Integrated Poultry Production As a Reservoir of Tet(x4) and Mcr-1.1 Encoding Escherichia Coli in Pakistan

Plasmid-mediated colistin and tigecycline resistance threatens last-resort therapeutic options. We investigated the farm-to-fork dissemination of Escherichia coli encoding mcr-1.1 and tet(X4) in a vertically integrated broiler production system. A total of 200 samples were collected in 2024, representing four sequential production stages: broiler breeders, day-old chicks, day-30 broilers and retail meat. All positive [mcr-1.1 and/or tet(X4)] isolates underwent Illumina short-read sequencing, with long-read sequencing of three representatives. The study was enriched and analysed with mcr-1.1 and tet(X4) encoding 213 publicly available E. coli genomes from Pakistan. Of 18 isolates, tet(X4) was detected in 11 and mcr-1.1 in 7 isolates with a single breeder isolate (ST-398) co-harbouring both on separate plasmids. tet(X4) predominated breeders and day-30 broilers, whereas mcr-1.1 was distributed across all four stages. ST-1011 exhibited a pattern of clonal farm-to-fork transmission of tet(X4) via IncFIB(AP001918)-IncFII providing molecular evidence suggestive of clonal transmission. Analysis of 231 genomes across 87 sequence types (STs) revealed contrasting evolutionary trajectories: mcr-1.1 with post-mobilization stabilization marked by complete absence of ISApl1 and highly conserved IncI2 plasmid (93%), whereas tet(X4) retained active transposition within IS26-bounded elements. tet(X4) carriage was also associated with elevated resistance gene burden, driven by the IncF megaplasmids. All 18 isolates exhibited adhesion to chicken intestinal epithelial cells (CHIC-8E11), with high adhesion capacity among tet(X4)-positive isolates. This study demonstrates that mcr-1.1 and tet(X4) disseminate through poultry production via distinct mechanisms. These findings highlight the need for integrated One Health surveillance of antimicrobial resistance dissemination.