PCR for virulence genes

PCR assay targeted 26 virulence genes was performed for all the S. aureus isolates, as follows: cytolysins (hla, hlg, pvl), SAgs (sea, seb, sec, sed, see, seg, seh, sei, sem, sen, seo, seu, tst), exfoliative toxins (eta, etb), adhesins (bbp, cna, ebpS, fnbA, fnbB, eap) and genes related to biofilm production (icaA and sasG). The PCR references and control strains are summarized in Table S1 (supplementary material). The primers HLA-1 (5ʹ-AATCCTGTCGCTAATGCC) and HLA-2 (5ʹ-CAGCAATGGTACCTTTCG) used in the PCR for the hla gene were designed previously by our group [¹⁹] and generated a fragment of 208 pb. Amplification conditions were: denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, with a final extension at 72°C for 5 min. The expression of the hld gene was assessed by measuring the hemolysin activity in blood agar, as described by Harigaya et al. [²⁰]. The RN4220 strain was used as control.