FeOOH-Catalyzed Removal of Extracellular Antibiotic Resistance Genes: Structural Effects on Kinetics and Mechanisms

Extracellular antibiotic resistance genes (eARGs), as critical drivers of antibiotic resistance transmission, exist in diverse structural forms, complicating their environmental persistence and propagation. Goethite (FeOOH), a naturally abundant catalyst, has shown great potential in catalyzing the hydrolysis of phosphate diester bonds. However, the FeOOH-catalyzed hydrolysis of eARGs, particularly the structural-dependent differences in the removal of eARGs, remains poorly understood. This study comprehensively explored the FeOOH-based hydrolysis of eARGs, with a special focus on how the structural forms of eARGs (supercoiled, nicked circular, and linear) influence their hydrolysis kinetics and underlying mechanisms. The results confirmed the effectiveness of FeOOH in catalyzing eARGs removal, with replication activity reduced by over 60% within 11 days. The FeOOH-catalyzed hydrolysis of eARGs exhibited structure-dependent behavior. Specifically, the removal rate of supercoiled eARGs was 1.47 times higher than that of nicked circular eARGs and 3.13 times higher than that of linear eARGs. Agarose gel electrophoresis and atomic force microscopy analyses revealed that supercoiled and nicked circular eARGs underwent both single-point cleavage and multisite damage, whereas linear eARGs primarily experienced multisite hydrolytic damage. qPCR amplification with different primers further verified that hydrolysis preferentially occurred in the nick-adjacent region. Moreover, longer phosphate backbones were more susceptible to hydrolytic cleavage.