Emergence of highly virulent multidrug and extensively drug resistant Escherichia coli and Klebsiella pneumoniae in buffalo subclinical mastitis cases

This study aimed to characterize virulence and antibiotic resistance genes in multidrug-resistant (MDR) and extensively drug-resistant (XDR) Escherichia coli and Klebsiella pneumoniae isolated from cases of subclinical mastitis (SCM) in buffaloes. A cross-sectional study was conducted on 1540 quarter milk samples collected from 385 buffaloes. Milk samples were screened using the California Mastitis Test and Modified Whiteside Test. Positive samples underwent bacterial culture, biochemical tests, biofilm detection and molecular analysis for pathogen identification and detection of virulence, resistance, and extended-spectrum beta-lactamase (ESBL) genes. The prevalence of SCM was 67.9% (1046/1540) at the quarter level and 80.8% (311/385) at the animal level. E. coli was identified in 9.5% (146/1540) of the samples, while K. pneumoniae was detected in 9.09% (140/1540). Virulence genes, such as stx1 (27.4%), and resistance genes, including aac(3)-iv (77.4%) and tetA (76.7%), exhibited higher prevalence. Additionally, β-lactamase genes, notably bla_TEM (67.1%), and ESBL genes, such as bla_CTX-M1, were detected. Biofilm formation was detected in 83.6% (122/146) of E. coli isolates and 75.7% (106/140) of K. pneumoniae isolates. Antimicrobial susceptibility testing revealed significant resistance to ampicillin, amoxicillin-clavulanic acid, and aminoglycosides. MDR was observed in 31.5% of E. coli and 39.3% of K. pneumoniae isolates, with XDR rates of 8.9% and 12.9%, respectively. These findings underscore the alarming spread of resistant pathogens in SCM-affected buffaloes, emphasizing the urgent need for ongoing surveillance and targeted intervention strategies.