ROS-Induced Oxidative Stress Causes Enrichment and Emergence of Antibacterial Resistance in Conventional Activated Sludge Process

Abstract

ABSTRACTConventional activated sludge (CAS) processes represent significant reservoirs of antibacterial resistance genes (ARGs). However, the mechanisms governing ARG proliferation (enrichment and emergence) in CAS remain poorly understood. We hypothesize that Reactive Oxygen Species (ROS), generated by oxygenation in aeration tanks, govern ARG proliferation. Shotgun-metagenomics assessment of ARG abundance across treatment stages of two plants handling hospital effluents revealed a substantial reduction in total ARGs in treated effluent. However, specific ARGs conferring resistance to beta-lactams, tetracyclines, aminoglycosides, rifamycins, macrolides, and other drug classes showed marked increase in CAS, exhibiting correlations with ROS stress response genes. Notably, many of these ARGs emerged in the CAS and were absent in upstream processes. Proof-of-concept experiments demonstrated increased ROS and ARG levels under high dissolved oxygen (8 mg/L) compared to low oxygen (2 mg/L) conditions. Both ESKAPE and non-ESKAPE host pathogens in CAS harbored multiple shared ARGs, indicating horizontal gene transfer facilitated proliferation. ROS-induced stress responses, including antioxidant defense activation, cell damage, DNA mutation, metabolic adaptation, efflux pump activation, and enhanced transformation, could contribute to ARG enrichment/emergence. This study highlights that oxygenation in CAS inadvertently promotes ARG proliferation, underscoring the need to manage oxygen levels to control ROS-induced ARG proliferation and environmental dissemination.Abstract Figure