Hospital Wastewater as A Reservoir of Antimicrobial Resistance Genes: Implications for Public Health and Genomic Surveillance in The United States

Abstract

Hospital wastewater represents a concentrated environmental reservoir of antimicrobial resistance genes (ARGs), resistant organisms, pharmaceutical residues, and mobile genetic elements. This quantitative longitudinal study examined ARG occurrence, abundance, diversity, mobility, and clinical associations across 12 hospitals in four United States census regions. Of 1,152 planned samples, 1,098 met quality requirements, producing a 95.3% inclusion rate. Samples were collected from hospital outlets, upstream community sewers, downstream locations, and municipal influent over 12 months. Quantitative polymerase chain reaction, droplet digital polymerase chain reaction, culture, and metagenomic sequencing were combined with antimicrobial-use, patient-activity, wastewater-flow, and clinical-resistance data. Hospital outlets had 1.43-fold higher total ARG abundance than upstream sites. Detection of blaKPC occurred in 56.5% of hospital samples and 25.2% of upstream samples, corresponding to an adjusted odds ratio of 4.12. Metagenomic analysis identified 412 ARGs across 18 resistance classes. Mean ARG richness was 176.4 genes per hospital sample compared with 132.7 upstream, while 41.7% of hospital ARGs were linked to mobility markers. A 10-defined-daily-dose increase in carbapenem use per 1,000 patient-days was associated with an 18.6% concurrent increase in wastewater blaKPC and a 22.4% increase after a one-month lag. Wastewater blaKPC was positively associated with clinical carbapenem-resistant Enterobacterales. Treatment reduced absolute ARG concentrations by 91.7%–99.8%, although viable resistant organisms declined more strongly than total detectable DNA. These findings characterized hospital wastewater as a measurable, clinically connected resistance reservoir and supported its integration with clinical microbiology, antimicrobial stewardship, and genomic surveillance.