Biofilm production and virulence traits among extensively drug-resistant and methicillin-resistant Staphylococcus aureus from buffalo subclinical mastitis in Bangladesh

Methicillin-resistant Staphylococcus aureus (MRSA) is a critical pathogen implicated in subclinical mastitis (SCM), a hidden threat to dairy productivity. This study investigated the prevalence, antibiotic resistance profiles, and virulence traits of MRSA from SCM-affected riverine buffaloes in Jamalpur, Bangladesh. A total of 344 milk samples were screened using the California Mastitis Test (CMT) and Modified Whiteside Test (MWST). Among the milk samples, 46.5% were positive for SCM by CMT. Culture, biochemical tests, and PCR confirmed 73 (21.2%) Staphylococcus spp., of which 30 (41.1%) were identified as S. aureus and 43 (58.9%) as non-aureus staphylococci (NAS). Among the 30 S. aureus-positive isolates, 10 (33.3%) were identified as methicillin-resistant S. aureus (MRSA), corresponding to a prevalence of 2.9% among the total milk samples. The MRSA isolates exhibited high multidrug resistance, especially to tetracycline (80%) and cefoxitin (80%), and commonly harbored resistance genes such as tetA (80%), aac(3)-iv (70%), and sul1 (50%). Virulence genes hla (66.7%) and sea (50%) were frequently detected, while icaA was found in 23.3% of MRSA. Notably, 60% of MRSA isolates were categorized as XDR based on international standard definitions, while 60% were biofilm producers with high MARI values up to 0.92, indicating severe resistance potential. These findings underscore a significant burden of MDR/XDR MRSA with virulence potential in buffalo SCM, posing serious risks to animal and public health.