Microbiota and resistome dynamics in untreated and treated wastewater: A ten-month study leveraging RNA-probe capture and subspecies-level metagenomics.

Wastewater is regarded as a hotspot for the acquisition and dissemination of antimicrobial resistance genes (ARGs) in bacteria, and wastewater treatment plants are key sites for studying and monitoring these phenomena. This study employed metagenomic approaches, with and without targeted ARG enrichment, to investigate the composition and dynamics of the microbiota, resistome, and mobilome in untreated (UWW) and treated (TWW) wastewater from a full-scale treatment plant serving municipal and hospital wastewater in Oslo, Norway. Over a ten-month period, we observed that wastewater treatment led to a significant reduction in the relative abundance of human gut-associated bacterial species and total load of coliform bacteria, alongside an increase in environmental bacterial taxa. This shift correlated with a significant reduction in the relative abundance and richness of ARGs and mobile genetic elements. Despite this, the effect of treatment on the relative abundance of key AMR-associated pathogens was highly inconsistent. Further subspecies analysis revealed several Escherichia coli and Klebsiella pneumoniae lineages persisted in UWW and TWW over multiple months, suggesting stable colonization and survival despite treatment processes. Targeted RNA probe-hybridisation enrichment detected clinically important ARGs in both UWW and TWW samples, including genes encoding extended-spectrum β-lactamases, carbapenemases, glycopeptide resistance, and colistin resistance. Most of these were undetectable by shotgun metagenomics alone, demonstrating the strength of this technique in high-sensitivity ARG surveillance. These findings highlight the value of combined metagenomic methods in wastewater AMR surveillance, the potential for monitoring high-risk bacterial lineages, and high-sensitivity detection of clinically important ARGs, in a low AMR prevalence setting.