Mechanistic insights into stereoselective effects of S-naproxen and R-naproxen on conjugative transfer of antibiotic resistance genes.

The dissemination of antibiotic resistance genes (ARGs) through plasmid-mediated conjugative transfer poses a serious public health threat. Although non-antibiotic pharmaceuticals are known to influence horizontal gene transfer (HGT), their stereoselective effects remain poorly understood. In this study, we investigated the enantioselective effects of naproxen (NAP) on RP4 plasmid-mediated conjugation between Escherichia coli and Pseudomonas aeruginosa. The R- and S-enantiomers of NAP (R-NAP and S-NAP) exhibited markedly different influences on antibiotic resistance gene (ARG) transfer, with R-NAP inducing a substantially higher conjugation frequency than S-NAP. This observation was consistent with transcriptomic and RT-qPCR analyses, which showed stronger upregulation of key conjugation-related genes (e.g., traF, traJ, trfAp, and trbBp) and membrane-associated genes (ompA, ompF, and oprF) under R-NAP exposure. Proteomic analysis further confirmed the upregulation of pilus- and membrane-associated proteins in response to R-NAP exposure. Molecular docking suggested that this stereoselective effect arises from distinct binding interactions between NAP enantiomers and conjugation-associated proteins, with R-NAP forming more stable conformations with core proteins such as TraF, TraJ, OmpA, and OprF. Collectively, these results demonstrate that R-NAP enhances RP4-mediated conjugative transfer by stereoselectively modulating gene and protein expression and strengthening protein-ligand interactions. This study highlights the overlooked role of chiral pharmaceuticals in accelerating ARG dissemination and underscores the need to consider stereoselectivity in environmental risk assessments of non-antibiotic pharmaceuticals.