Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species

The femA gene encodes a protein precursor which plays a role in peptidoglyc an biosynthesis in Staphylococcus aureus and is also considered as a factor influencing the level of methicillin resistance. A femA homologous gene wa s recently characterized in S, epidermidis, entailing the possibility of fe mA phylogenetic conservation in staphylococcal species. Accordingly, we ass essed the presence of femA homologous genes in S. hominis and S. saprophyti cus. Strategy for identification relied upon alignment of S. aureus and D, epidermidis femA sequences and upon identification of potentially conserved regions. Amplifications of portions of the femA genes were performed under permissive annealing conditions, by using several sets of primers designed to match the consensus regions. DNA sequencing of overlapping PCR fragment s led to the characterization of the entire femA genes of S. hominis and S. saprophyticus, and provided more precise information on the femA start cod on for all five species. The genomic organization of all these femA genes a ppeared highly conserved, with alternance of homologous and variable region s. On this basis, a consensus sequence of the femA gene was defined and int erspecies variations were exploited to design strategies for staphylococci species-specific identification, including multiplex PCR amplification and a reverse hybridization assay. (C) Elsevier, Paris.