Antibiotic-resistant bacteria sampled from metropolitan wastewater share genomic similarities with hospital-associated isolates

Wastewater surveillance is an effective approach for monitoring populations of antibiotic-resistant bacteria and tracking the spread of antimicrobial resistance (AMR) across clinical and environmental settings. In this study, hospital and municipal wastewater were collected monthly for 12 months from hospital and municipal locations in the greater Pittsburgh area to quantify the presence of antibiotic-resistant bacteria and investigate their genomic diversity. After quantitative culturing on six different selective medias, a total of 150 isolates were speciated by 16S rRNA sequencing, which revealed diverse pathogenic and non-pathogenic taxa, including Klebsiella spp., Pseudomonas spp. and Aeromonas spp. A subset of isolates belonging to clinically relevant species (n = 46) underwent whole-genome sequencing, which identified several antibiotic resistance genes of clinical concern, such as blaKPC, blaNDM, and blaIMP, and revealed genetic similarities between wastewater and clinical isolates collected from infected patients at a Pittsburgh-area medical center. In addition, analysis of plasmids carried by wastewater isolates revealed closely related plasmids present in isolates from different species and sampling locations. Overall, these findings demonstrate that both hospital and municipal wastewater act as interconnected reservoirs of antimicrobial resistance. Integrating wastewater surveillance with clinical and genomic data could enable the early detection of emerging resistance threats and support proactive infection-control strategies. IMPORTANCEAntibiotic-resistant bacteria persist within hospital environments and cause deadly infections. This study examined the prevalence of antibiotic-resistant bacteria in both hospital and municipal wastewater sampled from the Pittsburgh metropolitan area. Over a 12-month surveillance period, we observed a high abundance of antibiotic-resistant organisms, including pathogenic Gram-positive and Gram-negative species encoding diverse antibiotic resistance genes, many of which were plasmid-encoded. We also identified instances of closely related isolates sampled from wastewater and clinical sources, as well as closely related plasmids found in different species and sampled from different wastewater sites. Overall, this study demonstrates that wastewater surveillance is a viable method for non-invasive monitoring of resistant bacteria at a local scale.