Variation in biofilm formation among strains of Listeria monocytogenes.
Journal: 2004/April - Applied and Environmental Microbiology
ISSN: 0099-2240
PUBMED: 14660383
Abstract:
Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.
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Appl Environ Microbiol 69(12): 7336-7342

Variation in Biofilm Formation among Strains of <em>Listeria monocytogenes</em>

Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Department of Veterinary Microbiology and Pathology, Department of Civil and Environmental Engineering, Washington State University, Pullman, Washington 991643
Corresponding author. Mailing address: Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA 99164-6630. Phone: (509) 335-7407. Fax: (509) 335-8328. E-mail: ude.usw.demtev@ikcurobm.
Received 2003 Apr 24; Accepted 2003 Sep 12.

Abstract

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.

Abstract

Listeria monocytogenes is a gram-positive bacterium capable of causing morbidity and mortality in both humans and animals. Due to the ubiquitous nature and hardy growth characteristics of this bacterium, L. monocytogenes is able to contaminate and thrive in the food-processing environment (11). In particular, the psychrotrophic nature of L. monocytogenes allows replication in refrigerated, ready-to-eat food products that have been contaminated during processing and packaging. Consequently, L. monocytogenes is frequently associated with food-borne disease outbreaks that are characterized by widespread distribution and relatively high mortality rates.

Although L. monocytogenes is clearly a pathogenic organism, not all infections result in serious illness and healthy carriers from high risk groups have been identified (17). Additionally, most outbreaks are caused by serotype 4b, even though serotype 1/2a is more frequently isolated from food and environmental samples (13). A pathogenicity island has been identified in L. monocytogenes (8, 32), and inactivation of these genes results in attenuation (1, 5, 7, 12, 29, 31). All strains of L. monocytogenes have these virulence-associated genes, and the sequences of many of these genes are conserved (14, 33). Nevertheless, not all strains appear equally capable of causing disease. Therefore, it is likely that a number of factors are involved in the ability of a strain to cause food-borne outbreaks, including differential ability to persist in environments where contamination of food products may occur.

Many bacteria are able to attach to and colonize environmental surfaces by producing a three-dimensional matrix of extracellular polymeric substances (EPS) called biofilm (10). Biofilms allow microorganisms to persist in the environment and resist desiccation, UV light, and treatment with antimicrobial and sanitizing agents. In the case of L. monocytogenes, Djordjevic et al. (9) reported a possible relationship between phylogeny and the ability to form biofilms. On average, strains associated with Division I (serovars 1/2b and 4b) produced more biofilm than did strains from Division II (serovars 1/2a and 1/2c). In contrast, others (18, 21) have shown that serotype 1/2c is a better biofilm former than are 4b strains. There is also disagreement regarding the relationship between persistence and the ability to form biofilms (9, 21). Furthermore, Kalmokoff et al. (16) argue that L. monocytogenes does not form a classic biofilm but simply adheres to surfaces. These differences might be explained by the strains used in the studies, sample sizes, and assay formats. Nevertheless, we were intrigued by the hypothesis that the ability to form biofilms might be conserved within phylogenetic lineages, because this would be consistent with conservation of other phenotypic traits, such as serotype. Furthermore, we wanted to further examine the hypothesis that persistent strains of L. monocytogenes are better biofilm formers than are nonpersistent strains. This pattern would be a logical explanation for cases where a single isolate is repeatedly isolated from a given environment (e.g., bulk milk tank) whereas other isolates may be found rarely.

Acknowledgments

We gratefully acknowledge the excellent technical assistance provided by Edward Kuhn, Stacey LaFrentz, Edith Orozco, and the staff at the Washington State University Scanning Electron Microscopy Center. L. monocytogenes isolates were kindly provided by Peggy Hayes and Louis Graves (Centers for Disease Control and Prevention, Atlanta, Ga.), Jinxin Hu (Washington State Department of Health, Olympia, Wash.), Karen Jinneman (U.S. Food and Drug Adminisration, Bothel, Wash.), and Lisa Gorski (U.S. Department of Agriculture—Agricultural Research Service, Albany, Calif.).

Funding was provided by USDA-Agricultural Research Service CWU 5348-32000-017-00D; the Agricultural Animal Health Program (College of Veterinary Medicine, Washington State University); and the National Science Foundation through a Faculty Early Career Development award to Frank Loge (BES-0092312) and an Integrative Graduate Education and Research Training grant (NSF grant DGE-9972817) to Washington State University.

Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the supporting organizations.

Acknowledgments

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