Genome-resolved metagenomics uncovers antimicrobial resistance gene carriers in hospital and municipal wastewater environments

Wastewater-based epidemiology (WBE) is a powerful approach to study antimicrobial resistance (AMR) dynamics at the population level. Using genome-resolved metagenomics, we recovered 3978 metagenome-assembled genomes (MAGs) from archived metagenome sequences generated under the national wastewater surveillance programme across Wales, UK. Taxonomic profiling of MAGs revealed a diverse bacterial community, with significant compositional shifts observed across seasons and sample sources. Approximately 13.6 % of the MAGs carried one or more antimicrobial resistance genes (ARGs), with genes conferring resistance to tetracycline and oxacillin being the most prevalent within the wastewater microbiomes. We also recovered yet-uncultivated microbial genomes- often referred to as “microbial dark matter” harbouring clinically relevant ARGs, offering insights into previously uncharacterised resistance reservoirs in wastewater. ARG-host associations shifted between untreated influent and treated effluent, with effluent profiles also varying significantly between secondary and tertiary treatments, revealing the impact of treatment type on ARG host composition. This study represents the first comprehensive genome-resolved metagenomic characterisation of ARG carriers across both hospital and municipal wastewater in Wales, effectively bridging clinical and environmental compartments. Our findings highlight the need to integrate high-resolution genome-resolved metagenomic surveillance into national AMR monitoring frameworks to track emerging threats, characterise ARG reservoirs and inform targeted public health interventions. • Genome-resolved insights into ARG dissemination in hospital and municipal wastewater. • Accurate identification of ARG-carriers across complex wastewater environments. • “Microbial dark matter” identified as reservoirs of clinically relevant ARGs. • ARG-host dynamics shifted between WWTP influent and effluent. • High-resolution foundational framework for integrated AMR monitoring.