Humic acid’s sheltering effect paradoxically promotes antibiotic resistance gene dissemination during peroxymonosulfate water treatment

Advanced oxidation processes (AOPs) utilizing peroxymonosulfate (PMS) are increasingly deployed for water disinfection, yet their impact on the dissemination of antibiotic resistance genes (ARGs) remains poorly understood. Here, we conducted field surveys in three aquaculture ponds in eastern China and employed a Transwell-based horizontal gene transfer (HRT) sorting assay, metagenomics, and transcriptomics to investigate the underlying mechanisms. We found that PMS-treated waters harbored significantly higher intracellular ARG burdens compared to untreated or chlorinated systems. Mechanistically, the ubiquitous humic acid (HA) acts as a selective radical scavenger, protecting bacteria from lethal oxidative damage while stimulating type IV pilus-mediated uptake of extracellular ARGs. This interaction redirects HGT from conjugation toward natural transformation. Crucially, this protective effect is oxidant-specific: HA effectively quenches PMS-derived radicals but provides no defense against direct electrophilic attack by chlorine. Furthermore, predation experiments using Caenorhabditis elegans demonstrate that this “sheltering effect” facilitates the accumulation of ARGs in nematodes, decoupling resistance acquisition from oxidative stress intensity. These findings highlight a critical risk pathway in which some water treatment chemicals can promote the spread of resistance, necessitating a reassessment of oxidant selection based on their specific chemical mechanisms. In this study, the authors show that humic acid, a common organic substance, shields bacteria from peroxymonosulfate disinfection, inadvertently promoting the spread of antibiotic resistance genes via natural transformation in aquatic environments.