Insights into Fe0, Fe2O3, and Fe3O4-Mediated reduction of antibiotic resistance genes and horizontal gene transfer via reactive oxygen species during composting.

Reactive oxygen species (ROS) generated by Fe-based additives mitigate antibiotic resistance during composting, yet the associated patterns remain unclear. The objective of this study was to examine the specific roles of Fe0, Fe2O3, and Fe3O4 in ROS-mediated reduction of antibiotic resistance genes (ARGs) and associated host bacteria during composting of chicken manure (CM) and wine grape pomace (WGP). The results showed that Fe0, Fe2O3, and Fe3O4 achieved ARG attenuation efficiencies of 81%, 92%, and 83%, respectively, representing a 52-63% increase over the control (29%). Notably, Segmented linear regression analysis revealed a critical threshold: when potential pathogen horizontal gene transfer (HGT) frequency exceeded 0.084, metal resistance gene (MRG) abundance increased, indicating that pathogen-mediated transfer promotes rapid resistance accumulation. Concurrently, the abundances of mobile genetic elements (MGEs), host bacterial genera, and potential pathogen HGT frequencies decreased by 52-73%, 74-95%, and 75-80%, respectively. Moreover, ROS induced by Fe-based additives were identified as the primary driving force for ARG attenuation, with Fe2O3 triggering the highest level of ROS generation. Elevated ROS weakened the survival of high-risk bacterial genera (HBG) and reduced the transmission potential of ARGs by disrupting bacterial cell membrane integrity and inhibiting DNA repair processes. Functional pathway analysis further revealed that lipid damage repair (map00590) and nucleotide excision repair (map03420) were the primary metabolic response pathways of HBG under oxidative stress. These findings provide mechanistic insights into antibiotic resistance attenuation and can guide treatment of livestock manure for fertilizer application.