Biofilm production, quorum sensing system and analysis of virulence factors of Staphylococcus epidermidis collected from sheep milk samples

Intramammary infections are a serious problem in dairy sheep farming, and Staphylococcus epidermidis is one of the main etiological agents of ovine mastitis. In this work, 199 S. epidermidis isolates collected in Sardinia (Italy) from sheep milk samples and used for the preparation of inactivated autogenous vaccines were characterized with phenotypic and genotypic methods. The potential for biofilm production was evaluated phenotypically by assessing colony characteristics on Congo Red Agar (CRA), and genotypically by PCR for i) the icaA/D genes coding the polysaccharide intercellular adhesin (PIA) and ii) for the bhp, aap and embp genes coding biofilm accumulation proteins. Isolates were also investigated by PCR for the presence of autolysins (atlE and aae genes), microbial surface components recognizing adhesive matrix molecules (MSCRAMMs, sdrG and sdrF genes), enterotoxins (sea, seb, sec, sed and see genes), and the toxic shock syndrome toxin (tsst genes). In addition, the staphylococcal accessory gene regulator (agr), the most important locus responsible for the regulation of virulence factors, was characterized for the first time. As a result, only two isolates showed biofilm production on CRA and harbored the icaA/D genes. Four isolates, phenotypically classified as non-biofilm producers, possessed the icaA/D genes (n = 3) or only the icaA gene (n = 1). All isolates harbored the atlE, aae, embp and sdrG genes, but none harbored the genes encoding enterotoxins and tsst. Most isolates belonged to the agr-3(se) group (56.3%), followed by agr-1(se) (41.2%) and agr-2(se) (2.5%). All six isolates that harbored ica genes belonged to agr-1(se). In conclusion, this study provides a detailed characterization of the putative virulence genes present in S. epidermidis strains circulating in Sardinian sheep. Because of their role in ovine mastitis pathogenesis, these might represent valuable candidates for vaccine development.