Antibiotic resistance is a growing problem worldwide, with frequent transmission between pathogens and environmental organisms. Rural rivers can support high levels of recreational use by people unaware of inputs from treated wastewater, while wastewater treatment plants (WWTPs) can generate a small but significant portion of flow volume into a river surrounded by forest and agriculture.

Rivers in rural areas receive continual influx of wastewater carrying antibiotics originally administered to humans and livestock. The entry of antibiotic resistance genes (ARGs) into aquatic systems has been documented for large municipal wastewater treatment plants, but there is less study of the impact of smaller plants that are situated on small rural rivers. We sampled water metagenomes for ARG and taxa composition from the Kokosing State Scenic River, a small rural river in Knox County, Ohio. Samples were obtained upstream, a few meters downstream, and 6 km downstream from the effluent release of the Mount Vernon wastewater treatment plant (WWTP). Through all seasons, the metagenome just downstream of the WWTP effluent showed a substantial elevation of at least 15 different ARGs, including 6 ARGs from Acinetobacter baumannii such as msrE, mphE (macrolide resistance) and tet(39) (tetracycline resistance). The ARGs most prevalent near the effluent pipe persisted 6 km downstream. The taxa distribution near the effluent showed elevation of Acinetobacter species as well as gut-associated taxa, Bacteroides and Firmicutes. The ARG levels and taxa prevalence showed little dependence on seasonal chlorination of effluent. Nitrogen and phosphorus were elevated near the effluent pipe but had no consistent effect on ARG levels. We show that in a rural river microbiome, year-round wastewater effluent substantially elevates ARGs including those of multidrug-resistant A. baumannii.