An Ohio State Scenic River Shows Elevated Antibiotic Resistance Genes, Including Acinetobacter Tetracycline and Macrolide Resistance, Downstream of Wastewater Treatment Plant Effluent

The entry of antibiotic resistance genes (ARGs) into aquatic systems has been documented for large municipal wastewater treatment plants (WWTPs), but there is less study of the impact of smaller plants that are situated on small rural rivers. We sampled water metagenomes for ARGs and taxa composition from the Kokosing River, a small rural river in Knox County, Ohio, which has been designated an Ohio State Scenic River for retention of natural character. Samples were obtained 1.0 km upstream, 120 m downstream, and 6.4 km downstream from the effluent release of the Mount Vernon WWTP. ARGs were identified in metagenomes using ShortBRED markers from the comprehensive antibiotic resistance database (CARD) screened against UniPROT. Through all seasons, the metagenome just downstream of the WWTP effluent showed a substantial elevation of at least 15 different ARGs, including 6 ARGs commonly associated with Acinetobacter baumannii, such as msrE, mphE (macrolide resistance), and tet(39) (tetracycline resistance). The ARGs most prevalent near the effluent pipe persisted 6.4 km downriver. Using metagenomic phylogenetic analysis (MetaPhlAn2) clade-specific marker genes, the taxa distribution near the effluent showed elevation of reads annotated as Acinetobacter species as well as gut-associated taxa, Bacteroides and Firmicutes. The ARG levels and taxa prevalence showed little dependence on seasonal chlorination of the effluent. Nitrogen and phosphorus were elevated near the effluent pipe but had no consistent correlation with ARG levels. We show that in a rural river microbiome, year-round wastewater effluent substantially elevates ARGs, including those associated with multidrug-resistant A. baumannii. IMPORTANCE 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. There is little information on the rural impacts of WWTP effluent on the delivery and transport of antibiotic resistance genes. In our study, the river water proximal to wastewater effluent shows evidence for the influx of multidrug-resistant Acinetobacter baumannii, an opportunistic pathogen of concern for hospitals but also widespread in natural environments. Our work highlights the importance of wastewater effluent in management of environmental antibiotic resistance, even in high quality, rural river systems.