Trace antibiotics increase the risk of antibiotic resistance genes transmission by regulating the biofilm extracellular polymeric substances and microbial community in the sewer.

Sewer is considered a potential hotspot for antibiotic resistance, but the occurrence and proliferation of antibiotic resistance genes (ARGs) under trace antibiotics exposure have received little attention. This work evaluated the effects of tetracycline (TC) and sulfamethoxazole (SMX) individually and in combination in the sewer system and revealed the related mechanisms of ARG proliferation. The relative abundance of tetA and sul1 increased the most under TC and SMX stress, respectively, whereas sul1 increased the most under combined stress. Intl1 was abundant in both the liquid phase and the biofilm, and redundancy analysis confirmed that horizontal gene transfer was the main reason for the proliferation of ARGs. The increase in extracellular polymeric substances (EPS) secretion and the enhancement of the main hydrophobic functional groups facilitated the accumulation of biofilms, which promoted the proliferation of ARGs in biofilms. The relative abundance of most ARGs in the liquid phase was significantly correlated with EPS, protein and tryptophan-like substances. Furthermore, the microbial community structure and diversity affected the proliferation and spread of ARGs in the sewer. These findings contribute to our further understanding of the proliferation and development of ARGs in the sewer and lay the foundation for the front-end control of ARGs.