Persistence of mcr-1-carrying E. coli in rabbit meat production: Challenges beyond long-term colistin withdrawal.

Colistin, a last-resort antibiotic in human medicine, has been banned in European food animal production to mitigate antimicrobial resistance. This study investigates the long-term effects of the colistin ban on the occurrence and genomic features (WGS) of colistin-resistant, mcr-carrying Escherichia coli across intensive rabbit farms (8 farms, ~600 animals/farm, fecal and farm environmental samples) in the north and center of Portugal. Colistin-resistant E. coli was detected in 25 % of groups from three farms in pre-slaughter fecal samples, with mcr-1-positive strains found throughout the lifecycle (does, offspring, and feed) in all fecal samples from one farm. A polyclonal multidrug-resistant (MDR) E. coli population carrying mcr-1 persisted over three years, mostly in pre-slaughter rabbits but also in newly arrived younger does (GP). Comparative genomic analysis (cgMLST) revealed four clusters, with closely related strains between rabbit feces and feed (ST1196, ST40) and between feces and GP (ST1196), suggesting external reservoirs, biosecurity concerns, and cross-contamination. WGS also revealed high load and diversity in virulence (EPEC and ExPEC), antibiotic resistance and genes related to metal decreased susceptibility. All mcr-1 genes were located on similar IncHI2 multireplicon plasmids, carrying sil + pco (copper) co-located with antibiotic resistance genes, and circulating in global sources. These results highlight that, despite colistin withdrawal, MDR mcr-carrying E. coli clones persist over three years in a single farm, underscoring complex co-selection pressure and biosecurity gaps. The findings underscore food safety risks via the food chain and environmental contamination. Enhanced biosecurity, feed monitoring, and One Health surveillance are essential to mitigate AMR dissemination and safeguard public health.