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 bio®lm. In the three growth media studied, the percentage of adherent cells was reduced to nearly the same extent by the presence of 1-day bio®lms of Staph. sciuri for the two strains of L. monocytogenes studied. One-day bio®lms of Staph. sciuri exhibited the same exopolysaccharide content per square centimetre, although they colonized from 3Á5 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 bio®lms prevented the adherent L. monocytogenes population from increasing within the bio®lm, leading to an average logarithmic cfu difference of 0Á9±2Á7 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 bio®lm in preventing the adhesion of L. monocytogenes and in modifying the balance existing between its planktonic and bio®lm 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 bio®lms and involved in the decreased adhesion of L. monocytogenes could modify the balance existing between planktonic and bio®lm 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.

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 and N-acetyl-D-glucosamine or N-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.Biofilm is defined as a community of "cells immobilized on a substratum and frequently embedded in an organic polymer matrix of microbial origin" (7). The main polymers of this matrix are polysaccharides and proteins (10, 14) which could play a role in survival of biofilm bacteria to stresses (1,6,35).The most common methods developed to quantify exopolysaccharides are designed for those produced by planktonic bacteria. When adapted to biofilms, these techniques present some limits: successive steps (5, 33) may lead to loss of part of the material (3); solubilization of the exopolymers is dependent on the choice of the extraction fluid (30); and because the quantity of extracellular substances present in biofilm is small (microgram range), it is often necessary to increase the total area colonized by the cells in order to detect these products (5).Easier approaches involve the use of specific dyes directly applied to biofilms (13,32,34). However, these cationic dyes, whose specificity to polyanions was empirically established, are not always reliable as detectors of exopolysaccharides (12, 15). As underlined by Sutherland (29), there is a need for development of methods for the in situ analysis of small amounts of exopolysaccharides capable of detecting relatively minor differences.The binding specificity of lectins toward simple sugars appears to be a specific way to characterize and quantify exopolysaccharides. The specificity of lectins has been widely used in microbiology for the determination of components of microbial cells (17,22). The emergence of fluorochrome-conjugated lectins allowed for the direct visualization of the extracellular substances of biofilm by epifluorescence microscopy (12,23,24,25). More recently, Thomas and coworkers (31) successfully developed an enzyme-linked lectinsorbent assay (ELL...