We used a combination of shotgun metagenomics and RNA-probe target enrichment metagenomics to perform an in-depth 10-month survey of the microbiota, resistome, and mobilome of untreated and treated wastewater from a full-scale wastewater treatment plant in Oslo, Norway

Wastewater treatment plants (WWTPs) are hotspots for the acquisition and dissemination of antimicrobial resistance genes (ARGs) by bacteria and represent key sites for surveillance of antimicrobial resistance (AMR). We performed a ten-month metagenomic survey of untreated wastewater (UWW) and treated wastewater (TWW) from a full-scale WWTP serving municipal and hospital wastewater from Oslo, Norway. Wastewater treatment, which combines both chemical and biological treatment processes, led to a significant reduction in the relative abundance of human gut-associated bacterial species and total load of coliform bacteria, with a corresponding increase in environmental bacterial taxa. This was associated with a significant reduction in the relative abundance and richness of both ARGs and mobile genetic elements. Despite this, the effect of treatment on the relative abundance of several key AMR-associated pathogens, including multiple ESKAPE pathogens and Enterobacterales, was highly inconsistent. Subspecies analysis demonstrated multiple Escherichia coli sequence types (STs) and Klebsiella pneumoniae sub-lineages (SLs) persisted in both UWW and TWW samples over multiple months. This included a K. pneumoniae SL12 lineage that appeared in all UWW and TWW samples throughout the entire study period. Targeted RNA probe-hybridisation enrichment revealed the presence of multiple clinically important ARGs in both UWW and TWW samples, encoding extended-spectrum ß-lactamases, carbapenemases, glycopeptide resistance, and colistin resistance, which were undetectable by shotgun metagenomics. These findings demonstrate the utility of metagenomic methods in wastewater AMR surveillance, including the potential for monitoring high-risk bacterial lineages and high-sensitivity detection of clinically important ARGs, in a low AMR prevalence setting such as Norway.