulate the development of biofilms
order to eliminate the unattached cells. Sessile cells were detached by other organisms (O’Toole and Kolter 1998; Sanin and others
from chips in a sterile test tube containing 20 mL of sterile saline 2003), we decided to test various concentrations of BP (0.5 to
with a 20 Hz “Vibra Cell” sonicator (SONICS, Newcastle, Conn., 2.5 g/L) for their effects on the ability of Salmonella sp. to form
U.S.A.) for 3 min. Viable and cultivable cells were enumerated by biofilms. Moreover, a commonly used temperature in biofilm ex-
serial dilutions in 0.9% NaCl solution and plating on Tryptone periments with Salmonellae is 37 ◦ C, their optimum temperature
Soya Agar, incubated at 37 ◦ C for 24 h. Results were expressed as of growth. However, in the food production environment and
log CFU/cm2 . also in hospital environments, temperatures both below and above
37 ◦ C are relevant: this is why we tested different temperatures
Modeling and statistical analyses ranging from 20 to 40 ◦ C. For a similar reason, the effect of differ-
Each combination was performed twice with microbiological ent pH (from 4.5 to 7.5) on biofilm formation was investigated. To
analyses carried out in duplicate. The sessile cell load data of reduce the number of possible combinations to a manageable size
Salmonella sp. collected during the biofilm formation experiments using only a fraction of the total number of factor combinations
were modeled according to the Gompertz equation modified by for experimentation, the CCD approach was chosen.
Zwietering and others (1990): The obtained results highlighted that Salmonella sp. was able
to form biofilm on stainless steel surfaces in all tested combina-
M: Food Microbiology

y = k + A × exp{− exp[(μm a x × e /A) × (AB F − t ) + 1]} (1) tions: the evolution of sessile population showed a typical sig-
moidal trend; therefore, the collected data were modeled through
& Safety

where y is assumed as the biofilm bacteria concentration the Gompertz equation, as modified by Zwietering and others
(log CFU/cm2 ), k as the initial biofilm count equivalent to zero, (1990). The calculated fitting parameters for the 17 combinations
A as the maximum biofilm bacteria load attained at the station- of the CCD are shown in the Table 1. As can be observed, the ses-
ary phase (log CFU/cm2 ), μmax as the maximal adhesion rate sile population (parameter A) attained the maximum level (about
(log [CFU/cm2 ]/h), ABF as the aptitude to biofilm formation, 6.50 log CFU/cm2 ) in the combination 3 (T 25 ◦ C; pH 6.6; BP
defined as the time necessary to start adhesion on the surface (h), 1.0 g/L) and 14 (T 30 ◦ C; pH 5.9; BP 0.5 g/L). The most inter-
and t is the time. esting parameter is the aptitude to form biofilm (ABF), defined
Then, a 2nd-order model was fitted to the data to describe the as the time necessary to start adhesion on the surface (reported in
obtained ABF values as function of the independent variables of Table 1): the minimum value of ABF (3.96 ± 0.05 h) was ob-
the CCD: served in combination 1 (T 25 ◦ C; pH 5.2; BP 1.0 g/L) against
    the maximum value (11.54 ± 0.20 h) recorded for combination
Y= (Bi · xi ) + Bi i · xi2 + (Bi j · xi · x j ) (2) 10 (T 20 ◦ C; pH 5.9; BP 1.5 g/l). Data from Table 1 were used
as inputs for building a 2nd-order polynomial equation, able to
where Y is the dependent variable (ABF value); Bi , Bii , and Bij are show the effects of the 3 CCD factors (temperature, pH, and BP
regression coefficients of the model; and xi and xj are the indepen- concentration) on Salmonella sp. biofilm formation. The obtained
dent variables in coded values, which can be seen in Table 2. This best fit equation of the individual, quadratic, and interactive effects
2
model allowed to assess  the effects of linear (xi ), quadratic (xi ), of the independent variables on the ABF values was as follows:
and interactive terms ( xi x j ) of the independent variables on the
dependent variable. Terms with significance lower than 95% (P > AB F = 20.14[BP] + 4.77[pH] − 1.46[T]
0.05) were not included in the final model. A stepwise regression
with the backward selection procedure was used to determine the + 0.02[T]2 − 3.30[BP][pH] (3)
values of regression coefficients of Eq. (2). The significance of 2
(R , 0.97; F, 66.26; SE, 1.47)
the polynomial equation was evaluated through the coefficient of
determination (R2 ), the standard error (SE), and the Fisher test The R-value indicates the goodness of fit of the proposed model
value (F). to the experimental data; moreover, the F-test underlines the high
All statistical analyses were performed using Statistica for Win- significance of the derived equation (P < 0.00001). According to
dows (Statsoft, Tulsa, Okla., U.S.A.). this equation, the ABF was strongly depended on the BP concen-
tration, both as individual positive term and in negative interaction
with pH. The effects of pH and temperature were also significant.
Table 2–Coded levels of experimental design. Better evidence of these results can be seen in Figure 1 showing
Coded levels Temperature (◦ C) pH BP concentration (g/L) the Pareto chart of individual and interactive terms of the investi-
−α (−2) 20 4.5 0.5 gated factors on the ABF by Salmonella sp. This graph reports on
−1 25 5.2 1.0 the vertical axis the individual, quadratic, and interactive terms of
0 30 5.9 1.5 the studied variables and on the horizontal axis the values of stan-
+1 35 6.6 2.0 dardized coefficient, evaluated as the ratio between the regression
+α (+2) 40 7.5 2.5
coefficient of each term and the relative SE. The vertical line is

M14 Journal of Food Science r Vol. 76, Nr. 1, 2011

Salmonella sp. biofilm formation . . .
the significance breakdown (P = 0.05). This figure re-emphasizes that
the development of microbial association in biofilms by Salmonella
sp. is a complex process significantly influenced by all the investi-
gated factors and, in particular, by the nutritional status provided
(the standardized effect was in fact the highest, 3.578). A 3D sur-
face was developed to illustrate the effects of 2 factors on the
ABF; a constant value was imposed to the 3rd factor. Figure 2
and 3 show the effects of the interactions pH-T (for a BP con-
Figure 1–Pareto chart of individual and interactive terms of the inves-
tigated factors (temperature, pH, and BP concentration) on the ABF duction of fimbriae explained an increased biofilm production of
(aptitude to biofilm formation) by Salmonella sp. The vertical line is the Salmonella Typhimurium at 30 C. Research on L. monocytogenes
significance break-down (P = 0.05).
Figure 2–Three-dimensional plot of the effects of the interaction pH-T (for Figure 3–Three-dimensional plot of the effects of the interaction T-BP
a BP concentration of 1.5g/L) on the ABF by Salmonella sp.
centration of 1.5g/L) and T-BP concentration (at pH 5.9) on the
ABF by Salmonella sp., respectively. These graphs allow us to iden-
tify the conditions for the greater ABF: in poor media (0.5 to
1.0 g/L of BP), at temperature of about 32 ◦ C and pH around
6.0, Salmonella sp. cells were able to start adhesion on stainless steel
surfaces within only 6 h. This bacterium has been demonstrated
to form biofilms on biotic and abiotic surfaces (Hood and Zot-
tola 1997; Römling and Rohde 1999; Römling and others 2000;
Sinde and Carballo 2000; Joseph and others 2001). The 2 main
matrix components in Salmonella biofilms are thin aggregative fim-
briae (agf) (Tafi, amyloid fibers) and cellulose: both are involved in
adhesion to surfaces, cell aggregation, environmental persistence,
and biofilm development (Collinson and others 1993; White and
others 2006). The agf genes involved in Tafi biosynthesis are orga-
nized into 2 adjacent divergently transcribed operons (Collinson
and others 1996), whereas the cellulose is biosynthesized by other
4 different genes. Syntheses of both Tafi and cellulose are co-
regulated by a complex regulatory system. The co-expression of
thin agf and cellulose leads to the formation of a highly hydropho-
bic network with tightly packed cells aligned in parallel in a rigid
matrix. It is well documented that the Salmonella sp. multicellu-
lar behavior, associated with the complex phenomenon of biofilm
formation, is regulated by the environmental conditions that target
the agfD promoter (Gerstel and Römling 2001). For Salmonella
M: Food Microbiology
Typhimurium strains, it was shown that the maximal expression
of agfD promoter was in nutrient-limited media and that the
& Safety
production of thin agf took place in a temperature dependent
manner, at 28 ◦ C but not at 37 ◦ C (Gerstel and Römling 2001).
These observations could also explain the results obtained in this
study, that is, a more rapid transition from planktonic to sessile
mode of life observed at low BP concentrations and at 30 to
32 ◦ C rather than at more suitable conditions. The mechanisms
behind increased biofilm formation at suboptimal temperatures are
not known. Stepanovic and others (2003) proposed that the pro-
◦
concentration (at pH 5.9) on the ABF by Salmonella sp.
Vol. 76, Nr. 1, 2011 r Journal of Food Science M15
 Salmonella sp. biofilm formation . . .
has shown that temperature affects the bacterial hydrophilic sur-
face properties (Chavant and others 2002), with low temperatures
increasing the cells hydrophilic properties and altering the bacte-
ria’s ability to adhere to hydrophobic materials. Even pH can affect
the formation of multicellular status of Salmonella Typhimurium,
depending on the nutrient availability and promoter status (Ger-
stel and Römling 2001). The optimal pH for Salmonellae growth
as planktonics is 6.5 to 7.5; in this experiment, cells rapidly at-
tached to the surfaces even at pH 4.5. However, a short time was
necessary to start adhesion when pH was over 5.9 to 6.0. Loo
and others (2000) observed that biofilm formation by Streptococcus
gordonii was more sensitive to pH changes than bacterial growth
as planktonics. In contrast, pH had no effect on biofilm forma-
tion in Pseudomonas fluorescens (O’Toole and Kolter 1998). These
results suggest that pH effects, in terms of biofilm formation, may
differ from one bacterial species to another, thereby enabling each
bacterial species to efficiently colonize its preferred environment.
Conclusions
The results of the present study show that external conditions
exert an important influence on the aptitude to form biofilm by
Salmonella sp. with a more rapid transition from planktonic to
sessile mode of life in nutritionally limited environments, at 30
to 32 ◦ C and neutral pH. However, the obtained results indicate
M: Food Microbiology
the need of screening several Salmonella strains before drawing
general conclusions regarding biofilm formation, as the strain-to-
& Safety
strain variation could be considerable. Several genes are involved
in biofilm production, but the regulatory mechanisms are still
poorly understood. Our work confirms how several factors are
involved in biofilm formation. The finding that biofilm formation
may be promoted at conditions present in the food industry in-
dicates that food producers should be aware of the importance of
controlling biofilm formation by Salmonellae. Our results seem to
be misleading if compared to more realistic situations with mix-
tures of microorganisms, since they were obtained using a pure
culture and physicochemical conditions that approach those used
in a food industry. Nonetheless, the importance of this work lies
in its extension of our knowledge of the effect of different en-
vironmental conditions on Salmonella adherence to stainless steel
food-processing equipment, as a better understanding of biofilms
may provide valuable pathways in the prevention of biofilm for-
mation.
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