Dual-Pathway Inhibition of Antibiotic Resistance Genes by Ferrate (Fe(VI)): Oxidative Inactivation and Genetic Mobility Impairment in Anaerobically Digested Sludge

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are emerging environmental contaminants that threaten public health, highlighting the urgent need for effective control strategies. Ferrate (Fe(VI)), a strong and eco-friendly oxidant, shows great potential for this purpose. This study systematically evaluated the efficacy of Fe(VI) in mitigating ARGs and ARB in anaerobically digested sludge, with a particular focus on elucidating the underlying mechanisms by which Fe(VI) effects ARGs dissemination through both vertical gene transfer (VGT) and horizontal gene transfer (HGT). Result shows that Fe(VI) doses of 20 and 60 mg/g-TS reduce ARGs by 9.75% and 19.12%, respectively, while inactivating up to 24.7% of ARB at the higher dose. Pathogenic ARB, such as Escherichia coli and Shigella sonnei, are preferentially removed, with abundances decrease by 63.7% and 28.0%. Mechanistically, the structural disruption of bacterial cells caused by Fe(VI) in anaerobically digested sludge, as indicated by a 29% reduction in extracellular polymeric substances and a 23.7% increase in cell membrane permeability. Subsequently, a marked release of intracellular ARGs into the extracellular environment is also observed, where they are likely subjected to degradation by Fe(VI). This oxidative killing accounts for the observed ARB decrease, thereby limiting the VGT of ARGs. In addition, Fe(VI) impairs the HGT of ARGs by diminishing their mobility potential, reflected in the reduced co-occurence with mobile genetic elements. Meanwhile, sludge bacterial competence for DNA uptake and recombination is markedly reduced, as evidenced by a 9.8% decline in the abundance of related functional genes. These findings demonstrate that Fe(VI) effectively inhibits the dissemination of ARGs by targeting both primary transmission pathways. It suppresses VGT, thereby reducing the inheritance of ARB within populations, and limits HGT, curbing the spread of mobile ARGs among competent species. By disrupting these two critical routes, Fe(VI) shows strong potential as an effective strategy for mitigating ARGs propagation in sludge systems.