Abstract:Residual disinfectants in aquatic environments may influence the dissemination of antibiotic resistance genes (ARGs) by affecting horizontal gene transfer among microorganisms. The phenolic disinfectant p-chloro-m-xylenol (PCMX) has been widely used and is frequently detected in municipal wastewater and surface waters at concentrations ranging from ng/L to μg/L. However, its potential effects on plasmid-mediated ARG dissemination remain insufficiently understood. This study investigated the influence of PCMX on the conjugative transfer of ARGs and explored the potential mechanisms involved. An intergeneric conjugation model was established using Escherichia coli CHS56 carrying plasmid RP4-8 as the donor strain and Pseudomonas sp. TS44 as the recipient strain. The effects of PCMX on conjugative transfer were evaluated over a concentration range of 0.1–10 000 μg/L. Conjugation experiments were combined with reactive oxygen species (ROS) detection, cell membrane permeability measurements, and transcriptomic analysis to investigate the physiological responses and molecular mechanisms associated with PCMX exposure. The results demonstrated that PCMX promoted plasmid-mediated conjugative transfer within environmentally relevant concentration ranges. Specifically, at concentrations between 0.1 and 100 μg/L, PCMX enhanced the transfer frequency of plasmid RP4-8 between the donor and recipient strains. The highest conjugation frequency (7.22×10−6) was observed at 10 μg/L PCMX. When the concentration increased to 1000–10 000 μg/L, the conjugation frequency declined, which may be associated with the inhibitory effects of high PCMX concentrations on bacterial growth and cellular activity. Flow cytometry analysis using DCF-DA staining indicated that PCMX exposure increased intracellular ROS levels in the donor strain E. coli CHS56 at concentrations of 100 and 10 000 μg/L. In contrast, no significant ROS variation was detected in the recipient strain Pseudomonas sp. TS44. The addition of the ROS scavenger N-acetylcysteine (NAC) significantly reduced intracellular ROS levels in the donor strain but did not markedly decrease the conjugation frequency, suggesting that oxidative stress was not the dominant factor responsible for the enhancement of plasmid transfer under PCMX exposure. Membrane permeability analysis revealed that PCMX exposure altered the integrity and permeability of bacterial cell membranes, particularly in the recipient strain. Increased membrane permeability may facilitate physical contact between donor and recipient cells and promote plasmid transfer. Transcriptomic analysis further showed that PCMX exposure significantly affected the global gene expression profiles of both donor and recipient strains. Multiple genes associated with membrane transport and transmembrane processes were differentially expressed. Among them, the glycerol uptake facilitator gene involved in membrane transport was significantly upregulated, indicating enhanced transmembrane transport activity under PCMX stress. These results suggest that PCMX can enhance plasmid-mediated ARG conjugative transfer primarily by altering cell membrane permeability and transmembrane transport processes rather than by ROS-mediated oxidative stress. Considering that PCMX cannot be completely removed during conventional wastewater treatment, residual PCMX may persist in biological treatment units containing dense microbial communities, thereby increasing the potential risk of ARG dissemination. The findings provide experimental evidence for evaluating the ecological risks of disinfectant residues in wastewater treatment systems and aquatic environments. Close-