Abstract
This cross-sectional study, conducted from August to December 2024, investigated the antimicrobial resistance and biofilm-forming abilities of Staphylococcus aureus from clinical bovine mastitis in Chitwan, Nepal. Out of 134 California Mastitis Test-positive milk samples, 32 (23.9%) were confirmed as S. aureus by biochemical tests and species-specific nuc gene PCR. Antimicrobial susceptibility testing of the 32 isolates against 12 antibiotics revealed high resistance rates, particularly to ampicillin (78.1%), nalidixic acid (75.0%), and enrofloxacin (62.5%). The prevalence of multidrug resistance (MDR), defined as resistance to ≥3 antibiotic classes, was alarmingly high, with 27 (84.4%) isolates classified as MDR. Presumptive methicillin-resistant S. aureus (MRSA), detected via cefoxitin resistance, was identified in 14 (43.8%) isolates, all of which were also MDR. Biofilm-forming abilities were assessed qualitatively and quantitatively, with 5 (15.6%) isolates classified as strong biofilm producers. Fisher's exact test revealed no significant association between biofilm formation and overall MDR status (p > 0.05). However, a statistically significant correlation was found between strong biofilm formation and high-level MDR (resistance to ≥ 4 classes) (p < 0.05), as well as between strong biofilm formation and presumptive MRSA status (p < 0.01). These findings highlight the co-existence of high-level resistance and strong virulence phenotypes in S. aureus from bovine mastitis in Nepal, underscoring the urgent need for robust antimicrobial stewardship, enhanced surveillance, and the development of strategies to mitigate biofilm-associated treatment failures.