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.