Genomic characterization and diversity of tet(X4)-positive bacteria in poultry-associated fecal environments across multiple regions in China.

The emergence of tigecycline resistance genes, particularly tet(X) variants, has raised increasing concern as emerging antimicrobial resistance determinants in agro-environmental systems. Here, we conducted a large-scale environmental survey to characterize the burden and genomic features of tet(X)-positive bacteria in poultry-associated fecal matrices. A total of 514 chicken fecal samples were collected from fourteen poultry farms distributed across eight cities in five provinces of China, yielding 205 tet(X)-positive bacterial isolates. Escherichia coli was the predominant host species (72.2%), with tet(X4) identified as the dominant variant. Most isolates (90.2%) exhibited multidrug resistance, showing high resistance rates to florfenicol (98.0%) and enrofloxacin (83.4%), while resistance to meropenem remained low (0.5%). Conjugation assays showed that 43.9% of the isolates were capable of transferring tet(X), with transfer frequencies centered around 10-4. Whole-genome sequencing of 105 representative isolates revealed a high resistance gene load (average of 11 genes per genome) and diverse tet(X4)-carrying plasmid backbones, including IncFIA(HI1)/FII, IncX1, IncFIA(HI1)/HI1A, and IncFII. In addition, tet(X4) was detected on the chromosome of one isolate with an identical genetic context to its plasmid-borne counterpart. The tet(X4)-associated region displayed a conserved estT-tet(X4)-ISCR2 core structure. Overall, these results demonstrate that poultry feces represent an important agro-environmental reservoir of tet(X4), characterized by high host diversity, substantial resistance gene burden, and pronounced genetic mobility, underscoring the need for systematic environmental surveillance of antimicrobial resistance determinants in agricultural ecosystems.