Comparative analysis of planktonic and sessile microbiomes and associated resistomes in a freshwater ecosystem impacted by municipal wastewater treatment plant discharge

Abstract

Wastewater treatment plants (WWTPs) disrupt freshwater ecosystems and are recognised as hotspots for antimicrobial resistance. This study assessed the effect of treated municipal wastewater discharge on both planktonic and sessile freshwater microbial communities. A monitoring network was established in a Spanish urban river basin during 2022 dry season, with sampling points upstream and downstream of a WWTP. Artificial glass-based substrates enabled biofilm colonisation, while river water and effluent samples were collected during biofilm harvesting. Chemical exposure was assessed by characterising nutrients and antimicrobial concentrations in water, while shotgun metagenomics revealed taxonomic composition and resistome pools in both matrices. River water exhibited greater genus richness and pathobiome diversity than biofilms. Wastewater discharge shaped microbial composition, leading to shifts in less dominant groups, with partial attenuation of this effect downstream. Resistome richness was higher in river water compared to biofilm, though the latter displayed greater total abundances and distinct resistance profiles suggesting their role as genetic reservoirs. While no consistent resistome trends were linked to sampling location, higher total resistance abundances occurred in river water at sites impacted by wastewaters. Background resistance levels found upstream likely persisted downstream due to pollutant-driven selection. Lower resistome richness in effluent water suggests downstream abundances may also result from microbiome shifts and environmental factors. Assembly-based analysis revealed resistance genetic contexts and indicators of gene dissemination.