Nano-ferrous sulfide regulates conjugative transfer of antibiotic resistance genes: Synergistic effects of extracellular reactive oxygen species, Fe2+, and mineral particles.

Conjugative transfer of antibiotic resistance genes (ARGs) in aquatic environments severely exacerbates global health risks. While iron (oxyhydr)oxide minerals are well known to regulate ARGs conjugative transfer in bacteria, the roles of reduced iron minerals remain poorly understood. Here we demonstrate that low concentrations (25-50 mg/L) of nano-FeS improve ARGs transfer efficiency by up to 707.12 %. Nano-FeS and its oxidation-derived species, including extracellular reactive oxygen species (ROS), Fe2+, and mineral particles, trigger intracellular ROS production to initially strengthen bacterial adhesion. These factors further increase cell membrane permeability via physicochemical damage, thereby promoting plasmid transmembrane transfer. Meanwhile, moderate ROS accumulation also upregulates plasmid transport gene expression and provides metabolic energy for conjugation. Beyond these dominant physiological/biochemical responses, mineral particles bridge adjacent bacteria to facilitate intercellular contact. In contrast, high nano-FeS dosage (500 mg/L) reduces conjugation efficiency by 42.19 %-79.29 %. Nano-FeS and its oxidation-derived species impair cell adhesion and energy metabolism by inducing excessive intracellular ROS, and cause lethal membrane damage. Moreover, mineral particles exert a prominent coating effect that ultimately inhibits conjugative transfer. Notably, this bidirectional regulatory effect is consistently observed across various real aquatic matrices including paddy field pore water and pond bottom water. Our findings highlight that reduced iron minerals serve as redox-sensitive "switches" governing ARGs conjugation, providing new insights into iron chemistry for mitigating ARGs propagation in engineered and natural environments.