Trace Hydrogen Sulfide Enhances Plasmid-Mediated Antibiotic Resistance Gene Transfer under Anaerobic Sewer-Like Conditions: Associations with Contact and Energy-State Responses

The dissemination of antibiotic resistance genes (ARGs) in sewer systems poses a potential threat to public health. However, it is currently unclear how hydrogen sulfide (H2S) in anaerobic sewers affects plasmid-mediated ARG transfer. Here, we evaluated the effects of environmentally relevant concentrations (1–100 μmol·L–1) of H2S on ARG transfer across different sewer microbial communities. The results demonstrated that H2S significantly enhanced the transfer frequency of plasmid pKJK5 in sediment-derived (2.06–3.06-fold), sewage-derived (1.50–2.81-fold), and bioaerosol-derived (2.42–4.68-fold) recipient communities in standardized liquid anaerobic conjugation assays. In mixed-community models, H2S increased total acyl-homoserine lactone (AHL)-equivalent signals and was associated with selective enrichment of polysaccharides in extracellular polymeric substances (EPS), coinciding with enhanced microbial aggregation and contact. In single-strain models, H2S was associated with reductive-stress-like responses, lowered the NAD+/NADH ratio, enhanced phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase activities, strengthened proton motive force, and increased ATP content. Inhibitor experiments and structural equation modeling suggested ATP availability as the strongest intracellular predictor of enhanced transfer, with AHL-associated EPS changes acting as a potential extracellular contributor. These findings identify a concentration-dependent sulfide effect on conjugation under anaerobic conditions and support a testable pathway-level framework for future in situ sewer studies.