Characterization of Virulence Factors, Biofilm Production, and Antimicrobial Resistance in Mastitis-Associated Staphylococcus aureus Strains by Phenotypic and Genotypic Methods.

Staphylococcus aureus, a common cause of bovine mastitis, relies on several virulence factors, with biofilm formation being a key contributor to its pathogenicity. The present study investigated the occurrence of S. aureus as etiological agents in bovine mastitis with a focus on the existence of various virulence factors and antibiotic resistance status. Among 120 milk samples collected from West Bengal and Uttar Pradesh, 36 (30%) S. aureus strains were confirmed by conventional methods and PCR. Phenotypic analysis revealed hemolysin (55.55%) and coagulase production (36.11%), while molecular analysis revealed the presence of leukotoxin (luksF, 19.44%), hemolysin (hlb, 58.33%), coagulase (coa, 63.88%), and toxic shock syndrome toxin (tsst-1, 30.55%) genes. Biofilm production ability was detected in 97.22% (crystal violet assay) and 86.11% (Congo red agar assay) strains. Biofilm-associated genes, namely, icaA (80.55%, 29/36), icaB (75%, 27/36), icaC (69.44%, 25/36), icaD (86.11%, 31/36), and MSCRAMMs genes, namely, clfA (58.33%, 21/36), clfB (75%, 27/36), fnbA (75%, 27/36), fnbB (55.55%, 20/36), bap (38.88%, 14/36), bbp (83.33%, 30/36), ebps (69.44%, 25/36), eno (66.66%, 24/36), fib (41.66%, 15/36), and cna (8.33%, 3/36), were also detected. Antimicrobial resistance was observed in 88.88% isolates, with 72.22% exhibiting multidrug resistance (MDR). Among the isolates, 83.33% were methicillin-resistant S. aureus (MRSA), and mecA, femA, and femB genes were present either singly or in combination in 76.66% of the isolates. Efflux pump protein genes, namely, norA, norB, norC, mdeA, mepA, and sepA, were detected either singly or in combination in S. aureus isolates. 61.53% of MDR-MRSA isolates harbored all six efflux pump genes. According to this study, S. aureus of mastitis origin harbors various virulence, antibiotic resistance, biofilm-forming, and efflux pump genes. Bovine mastitis-derived MDR S. aureus isolates can pose a significant public health risk and need urgent attention to formulate strategies for their control and preventing transfer to the human food chain.