Clonality, Virulence Genes, and Antimicrobial Resistance of Dairy Ruminants in Mastitic Milk-Associated Staphylococcus aureus in Sicily

Background: Staphylococcus aureus is one of the most prevalent pathogens causing mastitis in dairy animals and represents a serious issue of public health concern due to its resistance against multiple antimicrobials. Objectives: This study assessed 101 S. aureus isolates obtained from quarter milk of animals with subclinical mastitis in the Ragusa area (Sicily, Italy). Methods: Antibiotic resistance against nine antibiotics was evaluated using the Kirby–Bauer method, and the Minimum Inhibitory Concentration (MIC) values were measured for oxacillin (OXA) and vancomycin (VA). Additionally, the isolates were genetically characterized through multiplex PCR to identify the presence of spa, mecA, mecC, pvl, vanA, vanB, and vanC genes, along with pulsed-field gel electrophoresis analysis and multi-locus sequence typing (MLST). Results: The highest rates of antibiotic resistance were found against gentamicin (47.5%) and erythromycin (29.7%), with 86.1% of strains exhibiting resistance to at least two antimicrobials and 33.7% showing resistance to three antimicrobial classes. Furthermore, the results indicated that the presence of antibiotic resistance genes was not correlated with phenotypic resistance, and a phylogenetic analysis revealed varying phenotypic resistance profiles even within the same PFGE cluster. Lastly, alongside a new allelic profile ST 9471, MLST analysis identified five additional STs clustered into three CCs, with CC5 originating from human ancestral strains through human-to-animal host transfers, making it the dominant group. Conclusions: This study provided valuable insights into regional trends, allowing for the identification of significant antibiotic-resistant patterns and offering an understanding of bacterial dynamics in these environments, underscoring the importance of routine resistance surveillance in dairy farms.