Comparative insights into molecular characterization and resistance profiling of methicillin-resistant Staphylococcus aureus across dairy livestock in Pakistan.

Antimicrobial resistance (AMR) is a monolithic issue, particularly the global emergence of Methicillin-resistant Staphylococcus aureus (MRSA). This study investigated the prevalence of MRSA, its association with resistant genes and virulence factors. Furthermore, phylogenetic analysis, antibiogram, and In-silico analysis were performed. For this, 1,132 subclinical mastitis-positive milk samples from livestock (cattle, buffalo, sheep, goat, camel) were collected using convenient sampling. Out of the total, 558 (49.29%) and 441 (38.96%) isolates were found to be positive for S. aureus based on phenotypic and genotypic information, respectively. Moreover, the highest MRSA prevalence was found in cattle 56 (53.33%), while the lowest was in buffalo 21 (18.75%), using the PCR tool. Also, the prevalence of enterotoxin and biofilm-producing icaA and icaD genes was evaluated. Antibiogram of MRSA isolates revealed that dairy animals exhibited high resistance to penicillin G, cefoxitin, and gentamicin, but remained susceptible to ciprofloxacin and moxifloxacin. MRSA of sheep origin showed distinct resistance patterns, showing more sensitivity towards gentamicin and amoxicillin. Also, out of 159 MRSA isolates, 111 were declared as MDR S. aureus. Phylogenetic analysis revealed that Pakistan MRSA isolates were closely related to each other, with goat and buffalo MRSA isolates showing a close evolutionary relationship. In-silico analysis showed that all dairy animals in Pakistan shared conserved nucleotide and amino acid motifs. This study concludes that MRSA represents a serious threat to livestock health due to its multidrug-resistant properties, and antibiogram profiling can provide a valuable tool for guiding effective therapeutic interventions against MRSA infections.