The flexible cotransfer of plasmids drives the dissemination of tet(X4) in swine Escherichia coli

The spread of the tet(X4) gene threatens the efficacy of tigecycline, a last-resort antibiotic. To understand this threat, we studied its prevalence and spread in Escherichia coli from pigs in China. Our investigation of 190 swine E. coli from Henan Province identified 26 (13.68%) as tet(X4)-positive. Whole-genome sequencing and phylogenetic analysis revealed considerable genetic diversity across multiple sequence types. Among these, we identified a globally epidemic O11:H25-ST457 clone. Conjugation assays confirmed high transferability, with 76.92% (20/26) of tet(X4)-harboring plasmids being successfully transferred, predominantly through independent transfer or cotransfer with other plasmids. We further elucidated, for the first time, the molecular mechanism of an IS15DI-mediated fusion between a tet(X4)-bearing IncHI1A/IncHI1B/IncFIA plasmid and an IncFIC(FII)/IncR plasmid. However, the resulting fusion plasmid was highly unstable without antimicrobial selection. Additionally, we report a tet(X4)-positive IncHI2/IncHI2A plasmid whose flexible multidrug resistance region readily underwent rearrangements during conjugation, generating heterogeneous plasmids in transconjugants. Our findings demonstrate that tet(X4) dissemination is primarily driven by the flexible cotransfer of plasmids rather than stable fusion events, highlighting an ongoing and dynamic threat of tigecycline resistance dissemination from livestock to the global health landscape.