Biofilm formation and virulence factor analysis of Staphylococcus aureus isolates collected from ovine mastitis

Abstract

Aims: To perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines.

Methods and results: The potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly-β and hly-γ), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 71·3% of the Staph. aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (61·6%) possessed all lukD-E/lukM/lukPV83 genes, 123 (47·7%) harboured both hly-β/hly-γ genes, whereas almost all (97·3%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (69·4%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins.

Conclusions: In this search, several putative virulence determinants have been identified in ovine Staph. aureus isolates collected in Sardinia.

Significance and impact of the study: Some of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis.

Keywords: MSCRAMM; Staphylococcus aureus; biofilm; ovine mastitis; toxins.