Evolutionary insights and antibiogram analysis of extended-spectrum β-lactamase producing Escherichia coli from bovine subclinical mastitis in Pakistan.

Extended-spectrum β-lactamase (ESBL) producing E. coli has become a significant challenge, limiting therapeutic options due to its structural adaptability and increasing resistance pattern. This study was targeted at understanding the molecular epidemiology of ESBL-producing E. coli involved in subclinical mastitis (SCM), accompanied by computational modeling of β-lactamase in conjunction with antibiogram analysis. Three eighty-four milk samples were examined for SCM and the identification of E. coli. The ESBL isolates were phenotypically tested using the double-disc synergy testing (DDST) followed by molecular detection of the blaTEM gene. Computational structural analysis was performed to identify β-lactamase sequence and structural characteristics, along with in vitro drug susceptibility to various antibiotics. A total of 173 (45.05%) were positive for subclinical mastitis, with E. coli confirmed in 43 (24.85%) of milk samples. Moreover, 13 (30.23%) of E. coli isolates showed results for DDST, while the blaTEM gene was detected in 16 (37.21%). Phylogenetic analysis of ESBL-producing isolates indicated close genetic association, even with diverse geographic origins. Computational structure analysis of β-lactamase revealed a significant degree of sequence conservation, several high-confidence phosphorylation sites, and moderate structural stability, indicating conservation of essential functional characteristics of β-lactamase protein. E. coli isolates revealed maximum resistance to amoxicillin and tetracycline, followed by doxycycline and ciprofloxacin. This study concluded that ESBL-producing E. coli is a significantly prevalent bacteria in SCM in bovines, and computational analysis gives crucial insights into the functional dynamics of β-lactamase protein, contributing valuable information for antimicrobial surveillance and control strategies.