Functional and evolutionary insights into the emerging tet(X4)-carrying non-O1/O139 Vibrio cholerae from retail pork

The tigecycline resistance gene tet(X4) is prevalent in Enterobacteriaceae, particularly in Escherichia coli. To our knowledge, no study has reported the dissemination dynamics of tet(X4) in Vibrio spp. Herein, we isolated and characterized a first tet(X4)-positive non-O1/O139 Vibrio cholerae isolate from retail pork. Genomic sequencing identified a novel tet(X4) variant in the V. cholerae chromosome, harboring a G568A nucleotide substitution that resulted in an Ala190Thr (A190T) amino acid substitution in Tet(X4). While this Tet(X4)-A190T variant conferred lower phenotypic resistance to tetracyclines (including tigecycline) than the wild-type Tet(X4), its overall catalytic efficiency against these antibiotics was paradoxically enhanced despite a reduced substrate affinity. Genomic comparisons revealed that two copies of ISCR2 flanked the variant gene, and the structure was ISCR2-hp-hp-abh-tet(X4)G568A -ISCR2, which is highly homologous to the reported E. coli plasmids carrying tet(X4). In addition, it confirmed the presence of an ISCR2-mediated circular intermediate, proving this modules capacity for horizontal transfer of the tet(X4)G568A variant. Furthermore, the ISCR2-tet(X4) genetic structure carrying the G568A substitution was integrated within a chimeric SXT/R391-like integrative and conjugative element (ICE), which is also serving as a vehicle for genetic dissemination. As per our knowledge, this is the first report on the emergence of SXT/R391-like ICE carrying tet(X4) in Vibrio strains. Our finding demonstrates that the clinically relevant tigecycline resistance gene tet(X4), previously confined mainly to Enterobacterales from humans and livestock, is now actively spreading into environmental Vibrio populations. This cross-species transfer highlights a previously underappreciated ecological and public health concern in aquatic ecosystems. ImportanceTigecycline serves as a vital last-resort antibiotic against severe multidrug-resistant bacterial infections, but its clinical efficacy is currently threatened by the rapid global dissemination of resistance genes like tet(X4). While land-based agriculture is a well-recognized reservoir for these genes, the role of aquatic ecosystems and environmental pathogens, such as V. cholerae, in harboring tet(X) determinants remains largely unexplored. In this study, we characterize a non-O1/non-O139 V. cholerae isolate from retail pork that harbors a naturally occurring, chromosomally integrated tet(X4)G568A variant. This novel variant exhibits elevated catalytic efficiency against tetracycline antibiotics. The tet(X4)G568A allele is embedded in a highly conserved structural module (ISCR2-tet(X4)-abh-hp-hp-ISCR2) flanked by two ISCR2 repeats, which is integrated into an SXT/R391-like ICE at the chromosomal prfC locus. These findings provide the first high-confidence genomic evidence of tet(X4) in V. cholerae, highlighting aquatic Vibrio species as critical environmental reservoirs for clinically significant antimicrobial resistance genes and emphasizing the urgent need for continuous genomic surveillance.