Mixed-species slaughterhouses represent potential ecological hotspots associated with IS1-driven plasmid evolution and dissemination of tet(X4).

The rapid dissemination of the plasmid-borne tigecycline resistance gene tet(X4) in food production systems poses a severe threat to global public health. While slaughterhouses are recognized as antimicrobial resistance (AMR) reservoirs, the specific ecological factors associated with tet(X4) evolution within these environments remain oversimplified. Challenging the assumption of uniform risk across processing facilities, this study systematically investigated how distinct operational settings (pig, chicken, and mixed-species slaughterhouses) influence plasmid-mediated horizontal gene transfer and host fitness adaptation. Among 410 isolates, a representative mixed-slaughterhouse-derived strain (DAR-EC600) demonstrated an exceptional ecological advantage, exhibiting significantly enhanced conjugation efficiency and remarkable plasmid stability (>95% retention over 14 days) compared to single-species facility isolates. Rather than suffering typical AMR fitness costs, multi-omics and in vivo analyses revealed that DAR-EC600 employs an immune modulation-driven colonization strategy. This is achieved through the coordinated up-regulation of motility (fliA/Z), adhesion (fimA/H), and nutrient transport (rbsA/B), effectively neutralizing fitness burdens and triggering pronounced intestinal inflammation and microbiota perturbation. Mechanistically, integrated plasmid and metagenomic profiling identified a highly plastic IncHI1 plasmid architecture (IS1-tet(X4)-blaTEM-1-intI1), where IS1-enriched recombination hotspots facilitate the synergistic co-selection of resistance and virulence determinants. Collectively, these findings suggest that mixed-species slaughterhouses may represent important ecological interfaces associated with enhanced plasmid adaptability, stability, and dissemination potential of tet(X4)-carrying plasmids. This study connects localized AMR surveillance to broader One Health discourses, underscoring the urgent need to target environmental transmission hotspots in global food safety frameworks.