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Listeria monocytogenes is involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation by L. monocytogenes isolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96 L. monocytogenes strains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improve L. monocytogenes biofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA). Listeria monocytogenes still represents an important risk for public health; 1,740 listeriosis cases were reported in the European Union (EU) in 2011 with a mortality rate of 12.7% (1). Listeriosis is particularly dangerous for pregnant women and elderly or immunocompromised people. Persistence of L. monocytogenes strains on food plant surfaces can occur due to maladapted design of equipment and biofilm formation (2, 3). L. monocytogenes is able to attach to and colonize various surfaces, such as stainless steel, glass, and polystyrene, and to contaminate food products during processing (4-6). Biofilms of L. monocytogenes are associated with important ecological advantages, such as protection against biocide action (7). Several molecular determinants, such as flagella, biofilm-associated proteins (Bap), SecA2, and cell-cell communication systems, have been shown to be involved in biofilm construction within the species (8, 9). While no exopolysaccharidic components have been evidenced in the L. monocytogenes biofilm matrix (8), extracellular DNA (eDNA) has been shown to participate in initial cellular adhesion and biofilm organization under specific growth conditions (10). Biofilm formation by the species is highly dependent on environmental conditions, such as variations in temperature, pH, and nutrients (11, 12). L. monocytogenes is structured into four major phylogenetic lineages, each of which is genetically heterogeneous and substructured into highly recognizable clonal complexes as defined by multilocus sequence typing (MLST) (13,14). Attempts to relate biofilm formation ...

To investigate if the primary function of the Agr system of Listeria monocytogenes is to monitor cell density, we followed Agr expression in batch cultures, in which the autoinducer concentration was uniform, and in biofilms. Expression was heterogeneous, suggesting that the primary function of Agr is not to monitor population density.Quorum sensing (QS) is the mechanism by which bacteria secrete signaling molecules called autoinducers that are sensed by neighboring cells in a population (30). The binding of these autoinducers to cognate receptors results in transcriptional regulation of gene expression. So far, for the species Listeria monocytogenes, one QS system, mediated by the agrBDCA operon, has been described (2, 7). Deletion of agrD or agrA results in impairment of major adaptive strategies, such as biofilm development (22, 23) and virulence (2, 21).Historically, the term QS was coined to illustrate that accumulation of autoinducers enables a coordinated control of gene expression resulting in a population-wide phenotype switch when the population reaches a threshold or quorum (6,8,18). However, recent reports indicate that adaptive functions of QS can be diverse and are not limited to population density sensing (20).For example, phenotypic heterogeneity of QS-regulated traits was reported in biofilms. Several subpopulations with distinct phenotypes organize Bacillus subtilis biofilms (13,14). Extracellular DNA release during the sessile growth of Enterococcus faecalis is directed by a fratricidal mechanism triggered by a quorum-responsive subpopulation (26). Heterogeneity was also observed in QS-regulated bioluminescence of Vibrio harveyi (1).Recent reports showed that confocal laser scanning microscopy (CLSM) associated with fluorescent reporter fusions may be used to trace the spatiotemporal expression of specific genes at a single-cell level within the overall biofilm structure (9, 12). When we traced Agr expression in biofilms, we detected green fluorescent protein (GFP) mainly in a network of elongated chains reminiscent of scaffoldings that surrounded densely populated microcolonies (22). This heterogeneous expression was surprising; indeed, maximum expression was expected within microcolonies, where the autoinducer concentration is maximum (19).Thus, the question of whether the function of this QS system was primarily to monitor population density arose. In order to test this hypothesis, a P agr -gfp fusion was integrated upstream of the agr locus of the L. monocytogenes EGD-e genetic background. This construct was designed to develop Agr expression reporters without affecting expression of the downstream agrBDCA operon (22).We followed GFP fluorescence by flow cytometry and microscopy during growth in batch homogenized liquid cultures, which represents environmental conditions prone to facilitate responses to cell density (confined cultures and no diffusion). Cells were collected by centrifugation (10 min at 8,000 ϫ g), washed, and diluted in 150 mM filtered NaCl solution before flow cytometry a...