Unexpected Synergy: Calcium Channel Blockers and Residual Chlorine Cooperatively Accelerate Antibiotic Resistance...

The dissemination of antibiotic resistance driven by non-antibiotic pharmaceuticals is an emerging concern warranting further investigation. Calcium channel blockers (CCBs), frequently detected in aquatic environments, have rarely been studied for their role in the spread of antibiotic resistance genes (ARGs). This study examines the impact of two representative CCBs, amlodipine (AML) and verapamil (VER), on conjugative horizontal gene transfer (HGT) under low-level chlorine conditions. A bacterial conjugation system was established using Escherichia coli harboring the conjugative plasmid RP4, under simulated residual chlorine exposure typical of water distribution networks (0.3 mg/L). Conjugation assays revealed that, in the absence of residual chlorine, AML and VER at environmentally relevant concentrations (0.01-100 μg/L) exerted negligible effects on ARG conjugative transfer. Similarly, residual chlorine alone did not significantly enhance HGT. However, the co-occurrence of CCBs and residual chlorine synergistically promoted HGT, yielding a maximal 15.2-fold increase in conjugation frequency. This promotional effect was not mediated by increased cell membrane permeability but was driven by elevated reactive oxygen species production, upregulation of efflux pumps and outer membrane porins, and modulated transcription of conjugation-related genes. Notably, the oxidative stress response gene rpoS was upregulated by over 20-fold, while korA/korB (negative regulators) and kilA/kilB (repressors of vertical transfer) were both downregulated, collectively relieving the repression on conjugative transfer. These findings demonstrate that environmental non-antibiotic pharmaceuticals can synergistically promote ARG dissemination under residual chlorine in water supply systems. This study provides a scientific basis for refining risk assessment frameworks for pharmaceutical contaminants in water.