Emerging Dissemination of bla_CTX-M-65 in Bovine E. coli in Spain Associated With IncHI2 Plasmids

Food-producing animals act as reservoirs of antimicrobial-resistant bacteria, which may serve as sources of infection in humans. A selection of ESBL/AmpC-producing E. coli isolates recovered from healthy (n = 37) and diseased animals (n = 26) were subjected to long-read whole-genome sequencing to characterize their genomic diversity, phylogeny, resistome, mobilome, and the genetic environment of antimicrobial resistance genes (ARGs). Isolates showed high genetic diversity (36 STs, seven phylogroups, 42 serotypes, 24 fimH types) and a diverse resistome, with CTX-M enzymes as the most prevalent ESBL type and bla_CTX-M-14 and bla_CTX-M-65 predominating. Most ARGs were plasmid-encoded (71.9%), with IncHI2 plasmids harboring the highest number of ARGs (8-20) and carrying complex multidrug resistance islands interspersed with integrons, transposons, and insertion sequences. All 15 IncHI2 plasmids were found exclusively in bovine isolates of multiple phylogroups (5) and STs (13), and most (13/15) carried bla_CTX-M-65. This finding suggests a dissemination of this ESBL gene through a shared plasmid rather than a single dominant clone. This study reveals an emerging circulation of bla_CTX-M-65 in bovine E. coli in Spain and identifies IncHI2 plasmids as major vehicles driving its spread. The presence of these plasmids in genetically diverse and potentially zoonotic linages reinforces their relevance from a One Health and food-chain perspective.