Antagonistic Effects of Probiotic <i>Lactobacillus</i> Strains on Multidrug‐Resistant <i>Staphylococcus aureus</i> : Role of <i>mecA</i> / <i>mecC</i> and <i>icaAD</i> Gene Variants

Food safety is increasingly challenged by Staphylococcus aureus, which causes mastitis and persists in raw milk, exacerbating public health risks. Among 128 samples of raw cow milk from Barak Valley, Northeast India, 30.46% tested positive for S. aureus according to the International Organization for Standardization (ISO)-recommended methods. The spa gene, indicating pathogenic potential, was detected in 51% of the isolates. 16S rRNA gene sequencing confirmed 93%-100% identity with known S. aureus strains, and novel strains were registered in GenBank. In addition to the appearance of 16 vancomycin-resistant strains, 97% were resistant to ampicillin and nalidixic acid, 74.3% exhibited multidrug resistance (MDR), with an MDR index of ≥0.2, and 61.5% were methicillin-resistant (mecA/mecC-positive), all of which are biofilm producers. The qualitative nature of the Congo red agar method likely contributed to the high biofilm development that was observed in 53.8% of the isolates. In contrast, the tissue culture plate (TCP) method identified 33.3% of the strains as strong biofilm producers, which was significantly correlated with the presence of icaAD genes (45%), suggesting that TCP offers a more accurate assessment. The co-occurrence of mecA + mecC (33.3%) and icaA + icaD (18%) indicates advanced resistance and genetic adaptation. Alternative antimicrobials-ZnONPs and honey-demonstrated promising antibacterial effects with average inhibition zones of 18.3 ± 0.58 and 22.3 ± 0.38 mm, respectively, and minimum inhibitory concentrations (MICs) ranging from 15 to 30 mg/mL. A total of 17.9% of the isolates were resistant to honey. All strains of lactic acid bacteria were effective against 97% of the isolates, with Lactobacillus plantarum demonstrating the strongest antimicrobial action (average of 14.1 ± 0.02 mm), indicating potential use in combating biofilm-associated, drug-resistant Staphylococcus aureus.