A Fitness Burden Drives the Reversion of in vitro Acquired Doxycycline Resistance in V. cholerae O1

Introduction: Antibiotic exposure in both clinical and environmental settings can select for resistant isolates with expanded cross-resistance, including last-resort drugs. In Vibrio cholerae , such patterns emerge under sustained doxycycline exposure; however, the evolutionary dynamics and long-term stability of this adaptation remain unresolved. Methods: Here, we applied long-term experimental evolution to investigate the induction, fitness-costs, and reversion of doxycycline resistance in V. cholerae O1. Isolates were exposed to sub-inhibitory doxycycline concentrations over 13 weeks, followed by phenotypic, genomic, and phylogenetic analyses. Resistance stability was assessed after a 2-week subculture period in the absence of doxycycline. Results: Doxycycline-susceptible V. cholerae O1 isolates from the 2022 Lebanon outbreak evolved high-level resistance within 13 weeks of sustained exposure. The evolved resistance extended to colistin, while showing collateral sensitivity to azithromycin. Resistance imposed a marked fitness burden characterized by impaired growth and reduced yield. Notably, drug withdrawal restored both doxycycline susceptibility and fitness. Whole-genome sequencing revealed recurrent high-impact mutations in tetR and LysR -family regulators and efflux-associated pathways. Revertants were characterized by the loss of these variants and the accumulation of compensatory mutations in metabolism and membrane structure. Phylogenetic analysis showed that resistance evolved within the original lineages, with evolved isolates clustering closely with globally distributed resistant clinical strains. Conclusion: Doxycycline resistance in V. cholerae O1 emerges rapidly under antibiotic pressure but represents a transient, high-cost adaptation that is reversible upon drug withdrawal. These findings highlight the fitness burden associated with resistance and demonstrate reproducible evolutionary trajectories, with important implications for antimicrobial stewardship strategies. Keywords: Vibrio cholerae , doxycycline resistance, collateral sensitivity, reversion, fitness cost