Abstract
Antimicrobial resistance in raw milk is a critical challenge for public health. This study aimed to investigate the in-vitro and in-silico analysis of antimicrobial resistance, mutations, virulence factors (VFs), mobile genetic elements, and diversity of Escherichia coli in cattle raw milk of Gujarat, India during May 2023- April 2024. 56 isolates were recovered from 50 raw milk samples. Among them, 26 were identified as E. coli and subjected to antimicrobial susceptibility testing. The extensively drug-resistant 10 isolates were further confirmed by 16s rRNA identification. Whole-genome Nanopore sequencing was performed on 10 representative strains, and data were further used in bioinformatics analysis. The results revealed that wide array of ARGs and mutations were detected. Plasmid-mediated ARGs were detected in three isolates that include blaTEM-1, sul2, dfrA14, aph6-Id, aph(3'')-Ib, and tet(B), responsible for resistance against antibiotic classes such as beta-lactam, folate-pathway, aminoglycosides, and tetracyclines, respectively. Additionally, the conserved VFs in isolates facilitated serum survival and toxin production. The unit transposons Tn2 and Tn10 were detected in two isolates. The isolates exhibited considerable diversity, belonging to different serotypes, sequence types (ST), and phylogroups. This research provides crucial insights for developing effective monitoring, mitigation, and educational strategies to prevent the spread of pathogenic Escherichia coli through the food chain.