Differential regulation of conjugative transfer and OMV-mediated ARG delivery by iprovalicarb and cycluron: Implications for pathway-specific risk assessment

Horizontal gene transfer (HGT) is a major driver of the rapid dissemination of antibiotic resistance genes (ARGs) among bacteria. Non-antibiotic compounds can promote plasmid-mediated conjugative transfer. In waterfowl farming, waterfowl frequently move among water, soil, and feces, which serve as reservoirs for pesticides and ARGs and thus potential hotspots for ARG spread. However, whether outer membrane vesicles (OMVs), an emerging ARG transmission pathway, are regulated by pesticides and respond in a manner consistent with conjugation is unclear. In this study, two pesticides detected in waterfowl farm environments, iprovalicarb and cycluron were selected to investigate their effects on conjugative transfer and OMV-mediated ARG dissemination. Both pesticides significantly promoted RP4 plasmid conjugation frequency at sub-inhibitory concentrations. This effect was associated with elevated bacterial reactive oxygen species (ROS), increased membrane permeability and fluidity, enhanced interbacterial contact, and altered energy metabolism. However, their effects on the OMV pathway diverged: iprovalicarb increased OMV production and vesicle size and was associated with more bla -positive colonies after OMV exposure, whereas cycluron inhibited OMV production. This decoupling suggests that pesticides can differentially regulate conjugative and OMV-mediated ARG transfer. However, the bla gene was not stably inherited in recipient bacteria. Overall, these findings highlight the need to evaluate these two pathways separately when assessing the risk of antimicrobial resistance.