Metagenomic Analysis of Antimicrobial Resistance in Wastewater and Its Environmental Impact

Abstract

Antimicrobial resistance (AMR) in wastewater is one of the most significant One Health threats, and wastewater treatment plants (WWTPs) are important hybrid reservoirs and dissemination routes for antibiotic resistance genes (ARGs) exploiting environmental resources. Shotgun metagenomics was used in this study to profile the resistome, mobile genetic elements (MGEs) and microbial hosts from influent and effluent samples across municipal and hospital-associated WWTPs. We identified hundreds of unique ARGs in each system, with multidrug, tetracycline, macrolide, aminoglycoside and β-lactam resistance determinants predominating and included clinically relevant genes i.e. blaCTX-M-15 sul1 tet(Q), qnr variants. While conventional treatment decreased total ARG loads, some ARGs abundant in raw influents became enriched in effluents and many resistance determinants persisted after treatment. Genome-resolved revealed that ARGs were primarily associated with Pseudomonadota and Bacillota, many plasmids contained more than one ARG, indicating multi-horizontal gene transfer events across IncF, IncQ, IncL/M and Col-type plasmids. The environmentally risk assessment shows that WWTP effluents are major contributors to surface-water resistomes, downstream riverine compartments harbour high-risk ARGs in pathogenic taxa, and biosolids drive soil contamination. These findings highlight the urgent need for improved treatment barriers, such as advanced oxidation processes, as well as widespread genomic surveillance to reduce the risk of AMR dissemination from wastewater into ecosystems.