Three bacterial strains (Kocuria sp. C714.1, Brevibacterium linens B337.1 and Staphylococcus sciuri CCL101) were grown together on stainless steel and were subjected daily to a commercial alkaline chlorine solution (22 mg l-1 of free chlorine, pH 11) over a period of 4 weeks. After the daily chemical shock, culture madia [1:20 dilution of tryptic soy broth (TSB-YE/20) or diluted whey] was deposited on the biofilms. The chemical shocks led first to a drop in the culturable population, followed by an increase and finally stabilization at around 106-107 CFU cm-2 by day 11 of the experiment. These changes in the microbial population can be attributed to a decreasing susceptibility to the antimicrobial agent with biofilm age, and to the consumption of free chlorine by biofilm exoproteins. The microbial composition appeared to be linked to the free chlorine concentration that depended on exoprotein production. At the end of the experiment, exoprotein production was greater for biofilms grown in TSBYE/20 than in whey. As a consequence, biofilms grown in whey did not neutralize the chlorine and the dominant strain was the one having the highest resistance to chlorine: K. varians. When biofilm were grown in TSBYE/20, chlorine was neutralized and the dominant strain was the one having the highest growth rate: S. sciuri. The presence of chlorine may also explain the distribution of S. sciuri cells as a ring around Kocuria sp. microcolonies. When chlorine was totally consumed by the biofilm during the chemical shock, S. sciuri was no longer grouped around Kocuria sp. microcolonies but was evenly scattered over the substratum as single cells or in small clusters, as it was before any chemical treatment. These findings strongly suggest protection of S. sciuri by Kocuria sp. microcolonies against the chlorinated solution. This phenomenon, added to the low susceptibility phenotype of the biofilm cells, could at least partly explain the survival of microbial cells in an adverse environment.

The adhesion and subsequent development of Listeria monocytogenes on stainless steel was studied in the absence and in the presence of a Staphylococcus sciuri biofilm. In the three growth media studied, the percentage of adherent cells was reduced to nearly the same extent by the presence of 1-day biofilms of Staph. sciuri for the two strains of L. monocytogenes studied. One-day biofilms of Staph. sciuri exhibited the same exopolysaccharide content per square centimetre, although they colonized from 3.5 to 35% of the stainless steel depending on the growth media. This suggests that extracellular substances rather than cell-to-cell interactions were involved in the decreased adhesion. After 3 days of culture, Staphylococcus biofilms prevented the adherent L. monocytogenes population from increasing within the biofilm, leading to an average logarithmic cfu difference of 0.9-2.7 between the pure and mixed culture. A competition for nutrients by Staph. sciuri was observed in one of the three media. A role for extracellular polysaccharides produced by the Staphylococcus biofilm in preventing the adhesion of L. monocytogenes and in modifying the balance existing between its planktonic and biofilm phase is hypothesized. A higher proportion of L. monocytogenes cells was observed in the planktonic phase in mixed cultures, suggesting that the extracellular substances produced by Staph sciuri biofilms and involved in the decreased adhesion of L. monocytogenes could modify the balance existing between planktonic and biofilm populations. In addition, co-cultures of L. monocytogenes and Staph. sciuri in broth showed competition for nutrients for Staph. sciuri in one of the three media.

ABSTRACT An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification and characterization of extracellular polysaccharides produced by 1- and 4-day biofilms of 10 bacterial strains isolated from food industry premises. Peroxidase-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) were used, as they specifically bind to saccharide residues most frequently encountered in biofilms matrices:d-glucose or d-mannose for ConA andN-acetyl-d-glucosamine orN-acetylneuraminic acid for WGA. The ELLA applied to 1- and 4-day biofilms colonizing wells of microtiter plates was able to detect that for Stenotrophomonas maltophilia and to a lesser extent Staphylococcus sciuri, the increase in production of exopolysaccharides over time was not the same for sugars binding with ConA and those binding with WGA. Differences in extracellular polysaccharides produced were observed among strains belonging to the same species. These results demonstrate that ELLA is a useful tool not only for rapid characterization of biofilm extracellular polysaccharides but also, in studies of individual strains, for detection of changes over time in the proportion of the exopolysaccharidic component within the polymeric matrix.

The survival of Listeria monocytogenes in a binary biofilm with a bacteriocin producer (Lactococcus lactis CNRZ 150) was investigated. Two situations were simulated: in the first, L. monocytogenes was deposited on a 1-day biofilm of Lactococcus lactis (deferred adhesion); in the second, L. monocytogenes was simultaneously mixed with Lact. lactis (simultaneous adhesion). Biofilms were cultivated in tryptic soy broth supplemented with 6 g l−1 of yeast extract (TSB-YE) and L. monocytogenes counts were followed for 48 h, both in co-culture with Lact. lactis and in pure culture. The influence of the mode of inoculation of L. monocytogenes (deferred or simultaneous adhesion) into the Lact. lactis biofilm, the size of the L. monocytogenes inoculum and the replacement of the culture medium at 20–24 h on the survival of L. monocytogenes was studied. Results showed that the antilisterial activity of the Lact. lactis started within the first 6 h of the deposition of L. monocytogenes. The log cycle reduction rate in number of L. monocytogenes in the mixed biofilm (compared to the pure biofilm) was greatly dependent on the inoculum size: when the smallest inoculum was used to colonise stainless steel coupons (106–107 CFU ml−1), the log cycle reduction was greater and L. monocytogenes was not detected after t=10 h (simultaneous adhesion) and t=24 h (deferred adhesion) in the adherent population as well as in the planktonic population. On the other hand, in the case of a greater supply of L. monocytogenes (108 CFU ml−1), the results showed that the early reduction of L. monocytogenes counts was relatively slow and was followed by a stabilisation of the population, leading to the establishment of a great number of resident cells in the biofilm (105 to 106 CFU cm−2). This population level was maintained during the 48 h of experimentation and replacement of the culture media with fresh medium at t=22 h (simultaneous adhesion) or t=24 h (deferred adhesion) did not modify the level of the population of L. monocytogenes within the biofilm.

The aim of this study was to examine the physicochemical surface properties and the ability to adhere to stainless steel of three strains of Listeria monocytogenes after different cultivation procedures. To this end, bacteria were cultivated at 37 degrees C after storage at two frequently used temperatures (4 degrees C or -80 degrees C) and were then transferred into the liquid medium (trypticase soy broth supplemented with 6 g liter(-1) of yeast extract, pH 7.3) between one and four times. In addition, the influence of supplementing the growth medium with lactic acid was explored, this organic acid being representative of both the dairy and cured meat industries. The hydrophobic/hydrophilic and electron-acceptor/electron-donor characteristics of the strains were evaluated by the microbial adhesion to solvents method. Using this technique, we recorded an increase in the hydrophobic properties of one strain stored at 4 degrees C, with an increasing number of transfers in the media (P < 0.05). Another plant-isolated strain appeared more hydrophobic and stuck better to stainless steel when cells were stored at 4 degrees C rather than at -80 degrees C. Preculturing L. monocytogenes in a lactic acid-supplemented medium increased the affinity of microbial cells to solvents and the bacterial attachment to stainless steel (P < 0.05).

The survival after chlorine stress of the biofilm form of Salmonella typhimurium both alone and in association with the biofilm form of Pseudomo fluorescens was investigated. P. fluorescens showed the best adhesion and more extended growth than S. typhimurium when the two strains were cocultured. The presence of P. fluorescens resulted in an increased resistance of S. typhimurium to chlorine. This phenomenon, which was already seen in 1-day biofilms, increased in 4-day biofilms. Viable but nonculturable cells were observed only in 4-day single-species S. typhimurium biofilms subjected to chlorine stress; only 50% of substrate-responsive bacteria (SRB) were culturable. When daily cycles of disinfection, neutralization, and culture medium supply were performed with S. typhimurium biofilms for 4 days, only 20% of the SRB remained culturable. The chlorine consumption of such biofilms was more than twice that of 4-day single-species S. typhimurium biofilms.