Significant effects of electrophilicity in oral antidiabetic drugs upon conjugative transfer of drug-resistance plasmids in activated sludge and the mechanisms

The pivotal role of plasmid-mediated conjugative transfer in the spread of antibiotic resistance genes (ARGs) is well established, and environmental chemicals exert considerable selective pressure on this process. Oral antidiabetic drugs (OADs) are frequently detected in aquatic environments at concentrations reaching the mg/L range. However, their influence on the conjugative transfer of plasmid remains largely unexplored. Here, we investigated the effects of eight widely used OADs on the transfer of the RP4 plasmid between Escherichia coli strains isolated from representative wastewater sources. The results showed that all tested OADs significantly enhance plasmid transfer ( P < 0.05), albeit with up to a four - fold variation in change. Notably, this phenomenon is independent of OAD degradation profiles, but strongly associated with bacterial biochemical responses ( P < 0.05) such as reactive oxygen species (ROS) production, cell membrane permeability, and ATP synthesis. Mechanistic analyses further identify OAD electrophilicity as a key chemical determinant, modulating both physiological processes (membrane permeability and ATP production) and molecular components—genes ( ybbA, mlaA , and ompF ) and their corresponding proteins (YbbA, MlaA, and OmpF)—involved in conjugative transfer. Furthermore, results demonstrate that OADs consistently increase ARG abundance and diversity in activated sludge systems, an effect strongly correlated with their electrophilicity ( P < 0.05). Collectively, our findings reveal, for the first time, that OAD electrophilicity is a critical driver of ARG dissemination in wastewater treatment systems, thereby providing molecular-level insights and a theoretical foundation for controlling ARG spread.