Screening of Escherichia coli Species Biodiversity Reveals New Biofilm-

ABSTRACT Bacterial biofilms often formmultispecies communities in which complex but ill-understood competition and coop-eration interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhe-sion or biofilm formation. We produced cell-free solutions extracted from in vitromature biofilms formed by 122 natural Esche-richia coli isolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20 % of the tested biofilm extracts containedmolecules that antag-onize bacterial growth or adhesion. We characterized a compound, produced by a commensal animal E. coli strain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its pro-duction reduced susceptibility to invasion and provided rapid exclusion of Staphylococcus aureus from mixed E. coli and S. au-reus biofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacte-rial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms onmedical devices