Fe⁰ drives tetracycline resistance genes reduction and resistance mechanism shift in activated sludge system

The proliferation of tetracycline antibiotic resistance genes (TC-ARGs) in wastewater treatment plants (WWTPs) poses environmental and health risks. This study investigates zero-valent iron (Fe⁰) in regulating TC-ARGs in activated sludge under co-exposure with tetracycline (TC). TC enriched tetA and tetC via efflux pump activation and horizontal gene transfer (HGT), with tetA/tetC increasing 0.19-3.57 log units and intI1 by 1-2 log units. Fe⁰ addition slightly affected absolute TC-ARGs abundance in the first 40 d, with a 4.02 % decrease after 40 d, while relative abundance dropped markedly by 77.81 % and 84.87 % in the first and last 40 d, respectively; intI1 relative abundance decreased by 78.28 %. Fe⁰ shifted resistance from efflux genes toward ribosomal protection (tetM/O) and enzymatic modification (tetX), reduced bioavailable TC by 39.8 %, and alleviated EPS inhibition by enhancing polysaccharide production, weakening antibiotic selection pressure. Microbial community restructuring enriched stress-tolerant taxa (e.g., Bacteroidetes). This study demonstrates that Fe⁰ primarily reduces the dissemination risk of TC-ARGs by lowering their proportion in microbial genomes. These results provide new insights into optimizing the application of Fe⁰ for controlling the spread of TC-ARGs in WWTPs.