Metatranscriptomic Analysis Reveals Actively Expressed Antimicrobial-Resistant Genes and Their Hosts in Hospital Wastewater.

Antimicrobial resistance is a major global concern and economic threat, necessitating a reliable monitoring approach to understand its frequency and spread via the environment. Hospital wastewater serves as a critical reservoir for antimicrobial-resistant organisms; however, its role in resistance gene distribution and dissemination remains poorly understood. This study integrates metagenomic and metatranscriptomic analyses, elucidating the dynamics of antimicrobial resistance in hospital wastewater. Integrated metagenomic and metatranscriptomic sequencing were used to identify actively expressed antimicrobial-resistant genes and antimicrobial-resistant bacteria, offering comprehensive insights into antimicrobial resistance dynamics in hospital wastewater. Liquid chromatography-tandem mass spectrometry analysis revealed the presence of ampicillin, sulbactam, levofloxacin, sulfamethoxazole, and trimethoprim in the sample, which could apply selective pressure on antimicrobial resistance gene expression. While multidrug resistance genes were the most prevalent sequences in both metagenome-assembled genomes and plasmids, plasmid-derived sequences showed a high mRNA/DNA ratio, emphasizing the presence of functionally expressed antimicrobial resistance genes on plasmids rather than on chromosomes. The metagenomic and metatranscriptomic analyses revealed MAG14 with high mRNA levels of antimicrobial resistance genes; moreover, multidrug-resistant sp., genetically related to MAG14, was isolated from the wastewater, supporting the phenotypic characterization of crucial antimicrobial-resistant bacteria and validating the genome analysis results. The findings underscore key genes and bacteria as targets for antimicrobial resistance surveillance in hospital wastewater to protect public and environmental health.