Sublethal Antibiotic Exposure Induces Microevolution of Quinolone Resistance in Pathogenic Vibrio parahaemolyticus

The microevolutionary pathways and molecular mechanisms by which the important pathogen <i>Vibrio parahaemolyticus</i> acquires resistance in the aquatic environment under continuous selective pressure from quinolone antibiotic residues are still unknown. Here, the study successfully simulated the long-term pressure of antibiotic residues in aquaculture by susceptible <i>V. parahaemolyticus</i> (VPD14) which was isolated from seafood, to a 30-day in vitro induction with sublethal concentrations of levofloxacin, which yielded the mutants (VPD14M). A phenotypic analysis revealed that VPD14M exhibited resistance to ampicillin, levofloxacin and ciprofloxacin, compared to VPD14. These changes were accompanied by adaptations, including a decreased growth rate and an enhanced biofilm formation capacity. Whole-Genome Sequencing identified that the acquired resistance was primarily attributable to key point mutations in three Quinolone Resistance-Determining Regions (QRDRs). Specifically, a G → T substitution at nucleotide position 248 in the <i>gyrA</i> gene, leading to a serine-to-isoleucine substitution at the 83rd amino acid position (Ser83Ile) of the DNA gyrase subunit A; a C → T substitution at position 254 in the <i>parC</i> gene, resulting in a serine-to-phenylalanine substitution at position 85 (Ser85Phe) of the topoisomerase IV subunit A; and a C → T substitution at position 2242 in the <i>gyrB</i> gene, causing a proline-to-serine substitution at position 748 (Pro748Ser) of the DNA gyrase subunit B. Collectively, the study demonstrated that sublethal antibiotic levels rapidly drive quinolone resistance in <i>V. parahaemolyticus</i>, and the specific mutations identified offer critical support for resistance monitoring and seafood safety alerts.