High-risk AMR genes blaCMY-42, blaCTX-M-15, and blaNDM-5 in raw milk: A foodborne transmission concern

Antimicrobial resistance in raw milk threatens public health by spreading multidrug resistance via horizontal gene transfer. This study investigates the prevalence and genetic diversity of ESBL and carbapenem-resistant Escherichia coli and Klebsiella pneumoniae in raw milk from Gujarat, India. The in-vitro and in-silico antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS) to assess antibiotic resistance genes (ARGs), virulence factors (VFs), mobile genetic elements (MGEs), phylogeny and genetic diversity was carried out. AST revealed both E. coli and K. pneumoniae were highly resistant to beta-lactam class of antibiotics including 3 rd & 4 th generation cephalosporins. WGS was employed to characterize 20 isolates (10 E. coli and 10 K. pneumoniae ) for the analysis of ARGs, VFs and MGEs. Based on WGS data, identified bla CTX-M-15 , bla NDM-5 , and bla CMY-42 genes as key drivers of resistance detected on both strains. Virulence genes linked to horizontal gene transfer, such as traT and traJ were detected. The study found that insertion sequences (IS), including ISEc9, IS3, and IS21, as well as composite transposons like Tn5403 and Tn6082, were mainly distributed across isolates. A diverse array of beta-lactam and carbapenem resistance genes were detected on plasmid associated contigs in E. coli and K. pneumoniae isolates from raw milk, underscoring the risk of food-borne transmission of resistance genes. Implementing a "One Health" approach is essential to address the interconnections between human, animal, and environmental health and prevent the further spread of MDR bacteria from raw milk to humans. • 3 rd and 4 th generation cephalosporins resistance was the most commonly observed phenotype. • Plasmid mediated ESBL and carbapenem ARGs: bla CTX-M-15 , bla CMY-42 , bla NDM-5 and bla TEM-1 . • Both bacterial species contained genes linked to horizontal gene transfer; traJ and traT. • IS26 flanked transposon found in E. coli & K. pneumoniae : Distributed globally in pathogens.