Listeria monocytogenes EGD-e biofilms: no mushrooms but a network of knitted chains

Abstract

Listeria monocytogenes is a food pathogen that can attach on most of the surfaces encountered in the food industry. Biofilms are three-dimensional microbial structures that facilitate the persistence of pathogens on surfaces, their resistance toward antimicrobials, and the final contamination of processed goods. So far, little is known about the structural dynamics of L. monocytogenes biofilm formation and its regulation. The aims of this study were, by combining genetics and time-lapse laser-scanning confocal microscopy (LSCM), (i) to characterize the structural dynamics of L. monocytogenes EGD-e sessile growth in two nutritional environments (with or without a nutrient flow), and (ii) to evaluate the possible role of the L. monocytogenes agr system during biofilm formation by tracking the spatiotemporal fluorescence expression of a green fluorescent protein (GFP) reporter system. In the absence of nutrient flow (static conditions), unstructured biofilms composed of a few layers of cells that covered the substratum were observed. In contrast, when grown under dynamic conditions, L. monocytogenes EGD-e biofilms were highly organized. Indeed, ball-shaped microcolonies were surrounded by a network of knitted chains. The spatiotemporal tracking of fluorescence emitted by the GFP reporter system revealed that agr expression was barely detectable under static conditions, but it progressively increased during 40 h under dynamic conditions. Moreover, spatial analysis revealed that agr was expressed preferentially in cells located outside the microcolonies. Finally, the in-frame deletion of agrA, which encodes a transcriptional regulator, resulted in a decrease in initial adherence without affecting the subsequent biofilm development.

FIG. 1.

Scanning confocal micrographs (upper lines) and 3D reconstruction of a z stack (lower lines) of biofilms formed by L. monocytogenes AR009 in TSB medium at 25°C under static conditions after 2, 24, and 48 h of incubation (A) and under flowing conditions after 1, 16, 24, and 40 h of incubation (B). (C) Biofilms grown under flowing conditions and observed at a higher magnification. Green indicates those cells expressing GFP from the reporter plasmid pGID128, which contains a fusion of the agr promoter region with gfp. The red cells are stained with the nucleic acid SYTO61 dye (cells lacking agr activity).

FIG. 2.

FLIP of intracytoplasmic GFP within chains. A fluorescent L. monocytogenes chain was repeatedly photobleached within a small region (indicated by a gray circle) for ∼60 s while the whole chain was continuously imaged. Single elongated cells being photobleached would gradually lose fluorescence due to the lateral movement of mobile GFP into this area. By contrast, the fluorescence in separate short rods near the area being bleached would not be affected.

FIG. 3.

Biovolume (A) and thickness (B) of L. monocytogenes AR009 biofilms in TSB medium at 25°C under static conditions after 2, 24, and 48 h of incubation (square on the gray line) and under flowing conditions after 1, 16, 24, and 48 h of incubation (diamond on the black line). For these quantitative analyses, images obtained by LSCM were analyzed using the PHLIP Matlab routine. The data represent the means and standard deviations from three independent experiments with five measurements for each point. Asterisks indicate statistically significant differences (P < 0.05).

FIG. 4.

Scanning confocal micrographs of biofilms formed by L. monocytogenes AR009 (A) and L. monocytogenes AR011 (ΔagrA) (B) in TSB medium at 25°C under flowing conditions after 1 and 16 h of incubation. The images were acquired using LSCM settings with a ×63 objective.