Sulfonamide metabolites enhance resistance transmission via conjugative transfer pathways

Human beings release thousands of antibiotics into the environment, which could generate the related transformation products (TPs), most of which have yet to be identified and lack rigorous microbial risk information. This study aimed to investigate the impact and mechanisms of 4-nitro sulfamethoxazole, N⁴-acetylated sulfamethoxazole, and N⁴-acetylated sulfadiazine, three typical sulfonamide (SAs) metabolites, on the risk of antibiotic resistance genes (ARGs) transmission. The results revealed that TPs significantly enhance the risk of conjugative transfer of RP4 plasmid at clinically and environmentally relevant concentrations (10 ng/L to 100 μg/L), with a maximum increase of up to 73-fold. These three metabolites' capabilities to enhance the conjugative transfer of ARGs are more pronounced than the parent sulfonamides. The induction mechanisms of TPs on ARGs transmission are also more complex, which primarily arise from the enhancement of reactive oxygen species, further increased cell membrane permeability and upregulated bacterial secretion systems. Transcriptomic analysis validated the aforementioned biological processes and showed that TPs also increased the activity of toxin-antitoxin system and bacterial intracellular transposon, thereby promoting the spread of ARGs. This research contributes to a better understanding of the antibiotic-like effects of TPs, which is crucial for improving our understanding of non-antibiotic drug-induced bacterial resistance risks.