S. In all, 4379 isolates from 35 products, including 24 artisanal cheeses, were surveyed with a view to identifying strains that could be used as starters in commercial dairy fermentations. Of the isolates, 38 % were classified as Lactococcus, 17 % as Enterococcus, 14% as Streptococcus thermophilus, 12 % as mesophilic Lactobacillus, 10% as Leuconostoc and 9 % as thermophilic Lactobacillus. Acid production by the isolates varied considerably. Of the 1582 isolates of Lactococcus and 482 isolates of mesophilic Lactobacillus tested, only 8 and 2 % respectively produced sufficient acid to lower the pH of milk to 5n3 in 6 h at 30 mC. In contrast, 53, 32 and 13 % of Str. thermophilus, thermophilic Lactobacillus and Enterococcus isolates respectively reduced the pH to 5n3. These isolates were found only in some French, Italian and Greek cheeses. Bacteriocins were produced by 11 % of the 2257 isolates tested and 26 of them produced broad-spectrum bacteriocins which inhibited at least eight of the ten target strains used, which included lactic acid bacteria, clostridia and Listeria innocua. The most proteolytic of the 2469 isolates tested wereStr. thermophilus from Fontina cheese followed by Enterococcus from Fiore Sardo and Toma cheese and thermophilic Lactobacillus from all sources. Exopolysaccharides were produced by 5n3 % of the 2224 isolates tested.In many Southern European countries cheeses are made from cows', goats', ewes'

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A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 h of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.

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Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.Listeria monocytogenes is a food-borne pathogen that causes serious illness, including meningitis, septicemia, and stillbirth, with a mortality rate of up to 30% (37). More recently, there have been reports of listerial gastroenteritis following the consumption of several different food types (16,34). A number of studies have demonstrated that this organism is able to persist in the food-processing environment for several months and even up to 10 years (23, 29). One of the major causes for concern about L. monocytogenes in these environments is its ability to attach to many different surfaces (2). Indeed, there is recent evidence to show that listerial biofilms formed inside the lumens of stainless steel tubes are able to withstand the shears generated by high-Reynolds-number flows (31). Biofilms, including those produced by L. monocytogenes, are more resistant to detergents and disinfectants (33) and also are a potential source of contamination within food-processing plants; hence, they pose a risk to the maintenance of product safety (27). Consequently, there is considerable interest in determining the mechanisms of attachment and biofilm formation.Our in silico analysis of the genome sequence of L. monocytogenes identified an open reading frame (lmo0435) for a protein with similarity to biofilm-associated proteins (Bap) believed to be important for the binding of staphylococci to abiotic surfaces (10). This Bap protein also has been implicated in the virulence of Staphylococcus aureus (10, 11). Thus, the aim of the current study was to establish if this protein (Lmo0435 [BapL]) of L. monocytogenes influenced biofilm formation and virulence and to determine the prevalence of the lmo0435 (bapL) gene within a selection of field isolates. MATERIALS AND METHODS Bacterial strains and plasmids.A list of the L. monocytogenes isolates and plasmids used in this study is given in Table 1. The strains were cultured in tryptone soya broth (TSB; Oxoid) or brain heart infusion agar (Oxoid) with shaking at 37°C unless otherwise stated. Escherichia coli JM...