Unravelling the resistome of carbapenem-resistant E. coli from bovine mastitis via whole-genome sequencing.

Carbapenem-resistant Escherichia coli (CREC) poses a growing threat to public health, particularly when emerging from animal reservoirs such as dairy cattle. This study aimed to characterize CREC isolates recovered from bovine mastitis cases in Gujarat, India, using a combination of phenotypic antibiotic susceptibility testing and whole-genome sequencing (WGS). Out of 130 confirmed E. coli isolates from 790 mastitic milk samples, 33 (25.38%) were resistant to imipenem. Of these, nine exhibited multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan-drug-resistant (PDR) phenotypes. WGS was performed on four representative isolates (SKN144, SKN685, SKN687, SKN926), revealing genome sizes ranging from 4.7 to 5.4 Mb and GC content between 50.4% and 50.8%. Annotation identified numerous resistance determinants, including carbapenemase genes (blaNDM, blaOXA-48, blaTEM, blaCMY, blaCTX-M), aminoglycoside-modifying enzymes (APH, AAC), macrolide resistance genes (mphA, ermB), and multiple efflux pump systems (AcrAB-TolC, EmrAB, MdtEF-TolC). Functional genes associated with replication, repair, stress response, and mobile genetic elements (integrases, transposases, CRISPR-Cas) were also detected, indicating high genomic adaptability. Phenotypic testing revealed alarming resistance to key antimicrobials, including ampicillin (56.15%), amikacin (55.38%), ceftazidime (53.08%), and colistin (79.23%, including intermediate strains). Subsystem analysis highlighted metabolic versatility, defence mechanisms, and virulence-associated pathways. Phylogenetic analysis indicated that all isolates clustered within the same clade, suggesting possible clonal dissemination within the bovine population. The presence of CRISPR-Cas elements, integrases, and transposases suggests ongoing horizontal gene transfer and genome plasticity. These findings underscore the alarming prevalence of CREC in dairy environments and the urgent need for enhanced AMR surveillance, prudent antibiotic stewardship, and implementation of a One Health approach to prevent zoonotic transmission. This study contributes valuable genomic insights into livestock-associated CREC and highlights their close genomic parallels with high-risk human clinical clones.