Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 ؎ 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.Listeria monocytogenes is a gram-positive facultative intracellular pathogen responsible for severe food-borne infections in humans and causes 20 to 50% mortality in susceptible populations, such as newborn children, the elderly, and immunocompromised persons (22,56). This bacterium is thought to be a saprophytic organism living naturally in the plant-soil environment, where it can survive for up to several months, being able to multiply in decaying vegetation but unlikely to multiply in soil (16). In the natural environment, biotic and abiotic factors shown to reduce L. monocytogenes survival are predation, high temperature, UV exposure, and low moisture (12,16,20). Soil texture also influences L. monocytogenes survival, but the availability of inorganic nutrients (nitrogen and phosphorus) does not influence survival (12).A possible agricultural route of human exposure is through the ingestion of uncooked food crops grown in soil irrigated with contaminated water and/or fertilized with Listeria-contaminated manur...

In this study, we investigated the agrBDCA operon in the pathogenic bacterium Listeria monocytogenes EGD-e. In-frame deletion of agrA and agrD resulted in an altered adherence and biofilm formation on abiotic surfaces, suggesting the involvement of the agr system of L. monocytogenes during the early stages of biofilm formation. Real-time PCR experiments indicated that the transcript levels of agrBDCA depended on the stage of biofilm development, since the levels were lower after the initial attachment period than during biofilm growth, whereas transcription during planktonic growth was not growth phase dependent. The mRNA quantification data also suggested that the agr system was autoregulated and pointed to a differential expression of the agr genes during sessile and planktonic growth. Although the reverse transcription-PCR experiments revealed that the four genes were transcribed as a single messenger, chemical half-life and 5 RACE (rapid amplification of cDNA ends) experiments indicated that the full size transcript underwent cleavage followed by degradation of the agrC and agrA transcripts, which suggests a complex regulation of agr transcription.Listeria monocytogenes is a gram-positive human pathogenic bacterium; it is the causative agent of listeriosis, a serious infection characterized by high mortality rates, in immunocompromised individuals and pregnant women (19). This pathogenic bacterium is widely spread in the environment (soil, vegetation, animals, farm environment, etc.). In connection with these extended reservoirs, L. monocytogenes is also a contaminant of the food industry. Its presence on working surfaces in food-processing plants is a major problem as a source of food contamination (1, 32). Like most bacteria, L. monocytogenes is able to colonize surfaces and form biofilms (sessile growth) while, in natural environments, free-floating cells (planktonic growth) are transitory (28). Several steps can be identified during biofilm development: after an initial step of reversible and then irreversible adherence, bacteria grow as microcolonies and spread on the surface. Finally, biofilms develop as complex, three-dimensional structures during the maturation step (17). Biofilm development and maturation requires complex cellular mechanisms in which cell-cell communication is involved (14, 30). To date, three major signaling systems have been identified; to regulate these systems, bacterial extracellular signaling molecules called autoinducers are produced (8). The acylhomoserine lactones have been identified as autoinducers in gram-negative bacteria (3, 13, 27, 46). The autoinducer 2 is found in both gram-negative and gram-positive bacteria (5,7,11,35,36,54). Finally, peptide-mediated signaling pathways have been characterized in gram-positive bacteria. Among these, the agr system has been described initially in Staphylococcus aureus (41); the production of many of its virulence factors (toxins, enzymes, and cell surface proteins) is regulated by this system (4). The role of the agr system during ...

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.Biofilms are communities of microorganisms attached to a surface (44). Several steps can be identified during biofilm development. After an initial step of reversible, then irreversible, adherence, bacteria grow as microcolonies and spread on the surface; also, biofilms develop as complex, three-dimensional (3D) structures during the maturation step (17). The microorganisms undergo profound changes during their transition from planktonic cells (free-floating organisms) to cells that are part of a complex, surface-attached community (sessile organisms) (44), and cells develop an increased resistance to antimicrobial agents (34,38,64). For this reason, the removal of established biofilms requires harsh treatments, with most using oxidizing biocides (18,20,25,54). The presence of biofilms raises safety issues in the food industry, especially when biofilms are located on food-processing surfaces and pipelines that are unreachable by washing agents (8). Clearly, biofilms can become a health hazard by harboring pathogenic bacteria such as Listeria monocytogenes (4). Moreover, L. monocytogenes is capable of attaching and developing biofilms on a variety of surfaces, such as stainless steel, polymers, and rubber gaskets (2,3,13,36,40). L. monocytogenes is a gram-positive human pathogen that is resp...

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