CHARACTERIZATION OF TRANSPOSON MUTANTS OF BIOFILM-PRODUCING STAPHYLOCOCCUS-EPIDERMIDIS IMPAIRED IN THE ACCUMULATIVE PHASE OF BIOFILM PRODUCTION - GENETIC IDENTIFICATION OF A HEXOSAMINE-CONTAINING POLYSACCHARIDE INTERCELLULAR ADHESIN

The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to production of Staphylococcus epid ermis biofilms, which are thought to contribute to virulence in biomat erial-related infections. We isolated Tn917 transposon mutants of biof ilm-producing S. epidermidis 13-1, which were completely biofilm negat ive. In pulsed-field gel electrophoresis no obvious deletions of the m utants were noted. The Tn917 insertions of mutants M10 and M11 were lo cated on different EcoRI fragments but on identical 60-kb SmaI and 17- kb BamHI chromosomal fragments. Linkage of transposon insertions of mu tants M10 and M11 with the altered phenotype was demonstrated by phage transduction, whereas the several other mutants apparently represente d spontaneous variants. In a primary attachment assay with polystyrene spheres, no significant difference between any of the mutants and the wild type could be detected. Cell clustering as an indication of inte rcellular adhesion, which is a prerequisite for accumulation in multil ayered cell clusters, was not detected with any mutant. These results demonstrate that the mutants were impaired in the accumulative phase o f biofilm production. Mutants M10 and M11 did not produce detectable a mounts of a specific polysaccharide antigen (Ii. Mack, N. Siemssen, an d R. Laufs, Infect. Immun. 60:2048-2057, 1992), whereas substantially reduced amounts of antigen were produced by the spontaneous variants. Hexosamine was determined as the major specific component of the antig en enriched by gel filtration of biofilm-producing S. epidermidis 1457 because almost no hexosamine was detected in material prepared from t he isogenic biofilm-negative transductant 1457-M11, which differentiat es the antigen from other S. epidermidis polysaccharide components. Ou r results provide direct genetic evidence for a function of the antige n in the accumulative phase of biofilm production by S. epidermidis by mediating intercellular adhesion.