Environmental residue-level doxycycline accelerates compensatory evolution and persistence of tet(X4)-mediated...

Environmental residues of tetracycline antibiotics are widespread in livestock and poultry environments and represent an important but underexplored driver of antibiotic resistance (AR) persistence. Here, we investigated how residue-level doxycycline influences the compensatory evolution of E. coli carrying the clinically critical tigecycline resistance gene tet(X4). Using long-term experimental evolution (∼500 generations) under antibiotic-free and environmentally relevant doxycycline conditions, combined with phenotypic assays, gene expression analysis, and whole-genome sequencing, we found that tet(X4) imposed a significant initial fitness cost, which was largely alleviated by ∼300 generations. Notably, fitness recovery occurred without observable plasmid loss or downregulation of tet(X4) under the tested conditions. Instead, adaptive evolution was associated with high-frequency mutations in chromosomal global regulators crp and arcA, which coincided with coordinated phenotypic remodeling, including enhanced motility, reduced reactive oxygen species accumulation, and decreased outer membrane permeability. Importantly, residue-level doxycycline exposure further stabilized plasmid maintenance and resistance levels, indicating that environmental antibiotic residues may contribute to the persistence of high-priority resistance genes during compensatory evolution. These findings suggest that low-level environmental antibiotic exposure may create a selective landscape associated with the stabilization of tet(X4), highlighting a previously underappreciated pathway by which environmental contamination may influence antibiotic resistance and pose potential risks to environmental and public health.