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J Korean Soc Appl Biol Chem (2013) 56, 207−220
DOI 10.1007/s13765-012-3253-4
Biofilm Formation, Attachment, and Cell Hydrophobicity of Foodborne Pathogensunder Varied Environmental Conditions
Na-Young Choi · Bo-Ram Kim · Young-Min Bae · Sun-Young Lee
Received: 8 November 2012 / Accepted: 20 February 2013 / Published Online: 30 April 2013
© The Korean Society for Applied Biological Chemistry and Springer 2013
Abstract Biofilm formation, attachment and cell hydrophobicity
of foodborne pathogens, including Listeria monocytogenes,
Pseudomonas aeruginosa, and Staphylococcus aureus were
investigated under various environmental conditions such as
sodium chloride (0.5–7.0%, w/v), glucose (0.25–10.0%, w/v), pH
(6.0–6.8), temperature (25 and 37oC), incubation time (24 and 6
h), and nutrients trypic soy broth (TSB) and diluted TSB (1:10).
Biofilm formation for 24 h at 25 and 37oC and attachment for 30
min and 6 h on the surface of polystyrene were measured by the
crystal violet staining method. Cell hydrophobicity of pathogens
for 6 and 24 h at 25 and 37oC was conducted using the modified
bacterial adherence to hydrocarbons method (mBATH). Biofilm
formation and attachment of pathogens were highly influenced by
the addition of glucose and sodium chloride compared to pH. The
biofilm of all pathogens formed in TSB was greater than that in
diluted TSB. Biofilm formations of S. aureus and P. aeruginosa at
37oC were greater than that at 25oC. However, biofilm formation
of L. monocytogenes was not significantly affected by temperature.
Levels of L. monocytogenes hydrophobicity were influenced by
adding glucose and sodium chloride at 37oC, whereas levels of
hydrophobicity for other pathogens were significantly different
depending on the glucose condition (p <0.05). The results demonstrate
that biofilm formation, attachment, and hydrophobicity of pathogens
were affected by environmental conditions such as the addition of
glucose and sodium chloride. However, factors affecting biofilm
formation and cell hydrophobicity differed depending on the
pathogen type.
Keywords attachment · biofilm formation · cell hydrophobicity ·
environment factor · foodborne pathogens
Introduction
Contamination of food with pathogenic bacteria arising from the
processing environment is a significant food hygiene and safety
issue. Numerous studies have shown that Listeria monocyteogenes,
Staphylococcus aureus, and Pseudomonas aeruginosa are capable
of adhering and forming biofilms in food-processing environments
(Sundheim et al., 1992; Blackman and Frank, 1996; Mettler and
Carpentier, 1998; Poulsen, 1999; Holah and Gibson, 2000).
Pseudomonas spp. are spoilage bacteria commonly found in the
food industry. P. aeruginosa, an opportunistic pathogen, has a high
tendency to form biofilms and is resistant to disinfectants (Poulsen,
1999). In our previous study investigating the biofilm formation of
various pathogens (not published data), L. monocyteogenes, S.
aureus, and P. aeruginosa were generally strong biofilm formers
compared with others (Escherichia coli O157:H7, Cronobacter
sakazakii, Salmonella Typhimurium, and Bacillus cereus). The
formation of biofilms creates major problems in the food industry,
because they represent an important source of contamination for
foodstuffs, leading to food spoilage or disease transmission.
Particularly, biofilms allow microorganism to persist in the
environment and resist desiccation, ultraviolet light, and treatment
with antimicrobial and sanitizing agents (Borucki et al., 2003;
Folsom and Frank, 2006). L. monocytogenes strains that persist in
food industry environments form thicker biofilms than isolates
found sporadically (Lunden et al., 2000) indicating that biofilm
formation is important for the survival of pathogens in the food
industry.
Environmental factors, including pH, temperature, nutrient
composition, and bacterial characteristics play important roles in
the phenotype change from planktonic cells to the sessile form
(Herald and Zottola, 1988). Bacteria have a natural tendency to
adhere to surfaces as a survival mechanism, and bacterial
colonization of solid surfaces has been described as a basic and
natural bacterial strategy in a wide variety of environments (Hunt
N.Y. Choi · B.R. Kim · Y.M. Bae · S.Y. Lee (�)Department of Food Science and Technology, Chung-Ang University, 72-1Nae-ri, Daedeok-myeon, Anseong-si 456-756, Republic of KoreaE-mail: nina6026@gmail.com, nina6026@cau.ac.kr
ORIGINAL ARTICLE
208 J Korean Soc Appl Biol Chem (2013) 56, 207−220
et al., 2004). Møretrø and Langsrud (2004) reported that
environmental factors, including temperature, sugar, salt, pH, and
nutrients, which are common in foods and food-processing
environments, have impacts on L. monocytogenes adhesion and
biofilm formation. Bacterial attachment to surfaces is influenced
by physicochemical properties of the environment (temperature
and pH), surface (hydrophobicity), and the microorganism
(hydrophobicity, flagellation, and motility) (Herald and Zottola,
1988; Hood and Zottala, 1995; Chavant et al., 2002; Gorski et al.,
2003; Moltz and Martin, 2005; Folsom et al., 2006). Cell surface
hydrophobicity responds to a wide variety of environmental
factors and appears to be involved in cell-to-cell interactions;
adherence of bacteria to solid surfaces; partitioning at liquid-
liquid, solid-liquid or liquid-air interfaces; and resistance of cells
to specific treatments by organic solvents or antibiotics (Rosenberg
et al., 1980; Rosenberg and Doyle, 1990; Vanhaeche et al., 1990),
which are essential to various technologies and natural processes.
Jana et al. (2000) demonstrated that hydrophobicity of Pseudomonas
fluorescens differs depending on culture age, pH, and temperature.
Pathogens such as L. monocyteogenes, S. aureus, and P.
aeruginosa are responsible for major foodborne outbreaks due to
their wide distribution in the environment and presence in the food
industry. However, no studies have evaluated cell hydrophobicity,
biofilm formation and attachment of these pathogens under
different environmental conditions. Therefore, the present study
was conducted to investigate biofilm formation, attachment, and
hydrophobicity of L. monocyteogenes, S. aureus, and P. aeruginosa
on the surface of polystyrene under various environmental conditions,
including different sodium chloride and glucose concentrations,
pHs, temperatures, incubation times, and nutrients.
Fig. 1 Biofilm formation of Listeria monocytogenes on the surface of polystyrene incubated at 25oC for 24 h in tryptic soy broth (TSB) (A) anddiluted TSB (1:10) (B), at 37oC for 24 h in TSB (C) and diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
J Korean Soc Appl Biol Chem (2013) 56, 207−220 209
Materials and Methods
Bacterial strains and growth conditions. Three pathogens of 2–
3 strains each, including P. aeruginosa (ATCC 10145 and ATCC
15692), L. monocytogenes (ATCC 7644, ATCC 19114, and ATCC
19115), and S. aureus (ATCC 49444, ATCC 12692, and ATCC
12600) were obtained from the bacterial culture collection of
Chung-Ang University (Anseong, Korea) for use in this study. P.
aeruginosa. L. monocytogenes, and S. aureus, which may be
present in food, beverages, and food processing facilities, were
cultivated in tryptic soy broth (TSB: Difco Laboratories, USA)
without dextrose and diluted TSB (1:10) broth supplemented with
various concentrations of sodium chloride (0.5–7.0%, w/v),
glucose (0.25–10.0%, w/v), and pH values (6.0, 6.4, and 6.8) for
24 h at 25 and 37oC. All cultures were maintained on tryptic soy
agar (Difco Laboratories) slants at 4oC and subcultured monthly.
Biofilm formation assay. Each strain of pathogens was cultured
individually in TSB and 1:10 TSB for 24 h at 25 and 37oC. Sterile
96-well polystyrene plates were filled with 90 µL TSB and diluted
TSB (1:10) and inoculated with 10 µL of overnight-cultured
pathogens (ca. 109 CFU/mL) to form biofilms on the surface of
96-well microtiter plates. Negative control wells containing only
TSB and diluted TSB (1:10) were included in each assay. The
pathogens were incubated at 37oC for 24 h. After discarding the
medium in the microtiter plate by inversion of the plate, the wells
were rinsed three times with distilled water (200 µL/well). After
air-drying, the wells were stained with 50 µL of 0.5% crystal
violet for 5 min. Excess stain was removed by washing (5×) with
distilled water (200 µL/well). Dye bound to adherent cells was
destained with or without 50 µL of 99% ethanol, and the optical
density of each well was determined at 595 nm using a spectro-
photometer (Specronic 20 Genesys, Spectronic Instruments, USA).
Fig. 2 Attachment of Listeria monocytogenes on the surface of polystyrene incubated for 30 min in tryptic soy broth (TSB) (A) and in diluted TSB(1:10) (B) for 6 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
210 J Korean Soc Appl Biol Chem (2013) 56, 207−220
Bacterial adherence to hydrocarbons (BATH) assay on
polystyrene microtiter plates. The modified BATH method
(Goulter et al., 2010) was used in this study. Three strains of each
pathogen were harvested at the exponential (6 h) and stationary
phases (24 h) at 25 and 37oC, collected by centrifugation (1000 ×
g for 10 min), washed twice, and resuspended in phosphate
buffered saline (PBS) (pH 7.2). The OD of the suspension was
adjusted with PBS to 1.0 (±0.2) at 595 nm. Fifty microliters of
bacterial cell suspensions (Ac) was measured using a spectro-
photometer, and 90 µL of bacterial cell suspension was overlaid
with 30 µL of n-nonane (Alfa Aesar, UK) as the hydrocarbon and
was added to ammonium sulfate (Kanto Chemical, Japan) at the
final concentration of 2 M. The suspensions were vortexed for 5
min (Ab). A 96-well plate containing 50 µL of the untreated cell
suspension was used as the control (Ac). The 96-well plate was
allowed to stand at room temperature for 30 min. Following the
incubation, 50 µL of the lower aqueous layer was removed, and
the OD600nm was measured. The ratio of the absorbance of the
bacterial assay tubes (Ab) to the control suspension (Ac) was
calculated as a percentage of bound cells to the hydrocarbon using
the following formula; cell hydrophobicity (%) = (Ac – Ab)/Ac ×
100
Attachment of pathogens to polystyrene. The growth of pathogens
was monitored by measuring the absorbance at 600 nm (A600)
using a spectrophotometer, and the cells were incubated until the
late exponential phase (A600 of 0.9). Five milliliters of the cultures
were centrifuged (1800 × g, 10 min), washed twice with PBS (pH
7.0), diluted to attain an A600 of approximately 0.1 with PBS,
followed by addition of 100 µL of the diluted cells to each well of
a 96-well microtiter plate. The microtiter plate was allowed to
stand for 30 min or 6 h at room temperature, after which the
supernatant, including the planktonic cells, was removed from
each well. Each well was gently washed twice using aliquots of
150 µL of deionized water. After washing, the plate was air-dried
for 5 min, followed by addition of 50 µL of 0.5% crystal violet
solution, and the cells were allowed to stain for 5 min. The excess
Fig. 3 Hydrophobicity of Listeria monocytogenes incubated at 25oC for 6 h in tryptic soy broth (TSB) (A) and diluted TSB (1:10) (B), at 25oC for24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
J Korean Soc Appl Biol Chem (2013) 56, 207−220 211
crystal violet solution was removed by gently washing the wells
four times with 150 µL of sterile deionized water. The attached
cells were air-dried for 10 min, and 50 µL of 95% ethanol was
placed in each well. Crystal violet was sufficiently eluted by
pipetting, and the initial adherence to each well was measured at
A595.
Statistical analysis. All experiments were repeated three times
with duplicate samples. Data were analyzed by analysis of
variance with the Generalized Linear Model procedure of SAS
(version 9.1, SAS Institute Inc., USA) for a completely randomized
design. Duncan’s multiple-range test was applied to establish
differences between means for each parameter.
Results
The ability of L. monocytogenes to produce biofilm on the surface
of polystyrene in TSB and diluted TSB (1:10) for 24 h at 25 and
37oC is presented in Fig. 1. Biofilm formation of L. monocytogenes
increased significantly in all media supplemented with glucose
(0.25–5%) (p <0.05). In particular, L. monocytogenes biofilm was
produced at significantly higher levels in all media supplemented
with 1% glucose than those in media without glucose (p <0.05).
At 25oC, the level of L. monocytogenes biofilm formation in
medium supplemented with 2% sodium chloride was higher than
that of medium without sodium chloride. A medium with pH of
6.0 had a significant effect on L. monocytogenes biofilm formation
(p <0.05). L. monocytogenes formed higher levels of biofilm in
TSB than those in diluted TSB (1:10). No significant difference in
L. monocytogenes biofilm levels was observed for the biofilms
formed at 25 and 37oC.
When L. monocytogenes was cultivated in TSB and diluted
TSB (1:10), attachment to the polystyrene surface was significantly
affected by attachment time (p <0.05) (Fig. 2). Attachment of L.
Fig. 4 Hydrophobicity of Listeria monocytogenes incubated at 37oC for 6 h in tryptic soy broth (TSB) (A) and diluted TSB (1:10) (B), at 37oC for24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
212 J Korean Soc Appl Biol Chem (2013) 56, 207−220
monocytogenes for 30 min on the polystyrene surfaces was greater
in TSB supplemented with (0.5 and 5%) sodium chloride and in
TSB (1:10) supplemented with 5% sodium chloride, 1% glucose,
and adjusted to a pH of 6.8 compared to that under other
conditions. Attachment of L. monocytogenes on the polystyrene
surface for 6 h was influenced by the addition of glucose in TSB
and diluted TSB (1:10). In addition, attachment in diluted TSB
(1:10) supplemented with sodium chloride for 6 h was significantly
higher than the other bacteria tested (p <0.05).
Levels of L. monocytogenes hydrophobicity differed depending
on sodium chloride, glucose, pH, temperature, incubation time,
and nutrients (Figs. 3 and 4). Similar to the biofilm formation
results, L. monocytogenes cell hydrophobicity levels were significantly
influenced by the addition of glucose to diluted TSB (1:10) at 25
and 37oC, regardless of incubation time (p <0.05). A high level of
hydrophobicity was observed in both TSB and diluted TSB (1:10)
supplemented with 0.5% sodium chloride after 6 h at 25oC (Fig.
3). In particular, when L. monocytogenes was incubated in TSB
supplemented with sodium chloride at 37oC for 24 h, the levels of
L. monocytogenes hydrophobicity were significantly higher than
those of the other bacteria (p <0.05) (Fig. 4).
The ability of P. aeruginosa to produce biofilm in TSB and
diluted TSB (1:10) for 24 h at 25 and 37oC is shown in Fig. 5. P.
aeruginosa produced higher levels of biofilm in TSB compared to
those in diluted TSB (1:10). On the other hand, biofilm formed by
P. aeruginosa in diluted TSB (1:10) at 37oC for 24 h was not
significantly different compared to that of the control. P. aeruginosa
biofilm formation was enhanced when the glucose concentration
was increased from 5 to 10% in all media except for diluted TSB
at 37oC. Biofilm formation of P. aeruginosa was influenced by the
Fig. 5 Biofilm formation of Pseudomonas aeruginosa on the surface of polystyrene incubated at 25oC for 24 h in tryptic soy broth (TSB) (A) and indiluted TSB (1:10) (B), at 37oC for 24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
J Korean Soc Appl Biol Chem (2013) 56, 207−220 213
addition of sodium chloride in all media except for diluted TSB
(1:10) at 37oC.
P. aeruginosa demonstrated high levels of attachment after 30
min in TSB supplemented with glucose (Fig. 6). Levels of attachment
of P. aeruginosa after 30 min were influenced by the pH level of
the media and showed higher levels of attachment at the pH of 6.4
than that of the other pHs tested. Attachment of P. aeruginosa for
6 h on the polystyrene surfaces was greater in diluted TSB (1:10)
supplemented with 0.5% sodium chloride and a pH of 6.8 than
that in diluted TSB (1:10). No significant difference in the P.
aeruginosa attachment levels were observed when incubated in
TSB for 6 h.
Hydrophobicity of P. aeruginosa incubated at 25oC for 6 and 24
h in TSB and diluted TSB (1:10) is shown in Fig. 7. Levels of P.
aeruginosa hydrophobicity incubated at 25oC for 6 and 24 h in
TSB and diluted TSB (1:10) were not significantly different
except for results after the 6 h incubation in diluted TSB (1:10).
Levels of P. aeruginosa hydrophobicity incubated for 6 h in diluted
TSB (1:10) were influenced by the addition of 1 and 5% glucose.
Levels of P. aeruginosa hydrophobicity in TSB supplemented
with glucose at 37oC were significantly higher than that of the
others except for P. aeruginosa incubated for 24 h in TSB (p
<0.05) (Fig. 8).
No significant differences in S. aureus biofilm formation were
observed at 37oC in any of the media (Fig. 9.). However, S. aureus
formed greater levels of biofilm compared with that of the control
at 25oC when sodium chloride was added at concentrations of 2–
7% into TSB, and glucose was added to diluted TSB (1:10) at
concentrations of 0.25–5%. Biofilm levels of S. aureus formed at
37oC were greater than those formed at 25oC. S. aureus produced
high levels of biofilm in TSB compared to those in diluted TSB
(1:10).
Fig. 6 Attachment of Pseudomonas aeruginosa on the surface of polystyrene incubated for 30 min in tryptic soy broth (TSB) (A) and in diluted TSB(1:10) (B), for 6 h in TSB (C) and in diluted TSB (1:10) (D), * means are significantly different compared to control (p <0.05).
214 J Korean Soc Appl Biol Chem (2013) 56, 207−220
Significant differences in attachment of S. aureus on the
polystyrene surface were observed when S. aureus was adhered in
TSB and diluted TSB (1:10) supplemented with sodium chloride
(p <0.05) (Fig. 10). In particular, S. aureus in diluted TSB (1:10)
adjusted to pH of 6.4 for 30 min and 6 h displayed high levels of
attachment on the polystyrene surface than other pH levels.
Biofilm formation and attachment of S. aureus were highly
influenced by the addition of sodium chloride compared to those
of other pathogens. Additionally, S. aureus incubated at 37oC for
6 and 24 h in TSB and diluted TSB (1:10) supplemented with
sodium chloride had significantly different hydrophobicity levels
(p <0.05) (Fig. 12). However, hydrophobicity levels of S. aureus
incubated at 25oC for 6 and 24 h were also influenced by the
addition of glucose in TSB and diluted TSB (1:10) (Fig. 11).
Hydrophobicity levels of S. aureus incubated at 25oC for 24 h in
diluted TSB (1:10) adjusted to pH of 6.0 were significantly
different (p <0.05) (Fig. 11). In particular, hydrophobicity of S.
aureus incubated at 37oC for 24 h in diluted TSB (1:10) was
influenced by all factors tested such as sodium chloride, glucose,
pH, temperature, incubation time, and nutrients (Fig. 12). Taken
together, biofilm formation, attachment, and hydrophobicity of the
pathogens differed depending on various environmental factors
and pathogen types.
Discussion
Biofilm formation, attachment, and cell hydrophobicity of L.
monocyteogenes, P. aeruginosa, and S. aureus under various
environmental conditions were evaluated. Several studies have
suggested that attachment and biofilm formation is modulated by
glucose present in the culture media (Stanley and Lazazzera,
2004; Pan et al., 2010). Our results demonstrated that biofilm
formation of pathogen was highly influenced by the addition of
Fig. 7 Hydrophobicity of Pseudomonas aeruginosa incubated at 25oC for 6 h in tryptic soy broth (TSB) (A) and in diluted TSB (1:10) (B), at 25oC for24 h in TSB (C) and diluted in TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
J Korean Soc Appl Biol Chem (2013) 56, 207−220 215
glucose and sodium chloride compared to pH. Pan et al., (2010)
reported that L. monocytogenes generally forms higher-density
biofilms in TSBYE supplemented with 1% glucose at 37oC than
that at other concentrations, and that the optimal salt concentrations
for L. monocytogenes biofilm formation are 5% at 22.5oC and 2%
at 30 and 37oC. Similar to these results, we found that L.
monocytogenes produced biofilm at significantly higher levels in
all media supplemented with 1% glucose and 2% sodium chloride
than those in medium without supplementation (Fig. 1). We also
showed that a suspending medium at pH of 6.0 had a significant
effect on L. monocytogenes biofilm formation (Fig. 1). Duffy and
Sheridan (1997) found that the pH of the suspending medium
significantly affects the adhesion of L. monocytogenes NCTC
11994 cells to the membrane, with a greater number of microbial
cells adhering at lower pH values. Briandet et al. (1999a) also
reported that preculturing L. monocytogenes in an acid-
supplemented medium (pH=6.0) increases bacterial attachment to
stainless steel, confirming the influence of pH on biofilm
formation of L. monocytogenes. However, biofilm formation of L.
monocytogenes was not influenced by all levels of pH tested,
because difference was found only at pH of 6.0.
In the present study, S. aureus formed biofilms with higher
density compared to those of the control at 25oC when 2–7%
sodium chloride was added to TSB and when 0.25–5% glucose
was added to diluted TSB (1:10) (Fig. 9). Attachment of S. aureus
was influenced by adding sodium chloride (Fig. 10). Moretro et al.
(2003) demonstrated that S. aureus forms the thickest biofilm at
the highest sodium chloride concentration (5.4%) tested at 37oC.
The glucose concentration required for maximal biofilm formation
ranged between 0.3 and 1.3%, depending on the S. aureus strain.
Chaieb et al. (2007) demonstrated that the adherence of S.
epidermidis cells is inhibited at acidic pH levels and enhanced by
alkaline pH values. Our study demonstrated that the attachment of
S. aureus was inhibited at acidic pH levels, and that diluted TSB
Fig. 8 Hydrophobicity of Pseudomonas aeruginosa incubated at 37oC for 6 h in tryptic soy broth (TSB) (A) and in diluted TSB (1:10) (B), at 37oC for24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
216 J Korean Soc Appl Biol Chem (2013) 56, 207−220
(1:10) adjusted to pH of 6.4 resulted in high level of attachment
when S. aureus was attached on the polystyrene surface for 30
min and 6 h (Fig. 10).
Other studies have also investigated the attachment of P.
aeruginosa. Marchall et al. (1971) suggested that attachment of
Pseudomonas spp. is favored when the glucose concentration is
0.7% and is reduced when 1.4 or 2.1% glucose is present. Our
results showed that P. aeruginosa levels of attachment in TSB
supplemented with 0.25 and 1% glucose were higher than those of
the other bacteria (Fig. 6). In agreement with the results obtained
by Stanley (1983), adhesion of P. aeruginosa increased in the
presence of 10 mM NaCl or CaCl2, when P. aeruginosa attaches
to a batch of 304 stainless steel, and maximal adhesion occurred
at neutral pH. Vanhaecke et al. (1990) demonstrated that adhesion
of P. aeruginosa varies depending on the bacterial strain and
conditions; however, the influence of the NaCl concentration on
the extent of bacterial adhesion appeared to be minimal. In our
study, attachment of P. aeruginosa was not influenced by sodium
chloride (Fig. 6), although P. aeruginosa biofilm formation was
influenced by sodium chloride concentration (Fig. 5).
Petel et al. (2011) reported that biofilm formation by E. coli
O157:H7 strains 4688, 1918, and 5279 was significantly higher in
diluted TSB (1:10) than that in TSB or Luria-Bertani medium.
Similar results have been reported for E. coli, P. aeruginosa, and
L. monocytogenes under different environmental conditions
(Briandet et al., 1999b). However, in our study, biofilm formation
of pathogens in TSB on the surface of polystyrene was higher
than that in diluted TSB (1:10). Similarly, Mai and Conner (2007)
reported that the attachment of L. monocytogenes to stainless steel
and cultivated in BHI was significantly higher at 30 and 37oC
Fig. 9 Biofilm formation of Staphylococcus aureus on the surface of polystyrene incubated at 25oC for 24 h in tryptic soy broth (TSB) (A) and indiluted TSB (1:10) (B), at 37oC for 24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
J Korean Soc Appl Biol Chem (2013) 56, 207−220 217
compared to cultivation in a minimum medium. They also found
that attachment of L. monocytogenes is greater at higher
temperatures (30 and 37oC) than at lower temperatures (4, 20, and
42oC). Pan et al. (2010) demonstrated that L. monocytogenes
forms high density biofilms as temperature increases. In our study,
S. aureus and P. aeruginosa biofilm formation at 37oC was higher
than that at 25oC. However, L. monocytogenes biofilm formation
was not significantly affected by temperature.
Bacterial cell hydrophobicity could affect the attachment and
biofilm formation of pathogens. In our previous studies (unpublished
data), pathogens except for E. coli O157:H7 and B. cereus exhibited
a high degree of correlation between biofilm formation and cell
hydrophobicity for 24 h (r=0.9934). Takashi et al. (2010) also
reported that a high degree of correlation was observed between
adherence on PVC and cell hydrophobicity of L. monocytogenes
(ca. r=0.87). Many studies have indicated that bacterial cell
hydrophobicity is influenced by environmental conditions, such as
the temperature, growth media, and cultivation phase (Doelle et
al., 1982; Nikovskaya et al., 1989; Aono and Kobayashi, 1997;
Perez et al., 1998; Walker et al., 2005). Zikmanis et al. (2007)
reported that cell surface hydrophobicity values of Zymomonas
mobilis increase proportionally with increases in cultivation
temperature and concentration of the carbon source (glucose or
sucrose) in the medium. In the present study, hydrophobicity
levels of L. monocytogenes incubated in diluted TSB (1:10)
supplemented with glucose at 25oC for 6 and 24 h were significantly
different (Fig. 3). At 37oC, L. monocytogenes hydrophobicity was
influenced by glucose and sodium chloride when incubated for 6
and 24 h, respectively (Fig. 4). Levels of hydrophobicity of P.
aeruginosa and S. aureus were significantly different depending
on glucose concentration, although hydrophobicity varied depending
on the temperature, growth media, and cultivation phase (Figs. 7,
Fig. 10 Attachment of Staphylococcus aureus on the surface of polystyrene incubated for 30 min in tryptic soy broth (TSB) (A) and in diluted TSB(1:10) (B), for 6 h in TSB (C) and in diluted TSB (1:10) (D), * means are significantly different compared to control (p <0.05).
218 J Korean Soc Appl Biol Chem (2013) 56, 207−220
8, 11, and 12). Petel et al. (2011) demonstrated that the E. coli
strain O157:H7 for hydrophobicity was affected by the individual
strain and the growth phase. Each strain in the log phase (5 h,
37oC) was significantly more hydrophobic than the corresponding
strain in the stationary phase (18 h, 37oC). Jana et al. (1999)
reported that P. fluorescens hydrophobicity is dependent on
culture age, pH, and temperature; early-to mid-log exponential
phase cells were more hydrophobic than those in the stationary
phase. Maximum cell surface hydrophobicity of P. fluorescens
was observed at pH 7.0–7.5, with a decreasing trend at higher and
lower pHs. Chavant et al. (2002) demonstrated that hydrophobicity
levels of L. monocytogenes in the stationary phase at 37oC are
higher than those in bacteria at the exponential phase. L.
monocytogenes hydrophobicity in the exponential phase at 20oC is
higher than those in the stationary phase. In our study,
hydrophobicity levels of L. monocytogenes in the exponential
phase (6 h) at 25oC were higher than those in the stationary phase
(24 h), whereas hydrophobicity levels of L. monocytogenes in the
stationary phase (24 h) were higher at 37oC than those in the
exponential phase (6 h).
Biofilm formation, attachment, and cell hydrophobicity of the
tested pathogens were affected by environmental factors such as
glucose, sodium chloride, pH, temperature, incubation time, and
nutrients. In general, biofilm formation, attachment, and cell
hydrophobicity of the pathogens were significantly influenced by
the addition of glucose and sodium chloride. The biofilms of all
pathogens in TSB and at 37oC on the surface of polystyrene were
stronger than those in diluted TSB (1:10) and at 25oC. However,
the effects of environmental conditions on biofilm formation,
attachment, and cell hydrophobicity of pathogens varied depending
on the pathogen.
Fig. 11 Hydrophobicity of Staphylococcus aureus incubated at 25oC for 6 h in tryptic soy broth (TSB) (A) and in diluted TSB (1:10) (B), at 25oC for24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).
J Korean Soc Appl Biol Chem (2013) 56, 207−220 219
Acknowledgment This research was supported by Basic Science Research
Program through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (No. 2012-0004225).
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