Persistence of environmental and wastewater-associated antibiotic resistance in river water

River water serves both as a recipient of anthropogenic antibiotic resistance (AR) via wastewater and as a reservoir of AR that can disseminate and pose risks to human and animal health. However, the fate of AR from anthropogenic and environmental sources in aquatic ecosystems remains poorly understood. In this microcosm study, we investigated the persistence of AR vectors over 10 weeks using river water from two European sites, with and without the addition of 5% (v/v) wastewater. We employed a combination of culture-based techniques, quantitative PCR, 16S rRNA gene sequencing, and genome sequencing of isolates to track both anthropogenic and environmental AR components. Our findings demonstrated the rapid decline of AR vectors associated with anthropogenic sources such as extended-spectrum beta-lactamase (ESBL)-producing E. coli, bla_TEM, and bla_CMY-2, which had a maximum half-life of 2.1 days and were below detection limits after a maximum of 20 days. In contrast, environmental markers such as ESBL-producing bacteria grown on R2A medium and sul1 persisted over extended periods of time: their half-lives were up to 9.9 and 11.6 days, respectively, and they were detected in all samples after 10 weeks at concentrations approximately tenfold lower than at day 0. Here, we identified and characterized for the first time ESBL-producing Pseudomonas environmental isolates that persisted for at least 10 weeks in river water in the absence of anthropogenic input, demonstrating the pivotal role of this genus in AR maintenance in aquatic ecosystems and the need to include it in surveillance strategies to improve the accuracy of risk assessments.