Biofilm-Forming Abilities of Listeria monocytogenes Serotypes Isolated from Different Sources

Abstract

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 revealed 62 (63.26%) strains as weak, 27 (27.55%) strains as moderate, and 9 (9.18%) strains as strong biofilm formers. Among the strong biofilm formers, 6 strains were of serotype 1/2a and 3 strains were of serotype 1/2b. None of the strain from 4b serotype exhibited strong biofilm formation. No firm correlation (p = 0.015) was noticed between any serotype and respective biofilm formation ability. Electron microscopic studies showed that strong biofilm forming isolates could synthesize a biofilm within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Cell enumeration of strong, moderate, and weak biofilm was performed to determine if the number of cells correlated with the biofilm-forming capabilities of the isolates. Strong, moderate, and weak biofilm showed 570±127× 103 cells/cm2, 33±26× 103 cells/cm2, 5±3× 103 cells/cm2, respectively, indicating that the number of cells was directly proportional to the strength of the biofilm. The hydrophobicity index (HI) analysis revealed higher hydrophobicity with an increased biofilm formation. Fatty acid methyl esterase analysis revealed the amount of certain fatty acids such as iso-C15:0, anteiso-C15:0, and anteiso-C17:0 fatty acids correlated with the biofilm-forming capability of L. monocytogenes. This study showed that different strains of L. monocytogenes form biofilm of different intensities which did not completely correlate with their serotype; however, it correlated with the number of cells, hydrophobicity, and amount of certain fatty acids.

Fig 1. Biofilm formation and growth ability of L. monocytogenes strains of different serotypes.

(a) OD₅₉₅ of the biofilm after staining with crystal violet, and growth turbidity of the strains belonging to serotype 1/2a; (b) OD₅₉₅ of the biofilm after staining with crystal violet, and growth turbidity of the strains belonging to serotype 1/2b; (c). OD₅₉₅ of the biofilm after staining with crystal violet, and growth turbidity of the strains belonging to serotype 4b.

Fig 2. Scanning Electron Microscopy (SEM) of L. monocytogenes biofilm formation. Listeria monocytogenes strain was grown at 28°C in BHI on glass slides and observed after.

(a) 2 h, adherence of Listeria monocytogenes to glass surface; (b) 6 h, adherent cells multiplied and began multilayer formation within 6 h of deposition; (c) 12 h,a mature biofilm was observed; (d) 24 h, cells surrounded by matrix could be seen.

Fig 3. SEM of Listeria monocytogenes ILCC306 on different industrially important surfaces.

(a) L. monocytogenes ILCC306 on PVC pipe after 24 h. Multilayered and mat-like biofilms were observed; (b) L. monocytogenes ILCC306 on ceramic tiles after 24 h. The cells were found to be aggregated all over the surfaces of ceramic tiles; (c) L. monocytogenes ILCC306 on stainless steel (SS304) after 24 h. Biofilm aggregated near suture; (d) L. monocytogenes ILCC306 on stainless steel suture (artificially made) after 24 h; (e) L. monocytogenes ILCC306 on HDPE plastic after 24 h. The circled area shows biofilm rooted in the sutures; bacterial growth can be seeninside the sutures and aggregates formation toward the surfaces.