Background: Hospitals are recognized as point sources of antimicrobials in urban wastewater systems; however, comprehensive evaluations of their discharge profiles have not yet been conducted. Methods: This study presents a multi-site investigation of residual antimicrobial concentrations in effluents from five general hospitals and a commercial facility in the metropolitan area of Japan. Over a 12-week period (December 2023–March 2024), extensive sampling was conducted. Fifteen antimicrobials from multiple classes were quantified using high-throughput analysis. Results: The results revealed consistently higher concentrations in hospital effluents, particularly for levofloxacin, vancomycin, and ampicillin, than in non-clinical sites. Distinct facility-specific and temporal patterns suggest strong links between local prescribing practices and the effluent composition. Some compounds, such as clarithromycin and minocycline, showed dual contributions from both hospital and commercial sources. Conclusions: These findings highlight the need for source-targeted monitoring and antimicrobial pollution control strategies and provide a foundation for expanding surveillance efforts and informing environmental policies related to antimicrobial resistance (AMR).

Background/Objectives: Comparative analysis of antimicrobial resistomes in hospital and community wastewater can provide valuable insights into the diversity and distribution of antimicrobial resistance genes (ARGs), contributing to the advancement of the One Health approach. This study aimed to characterize and compare the resistome profiles of wastewater sources from a hospital and community. Methods: Longitudinal metagenomic analysis was conducted on wastewater samples collected from the National Center for Global Health and Medicine (hospital) and a shopping mall (community) in Tokyo, Japan, between December 2019 and September 2023. ARG abundance was quantified using reads per kilobase per million mapped reads (RPKM) values, and comparative analyses were performed to identify the significantly enriched ARGs in the two sources. Results: A total of 46 monthly wastewater samples from the hospital yielded 825 unique ARGs, with a mean RPKM of 2.5 across all detected genes. In contrast, 333 ARGs were identified in the three shopping mall wastewater samples, with a mean RPKM of 2.1. Among the ARGs significantly enriched in the hospital samples, 23, including genes conferring resistance to aminoglycosides (nine groups) and β-lactam antibiotics (eight groups), exhibited significantly high RPKM values. No ARGs were found to be significantly enriched in the community wastewater samples. Conclusions: This study highlights the higher diversity and abundance of ARGs, particularly those conferring resistance to aminoglycosides and β-lactam antibiotics including carbapenems, in hospital wastewater than in community wastewater. These findings underscore the importance of continuous resistome monitoring of hospital wastewater as part of the integrated One Health surveillance strategy.