Phenotypic Metagenomics tracks Wastewater-Associated Clinically Important Beta-lactam Resistant Bacteria Invading River Habitats

Abstract Background The abundance of clinically important antibiotic resistance determinants in wastewater has raised concerns regarding their dissemination into the environment and transmission to animals and humans. However, the high bacterial diversity and complexity of environmental microbiomes makes it challenging to comprehensively detect and quantify clinically relevant resistance determinants. To tackle this outstanding technical bottleneck, classic phenotype-based approaches vastly underestimate the diversity of clinically relevant resistance in environmental reservoirs, while increasingly applied genotyping-based approaches alone usually fail to capture information on the resistance phenotype, viability, and activities of resistant bacteria. Results Here, we developed phenotypic metagenomics as a novel high-throughput approach to couple selective cultivation (phenotyping) and metagenomics (genotyping) to overcome these difficulties of classic phenotype or genotype-based approaches. Using this new method, we were able to track the invasion of wastewater-born multi-resistant opportunistic pathogens into river sediment and epilithic biofilm in 9 Swiss rivers downstream of wastewater treatment plants (WWTPs). The results show that WWTPs release clinically important carbapenem-resistant opportunistic pathogens and promotes the spread of extended-spectrum beta-lactamase resistance genes in the receiving river water. Especially, epilithic biofilms were identified as an unrecognized environmental reservoir of wastewater-associated carbapenem resistant bacteria, which appear to also invade the gut of amphipods, a group of generalist invertebrate in local rivers. We further used metagenome-assembled genomes to characterize phenotypically resistant bacteria and their extended-spectrum beta-lactamase and carbapenemase resistance genes to confirm our finding that wastewater effluent increases resistance levels of extended-spectrum beta-lactamases in the receiving river water. Conclusions This study demonstrates that where culture plating and culture-independent metagenomics fail individually, their complementary combination allows tracking multi-resistant bacteria as they invade river habitats. The proof-of-concept approach that couple resistance phenotyping and genotyping provides a roadmap for sensitive, standardized, and cost-effective surveillance of the clinically relevant aspects of environmental resistomes. The findings call for a thorough life cycle assessment of the selection and transmission of last-resort antibiotic resistance determinants from clinical to environmental reservoirs.