Multidrug-Resistant Staphylococcus aureus from Raw Bovine Milk in Haryana, India: Resistance Determinants, Virulence Factors and Dissemination Potential.

This present study investigated antimicrobial resistance (AMR) and virulence in Staphylococcus aureus isolated from raw bovine milk collected from unorganized dairy farms in Haryana, India. A total 121 S. aureus strains from 153 raw milk samples were confirmed by PCR targeting the nuc gene and further screened for antimicrobial susceptibility. These isolates exhibited the predominant resistance against oxacillin (80.16%) followed by cefoxitin (60.33%), with lower resistance to tetracycline (14.04%) and gentamicin (3.30%). Among these resistant isolates, 40.5% were classified as multidrug-resistant (MDR), while 66.7% exhibited a multiple antibiotic resistance (MAR) index >0.2. The MIC testing demonstrated that 75.9% of isolates were vancomycin resistant (MIC ≥16 μg/mL). Further, Genotypic characterization identified methicillin resistance genes, with mecA detected in 89.2% and mecC in 10.8% of S. aureus isolates. Similarly, resistant determinants were found at high frequencies in isolates such as tetracycline resistance genes (tetM, 76.5%; tetK, 23.5%), macrolide resistance genes (ermC, 62.5%; ermB, 37.5%) while ermA was absent. The aminoglycoside resistance gene aacA-aphD was detected in 5.8% of isolates. Additionally, S. aureus isolates showed presence of virulence determinants such as coa (72.7%), luk-PV (15.7%), icaA/icaD (42.1%) and enterotoxin genes sec (39.6%), sed (34.7%) and sea (5.8%) while seb was not deteceted. Mobile genetic elements (MGEs), notably Tn916 was detected in 66.7%, class 1 integrons in 41.7% isolates and staphylococcal cassette chromosome mec (SCCmec) type IV among methicillin-resistant Staphylococcus aureus (MRSA). Overall, these findings highlight a high prevalence of AMR, virulence-associated genes and transmissible resistance elements in dairy-derived S. aureus, emphasizing the need for continuous surveillance and strengthened One Health strategies to limit the spread of resistant and potentially pathogenic strains.