Identification of methicillin resistant and biofilm producing S. aureus, S. intermedius and Coagulase-Negative Staphylococci isolated from clinical bovine mastitis by multiplex-PCR

Objective: S. aureus (SA) and coagulase negative staphylococci (CNS) are frequent causes of bovine mastitis, while S. intermedius Group (SIG) is infrequently described. Methicillinresistant staphylococci (MRS) carrying mecA gene are an emerging health problem in humans. Biofilm formation by SA at the site of infection could explain the therapy resistance in bovine mastitis. icaA gene has been related to the ability of SA to produce biofilm. In this study, the presence of SA, SIG and CNS in cows with clinical mastitis and their methicillinresistance and biofilm-production was evaluated by multiplex- PCR. Methods: The DNA of 326 Staphylococcus spp. strains cultured from milk of cows with clinical mastitis was tested by multiplex-PCR to detect the Staphylococcus spp.16S rDNA (252bp), the nuc gene of SIG (125bp) and SA (420bp), the mecA gene (532bp) and the icaA gene (188bp). Antimicrobial susceptibility testing for methicillin and qualitative biofilm evaluation was performed on isolated SA by conventional antibiotic disc diffusion test (ADDT) and Congo red agar method (CRAM) respectively. Results: Out of 326 Staphylococcus spp., 163 (50%) SA, 19 (5.8%) SIG and 144 (44.2%) CNS were identified. mecA was present in 19 SA strains (11.7 %), in 19 (13.2%) CNS, and was absent in SIG strains. icaA was detected in all SA strains and in 30 (20.8%) CNS, but not in SIG strains. In CRAM, 129 (79.1%) SA produced black colony (high biofilm producer) and 34 (20.9%) produced bordeaux colonies (weak biofilm producers). No red colonies (no biofilm production) were grown. All the mec A positive strains were resistant to methicillin, while other strains were susceptible by ADDT. Conclusion: Multiplex-PCR is a fast and reliable method for the detection of the methicillin-resistant and biofilm producing staphylococci and for the species identification. Although SA is a frequent cause of bovine mastitis, SIG also has to be considered. MRS are present in bovine clinical mastitis and constitute a therapeutic problem in veterinary medicine, but cattle may also be a reservoir for the mecA gene. All SA were icaA positive, demonstrating the high ability of clinical strains to produce biofilm. Furthermore, for the first time the presence of icaA has been demonstrated also in CNS isolated from cows. In conclusion, the gene-related antibiotic resistance and the biofilm production in staphylococci have to be considered an emerging health problem not only in human medicine but also in veterinary science.