Ecology of mixed biofilms subjected daily to a chlorinated alkaline solution: spatial distribution of bacterial species suggests a protective effect of one species to another

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.