This investigation assessed antimicrobial resistance (AMR), virulence determinants and transmissible genetic elements in Staphylococcus aureus recovered from raw bovine milk collected at unorganized dairy farms in Haryana, India. A total of 153 raw milk samples were processed; 121 S. aureus isolates were confirmed by PCR targeting the nuc gene. The study combined phenotypic antimicrobial susceptibility testing, minimum inhibitory concentration (MIC) determinations for vancomycin, and genotypic screening for resistance, virulence genes and mobile genetic elements (MGEs).
Phenotypic testing showed high resistance to beta-lactams: oxacillin resistance was observed in 80.16% of isolates and cefoxitin resistance in 60.33%. Resistance to tetracycline and gentamicin was substantially lower, at 14.04% and 3.30% respectively. Using established criteria, 40.5% of isolates met the definition of multidrug-resistant (MDR). A large proportion, 66.7%, exhibited a multiple antibiotic resistance (MAR) index greater than 0.2, indicating frequent exposure to antibiotics or selection pressure in the sampled environment.
MIC testing for glycopeptide susceptibility revealed that 75.9% of isolates had vancomycin MIC values ≥16 μg/mL, meeting the study's criteria for vancomycin resistance. These phenotypic data highlight substantial resistance to both methicillin-class agents and vancomycin among dairy-derived isolates in this sample set.
Molecular screening identified methicillin-resistance determinants in a majority of isolates: mecA was detected in 89.2% of S. aureus isolates and mecC in 10.8%. Tetracycline resistance genes were common, with tetM present in 76.5% and tetK in 23.5% of isolates. Macrolide resistance genes showed a high frequency for ermC (62.5%) and ermB (37.5%); ermA was not detected. The aminoglycoside-modifying enzyme gene aacA-aphD was identified in 5.8% of isolates. These genotypic findings correspond to the observed phenotypic resistance patterns and indicate multiple coexisting resistance mechanisms within the dairy-associated S. aureus population.
The isolates also harbored several virulence-associated genes. Coagulase gene coa was present in 72.7% of isolates. The Panton–Valentine leukocidin gene luk-PV was found in 15.7%. Biofilm-associated genes icaA and icaD were detected in 42.1% of isolates, which may contribute to persistence and colonization in dairy environments. Enterotoxin genes were variably present: sec (39.6%), sed (34.7%) and sea (5.8%) were detected; seb was not detected. The combination of toxin genes and biofilm-associated determinants suggests potential for both pathogenicity and environmental persistence of these strains.
The study screened isolates for mobile genetic elements linked to resistance dissemination. Transposon Tn916 was identified in 66.7% of isolates, and class 1 integrons were present in 41.7%. Among methicillin-resistant isolates, staphylococcal cassette chromosome mec (SCCmec) type IV was reported. The detection of these MGEs indicates the potential for horizontal gene transfer of resistance determinants within and possibly beyond the bovine milk-associated S. aureus population, supporting the concern for spread to other bacteria or animal and human hosts.
Collectively, the high frequency of phenotypic resistance to oxacillin and cefoxitin, the large proportion of isolates classified as MDR, widespread detection of mecA/mecC, elevated vancomycin MICs, multiple resistance genes, virulence determinants and MGEs point to a substantial reservoir of potentially transmissible, pathogenic S. aureus in raw bovine milk from the sampled region. The authors emphasize the need for continuous surveillance of dairy-derived S. aureus, and recommend strengthened One Health approaches to monitor and mitigate dissemination of resistant and virulent strains between animals, food chains and humans.
Details beyond the abstract about sampling design, temporal distribution of sampling, full methodological specifics, statistical analyses and broader geographic representation were not reported in the abstract. The authors disclosed that Diwas Pradhan reported financial support from the All-India Network Project on Antimicrobial Resistance in Fisheries and Livestock (AI-NP-AMR), India. Other authors declared no known competing financial interests or personal relationships that could have influenced the work, as reported in the conflict of interest statement.
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