Variation in biofilm formation among strains of Listeria monocytogenes

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.

FIG. 1.

L. monocytogenes biofilm formation measured by microtiter plate assay (crystal violet destaining). Bars represent average OD₅₉₅ values and standard errors. Persistent strains are represented by gray bars, nonpersistent strains are represented by white bars, and all other strains have black bars. Division I consists of serotypes 1/2b and 4b, Division II consists of serotypes 1/2a and 1/2c, and strains belonging to serotypes 4c and 3a are listed as Other Serotypes.

FIG. 2.

Ruthenium red carbohydrate stain of L. monocytogenes strain M39503A on glass (magnification, ×100). Crystal violet (purple) was used to stain bacterial cells, and ruthenium red (pink) was used to stain extracellular carbohydrate. The presence of pink stain between cells is consistent with an EPS matrix.

FIG. 3.

SEM images of strain M39503A (high biofilm former) on stainless steel (A) and PVC (C) and strain M35584A (low biofilm former) on stainless steel (B) and PVC (D). The cracks visible under cells in panel B are artifacts in the stainless steel surface. Scale bars, 8.6 μm.