133 Journal of Food Protection, Vol. 77, No. 1, 2014, Pages 133–140 doi:10.4315/0362-028X.JFP-13-074 Copyright G, International Association for Food Protection
Biofilm Formation under Different Temperature Conditions by a Single Genotype of Persistent Listeria monocytogenes Strains YOSHITSUGU OCHIAI,1* FUMIYA YAMADA,1,2 MARIKO MOCHIZUKI,3 TAKASHI TAKANO,1 RYO HONDO,1 FUKIKO UEDA1 1Department
of Veterinary Science and 3Department of Veterinary Nursing and Technology, Nippon Veterinary and Life Sciences University, Tokyo 180-8602, Japan; and 2Saitama Institute of Public Health, Saitama 338-0824, Japan MS 13-074: Received 25 February 2013/Accepted 6 September 2013
ABSTRACT Some Listeria monocytogenes strains, termed persistent strains, originate from the same processing plant and have the ability to survive and grow over extended periods of time at contamination sources. In order to evaluate biofilm formation by such persistent strains, we isolated the pathogen from chicken samples collected from the same retail shop in repeated visits over 6 months. Strains that were of serotype 1/2b and were assigned to the same genotype by multi-virulence-locus sequence typing analysis were isolated on repeated occasions from December 1997 to June 1998 and thus were defined as persistent strains. In the present study, biofilm formation by the persistent strains was evaluated using microplates at 30 and 37uC. The biofilm-forming capability was measured after cells attaching to the microplate well were stained with crystal violet. Comparison of biofilm formation at 30uC among the persistent strains showed that a significantly higher amount of the stain was obtained from the persistent strains isolated from December to March than from those isolated from April to June. However, no significant difference in biofilm formation at 30uC was observed between persistent and nonpersistent groups of L. monocytogenes strains. In contrast, biofilm formation at 37uC was consistent among the persistent strains, and they produced significantly more biofilm at 37uC than did the nonpersistent strains. The persistent strains were also found to change their biofilm-forming ability in a temperature-dependent manner, which may suggest that the persistent strains alter their biofilm formation in response to changing environmental factors.
Listeria monocytogenes is recognized as an important foodborne pathogen. Patients with listeriosis, primarily in the immunocompromised population, exhibit severe manifestations of invasive disease, including neurological infections such as encephalitis and meningitis, septicemia, and abortion (30). This pathogen can survive and grow over a wide range of temperatures (approximately 0 to 45uC) and pH values (approximately 4.4 to 9.6) and at high concentrations of sodium chloride (10 to 12%) (26). These characteristics are associated with the ubiquitous distribution of this bacterium in the environment, including food products and foodprocessing environments. Its ubiquitous nature increases exposure of human hosts to L. monocytogenes, as evidenced by a report indicating that this pathogen is associated with the greatest number of food product recalls in the United States (32). The prevalence of L. monocytogenes in foods in Japan is almost comparable to that reported for other countries (20). Therefore, eradication of L. monocytogenes from food products and food processing environments is important for the control of listeriosis. It has been suggested that L. monocytogenes forms biofilms in food industrial environments, which is a major factor in the contamination of food products (15, 24). * Author for correspondence. Tel: z81-422-31-4151; Fax: z81-42230-7531; E-mail: [email protected]
Biofilms are composed of bacterial cells and self-generated extracellular polymeric substances, which protect the organisms from desiccation, antibacterial agents, disinfectants, and host immune response (9, 12, 13, 25). Several studies have shown that environmental factors, including temperature, pH, nutrient conditions, and the presence of substances that affect bacterial survival and growth, have an influence on the biofilm formation of numerous bacteria, including L. monocytogenes (10, 14, 17, 21, 22). Moreover, comparative studies have reported variability in biofilm formation by L. monocytogenes strains and a correlation between this ability and specific groups, classified according to serotype or phylogenetic lineage (1, 3, 7, 10, 21). However, no conclusions regarding this correlation have been reached, because the studies involved presented conflicting results. Conflicting results have also been reported for persistent and nonpersistent strains (1, 7). It has been suggested that persistent strains have specific traits, including resistance to heat and acids, adherence to surfaces, and the ability to survive in certain environments over extended periods of time (8, 29). Therefore, elucidation of the marked characteristics that persistent strains exhibit during biofilm formation is necessary and may lead to a better understanding of the ecological advantages of the biofilms formed by persistent strains.
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To evaluate biofilm formation by L. monocytogenes, an assay using microtiter plates has been developed (7). This is a rapid and simple method and, therefore, has been used for comparative studies of biofilm formation among L. monocytogenes strains or variants (1, 7, 9, 14, 17, 21, 29). To investigate the characteristics of biofilm formation by persistent strains, we evaluated biofilm formation by a series of 30 persistent strains that were isolated from chicken samples on repeated occasions over 6 months and that were serologically and genetically identical using the microplate assay. Biofilm formation by the persistent strains was compared with that by L. monocytogenes strains that were isolated previously from various classes of meat (beef, chicken, and pork) collected from different retail shops (19). Given that differences in relative biofilm production among strains have been reported under different growth conditions (10), it has been suggested that evaluation of biofilm formation under multiple growth conditions is needed. Therefore, the present evaluation was performed under multiple temperature conditions, including at 30 and 37uC, because temperature is one of the critical factors influencing L. monocytogenes biofilm formation (15). MATERIALS AND METHODS Isolation of L. monocytogenes. Persistent strains were isolated from samples of minced chicken processed from breast meat that was obtained on repeated visits to the same retail shop in Tokyo. A total of 21 visits to obtain the samples (dates listed in Table 1) were made from December 1997 to June 1998. All meat samples were collected from the same area in the store and were confirmed to have been processed in the same plant according to the labeling. The samples were stored at 220uC before isolation of L. monocytogenes, as described previously (19). Isolation of L. monocytogenes from all the chicken samples was performed in 1998. For the enrichment procedure for isolation of L. monocytogenes, 10 g of a meat sample was added to 90 ml of University of Vermont–modified Listeria enrichment broth (Nippon BD, Tokyo, Japan) and incubated at 30uC for up to 48 h, as described previously (19). The culture was streaked onto PALCAM Listeria-selective agar (Merck Japan, Tokyo) and incubated at 37uC for 48 h. Listeria-like colonies on the agar plate were selected for further analyses. Beta-hemolytic activity, Gram staining, and biochemical properties were used for bacterial identification. Serotyping of the L. monocytogenes isolates was performed using a commercial kit (Denka Seiken, Tokyo). The isolates obtained were stored at 280uC in brain heart infusion broth containing 10% glycerol. The isolates stored were passaged twice using brain heart infusion agar before use in the present study. For the nonpersistent strains of L. monocytogenes, 25 strains of serotype 1/2b were used for comparison. These strains were previously isolated from different classes of retail meat, including beef, chicken, and pork, from shops in the Tokyo metropolitan area between 1998 and 2003 (19). The isolates of L. monocytogenes used are listed in Table 2. Multi-virulence-locus sequence typing. DNA extraction and purification were performed as described previously (28). DNA amplification of the six virulence genes (prfA, inlB, inlC, dal, clpP, lisR) was performed as described by Zhang et al. (34). However, an annealing temperature of 47uC was used for the amplification of the partial prfA and dal genes. The amplified products were purified with a QIAquick PCR purification kit or a QIAEX II gel extraction kit (Qiagen, Tokyo) according to the
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TABLE 1. Isolation of L. monocytogenes or other Listeria spp. from chicken samples collected from a retail shop on repeated visits Isolation of Listeria monocytogenes Sampling date
2 Dec. 1997 14 Dec. 1997 9 Feb. 1998 10 Feb. 1998 11 Feb. 1998 12 Feb. 1998 10 Mar. 1998 11 Mar. 1998 12 Mar. 1998 13 Mar. 1998 14 Mar. 1998 13 Apr. 1998 15 Apr. 1998 16 Apr. 1998 17 Apr. 1998 18 May 1998 20 May 1998 21 May 1998 22 May 1998 10 June 1998 12 June 1998 a
z z za zb z z z z z z z z z z z z
Isolation of other Listeria spp.
z z z z z
Three L. monocytogenes strains isolated from chicken collected on 9 February were assigned to the persistent genotype, while the other two strains were assigned to a different genotype. All L. monocytogenes strains isolated from chicken collected on 10 February were assigned to a genotype other than the persistent genotype.
manufacturer’s instructions. DNA sequencing was conducted by Operon Biotechnologies (Tokyo). Sequences for the reference strains were obtained from reports by Chen et al. (5) and Zhang et al. (34). Editing of the concatenated sequences and genetic distance estimation was performed using DNAsis Pro (version 2.0, Hitachi Software Engineering, Tokyo). Confidence intervals for the phylogenetic tree were obtained using 1,000 neighbor-joining bootstrap replications. Isolates within the same multi-virulence-locus sequence typing cluster were considered to be the same genotype. Microplate biofilm formation assay. Each of the L. monocytogenes strains was cultured overnight at 37uC in brain heart infusion broth and diluted 1:50 in modified Welshimer’s broth with reduced phosphate concentration (4.82 mM potassium dihydrogen phosphate and 11.55 mM disodium hydrogen phosphate) and 100 mM 3-(N-morpholino)propanesulfonic acid (pH 7.4) (23, 27). Aliquots (150 ml) of this dilution were inoculated into six wells of a flat-bottomed, 96-well polystyrene tissue culture plate (ref. 353072; BD Falcon, Tokyo). The outermost wells were not used for the assay, because these wells suffer edge effects. Modified Welshimer’s broth without L. monocytogenes was placed in six wells of each plate as control wells. The plates were incubated at 30 or 37uC for 48 h under humid conditions to prevent evaporation of the medium. After the incubation, the medium was removed from each well and the plates were washed three times with sterile distilled water. The plates were air dried and stained with 1% (wt/vol) crystal violet for 30 min at room temperature. After the plates had been washed three times with sterile distilled
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TABLE 2. L. monocytogenes strains used in the present study Strain
water, 150 ml of 95% ethanol was added to each well to destain the crystal violet. The amount of biofilm was measured as the absorbance at 595 nm (A595 nm) using a microplate reader (iMark Microplate Reader, Bio-Rad, Tokyo). The absorbance of the control wells was measured and subtracted from that of the wells inoculated with each strain to obtain the value for biofilm formation by L. monocytogenes. Two independent experiments were performed for each strain. Statistical analyses. Statistical analyses, including Fisher’s exact test, Mann-Whitney U test, and homoscedastic analysis by F test, were performed using Excel Statistics 2010 for Windows (SSRI Inc., Tokyo). The significance level was set at P , 0.05. Nucleotide sequence accession numbers. The DNA Data Bank of Japan accession numbers for the nucleotide sequences determined in the present study are AB750993 to AB751196.
RESULTS Isolation of L. monocytogenes and other Listeria spp. from chicken samples. L. monocytogenes serotype 1/ 2b strains were isolated from 17 (81%) of 21 chicken samples, which were collected from the same retail shop on repeated occasions from December 1997 to June 1998 (Table 1). In addition, a serotype 1/2a strain was sporadically isolated from a sample collected on 11 March. Other Listeria spp. strains were isolated from 11 (55%) of 21 chicken samples. Notably, the prevalence of other Listeria spp. in chicken samples collected from December to March was 4 (36.4%) of 11 samples, whereas the prevalence in those collected from April to June increased to 7 (70%) of 10 samples. However, the observed difference between the first and last half-periods was not significant (P ~ 0.20, Fisher’s exact test). Genetic classification. Isolated L. monocytogenes serotype 1/2b strains were classified genetically using MLVST. Two representative strains from each sample were used for genetic classification, except for three samples collected on 2 and 14 December and 12 March, for which one strain was used because only a single strain was isolated. Nucleotide sequences for the six MLVST genes were identical among all strains, except for strains isolated from chicken samples collected on 9 and 10 February (Table 1), although a nucleotide substitution in the prfA gene was found in the sequences of strains 77C2 and 89C6. Phylogenetic analysis also showed that the majority of the strains, including strains 77C2 and 89C6, constituted a single cluster (data not shown). Therefore, these strains were assigned to a single genotype. We defined this as a persistent genotype, as the strains assigned to this genotype were isolated repeatedly during the study period. Genetic classification was also employed for additional strains of L. monocytogenes from samples collected on 9 and 10 February, which were isolated strains assigned to a different genotype from the persistent genotype. All three strains isolated from a chicken sample collected on 9 February, strains 72C3, 72C4, and 72C5, were classified into the persistent genotype (Table 1). However, an additional strain isolated from a chicken sample collected
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on 10 February was assigned to a different genotype from the persistent genotype. Biofilm formation by persistent strains. Biofilm formation was evaluated at 30 and 37uC. The values of biofilm formation obtained from 30 persistent strains at 30uC ranged from 0.287 to 0.975, with a mean of 0.603 (Fig. 1A). The figure shows that the majority of strains producing more biofilms at 30uC were isolated from December to March. To confirm this, biofilm formation at 30uC was compared between a group of strains isolated from December to March and another isolated from April to June. The means of the former and latter groups were 0.676 and 0.494, respectively, and statistical analysis using the Mann-Whitney U test showed that biofilm formation by the former group was significantly greater (P , 0.01) than that by the latter group. On the other hand, values for biofilm formation at 37uC ranged from 1.028 to 1.652, with a mean of 1.276 (Fig. 1B). In contrast to the observations at 30uC, consistent biofilm formation at 37uC was observed among strains isolated during the entire period of this investigation. These findings are supported by the data indicating no significant differences (P ~ 0.18, Mann-Whitney U test) between a group of strains isolated from December to March and a group isolated from April to June. In addition, the ratio of the mean value of biofilm formation at 37uC to that at 30uC (the 37uC/30uC ratio) was calculated for each persistent strain. The 37uC/ 30uC ratio of the persistent strains ranged from 1.27 to 4.4 (Table 2). Almost 90% of the persistent strains were observed to have a 37uC/30uC ratio of .1.5, which shows a temperature-dependent response in biofilm formation. Comparison of biofilm formation by L. monocytogenes strains. In order to evaluate the ability of persistent strains to form biofilms, comparative examinations were performed. We used 25 strains of serotype 1/2b, isolated previously from various meat classes collected from different retail shops, as a nonpersistent group for this comparison (19). The reasons why these strains were used in the comparison are described below. First, this comparison was designed to be performed between persistent and nonpersistent strains of the same serovar. There are some reports of a correlation between serovar and the ability to form biofilms, although this correlation is limited to serotypes 1/2a and 4b (10, 22). Therefore, we aimed to exclude a possible effect caused by comparison between different serovars. In addition, the nonpersistent strains were isolated by the same procedure and stored under the same conditions as the persistent strains used in the present study (19). Biofilm formation by the nonpersistent strains is shown in Figure 2. The means obtained for biofilm formation at 30uC by the group of persistent and nonpersistent strains were 0.603 and 0.689, respectively. And the observed difference was not statistically significant (P ~ 0.18, MannWhitney U test). In addition, no significant difference was observed between the variances obtained for biofilm formation at 30uC by the persistent and nonpersistent groups (P ~ 0.18, F test). On the other hand, the mean value obtained for biofilm formation at 37uC by the group of
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FIGURE 1. Biofilm formation by persistent strains of L. monocytogenes at 30uC (A) and 37uC (B). Amount of biofilm formation was measured as absorbance (A595 mn) of crystal violet destained from L. monocytogenes biofilms in 96-well microplates. The bars indicate the mean values ¡ standard deviations, which were obtained from six replicates from each of two independent experiments (n ~ 12).
persistent strains was higher (P , 0.001, Mann-Whitney U test) than that of the nonpersistent strains (1.276 and 0.939, respectively). The variance obtained for biofilm formation at 37uC by the persistent group was significantly lower (P , 0.001, F test) than that by the nonpersistent group. Furthermore, the 37uC/30uC ratio of the nonpersistent strains ranged from 0.6 to 3.1 (Table 2). In contrast to persistent strains, half of the nonpersistent strains were observed to have a 37uC/30uC ratio from 0.75 to 1.25, which appears to show consistent biofilm formation between the two temperature conditions. However, the other nonpersistent strains showed a temperature-dependent response in biofilm formation, as observed in the majority of persistent strains. DISCUSSION Persistent strains, which were isolated from chicken samples from the same retail shop on repeated occasions during the study period and were assigned to the same serological and genetic type, were isolated from 16 (76.2%)
of 21 samples. The prevalence was significantly higher (P , 0.001, Fisher’s exact test) than that previously reported for L. monocytogenes, including not only serotype 1/2b but also other serotypes, isolated from chicken collected from various retail shops (46 [35.7%] of 129 samples) (19). This suggests that contamination of samples by the persistent strains used in the present study occurs repeatedly and with high frequency. A comparison of biofilm formation at 37uC between the groups of persistent and nonpersistent strains showed that the variance obtained from the former was significantly lower than that from the latter. The consistent biofilm formation among the persistent strains supports the hypothesis that they were derived from a common source of contamination. In contrast, persistent strains isolated from December to March produced more biofilms at 30uC than those from April to June. These findings suggest that the persistent strain changed between March and April, with regard to the biofilm phenotype observed at 30uC. This may
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FIGURE 2. Biofilm formation by nonpersistent strains of L. monocytogenes, which were of the same serotype as the persistent strains and were isolated from various meat classes from different retail shops, at 30uC (A) and 37uC (B). The bars indicate the mean values ¡ standard deviations, which were obtained from six replicates from each of two independent experiments (n ~ 12).
show a change in the environment that induced the phenotypic change in the persistent strain, although we do not have sufficient information on the background of the isolates to explain the change in biofilm formation. However, we found that other Listeria spp. showed a greater frequency of isolation from April to June, which suggests the presence of increased opportunities for microbial interactions among the Listeria species. Therefore, the variability in biofilm formation by the persistent strains observed at 30uC may reflect the influence of microbial interactions (11). However, we were unable to identify where these interactions might have occurred, such as in the biofilm at the contamination source, on the surface of the chicken sample, or during the propagation step using University of Vermont–modified broth in the process of bacterial isolation. Biofilm formation at 30uC by the series of persistent strains was almost equivalent to that of nonpersistent strains. These data are inconsistent with the report by Borucki et al. (1), in which persistent strains produced more biofilms than nonpersistent strains at the same temperature. However, some persistent strains used by the authors were observed to form equivalent biofilms to nonpersistent strains or those classified as neither persistent nor nonpersistent. Therefore, we do not consider our data to be in conflict with those of Borucki et al. (1). However, it is notable that the serotypes
used as the persistent strains differed between our study and that of Borucki et al. (1). They used persistent strains of serotype 1/2a assigned to lineage 2, whereas our persistent strains were of serotype 1/2b, and some of these were confirmed as lineage 1 in a previous study (18). It has been suggested that lineage 2 may be an environmentally adapted lineage, whereas lineage 1 may be a host-adapted lineage (16, 33). Therefore, several persistent strains of serotype 1/2a may have the ability to form higher amounts of biofilm at 30uC, which approaches environmental temperature conditions. In addition, there is no evidence that some of the nonpersistent strains used in the present study might not be persistent in other processing plants. Therefore, the nonpersistent group may be composed of some persistent strains, which may influence the comparative study between the persistent and nonpersistent groups. Furthermore, we cannot exclude the possibility that differences in biofilm formation may result from variations in the definition of persistence, as described elsewhere (1, 2). Evaluation of biofilm formation at 37uC showed that the group of persistent strains produced more biofilms than did the nonpersistent strains. Several studies have shown that biofilm formation at 37uC was greater than that at other temperatures (4, 6, 14, 21). With regard to the growth medium, almost all strains of the pathogen show more
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biofilm production in modified Welshimer’s broth, as used in the present study, than in nutritionally rich media (7, 14). Furthermore, exposure to stress, including high concentrations of salt and ethanol, has been reported to reduce the amount of biofilm produced by L. monocytogenes (21). The isolates in the present study were not exposed to stress. These data suggest that the biofilm formation observed at 37uC in the present study demonstrated the maximal ability of L. monocytogenes strains to form biofilms. Therefore, it is suggested that the persistent strains used have a greater maximal ability to form biofilms than other strains, although they do not always show this ability under all growth conditions. Enhanced biofilm formation was observed in almost all the persistent strains when temperature conditions were changed from 30 to 37uC. These findings suggest that the persistent strains used in this study regulate biofilm formation, depending on the temperature conditions. On the other hand, various effects of growth temperature on biofilm formation were observed in the nonpersistent strains. Although some studies have shown that L. monocytogenes produced more biofilm at 37uC than at lower temperatures (4, 6, 14, 21), our data suggest that half of the nonpersistent strains have no or only poor ability to regulate biofilm formation in a temperature-dependent manner. Therefore, the persistent strains used may have the ability to alter biofilm formation remarkably in response to changing environmental factors, including temperature conditions, which is necessary for persistent strains to survive and grow in changing environments over extended periods of time. This conclusion is consistent with that of Verghese et al. (31), who suggested that L. monocytogenes strains that contain a comk prophage showed rapid adaptation, biofilm formation, and persistence in different food processing environments. Therefore, it would be interesting to know if the persistent strains used in the present study contained the comk prophage. Further research would be needed to discover whether other genotypes of persistent strains have the ability to form biofilm that we observed in the present study. In addition, a study has shown that the ability of L. monocytogenes strains to form biofilm differs according to culture medium (10). A recent study reported that L. monocytogenes strains produced high cell densities and formed biofilms frequently when they grew on the foods from which they were originally isolated (31). Therefore, evaluation of biofilm formation using medium containing chicken extract would further the understanding of biofilm formation by the persistent strains that were isolated from chicken samples. REFERENCES 1. Borucki, M. K., J. D. Peppin, D. White, F. Loge, and D. R. Call. 2003. Variation in biofilm formation among strains of Listeria monocytogenes. Appl. Environ. Microbiol. 69:7336–7342. 2. Carpentier, B., and O. Cerf. 2011. Review—persistence of Listeria monocytogenes in food industry equipment and premises. Int. J. Food Microbiol. 145:1–8. 3. Chae, M. S., and H. Schraft. 2000. Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains. Int. J. Food Microbiol. 62:103–111.
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Disinfectant-tolerant Listeria monocytogenes biofilms can colonize surfaces that come into contact with food, leading to contamination and, potentially, food-borne illnesses. To better understand the process of L. monocytogenes biofilm formation and
Listeria monocytogenes is a dangerous bacterium that causes the food-borne disease listeriosis and accounts for nearly 20% of food-borne deaths. This organism can survive the body's natural defences within the digestive tract, including acidic condit
γ-Decalactone is a flavour compound that when obtained by biotechnological production using microorganisms is classified as natural. The aim of this study is to evaluate various conditions for γ-decalactone production by tropical yeast strains Yarrow
This study investigated the growth and survival of three foodborne pathogens (Clostridium perfringens, Campylobacter jejuni, and Listeria monocytogenes) in beef (7% fat) and nutrient broth under different oxygen levels. Samples were tested under anox
Biofilm formation is a major virulence factor in different bacteria. Biofilms allow bacteria to resist treatment with antibacterial agents. The biofilm formation on glass and steel surfaces, which are extremely useful surfaces in food industries and
A total of 98 previously characterized and serotyped L. monocytogenes strains, comprising 32 of 1/2a; 20 of 1/2b and 46 of 4b serotype, from clinical and food sources were studied for their capability to form a biofilm. The microtiter plate assay rev
We study a previously introduced mathematical model of amensalistic control of the foodborne pathogen Listeria monocytogenes by the generally regarded as safe lactic acid bacteria Lactococcus lactis in a chemostat setting under nutrient rich growth c
Listeria monocytogenes, a food-borne pathogen, has the capacity to maintain intracellular pH (pHi) homeostasis in acidic environments, but the underlying mechanisms remain elusive. Here, we report a simple microplate-based fluorescent method to deter