Functional resistomes in municipal wastewater treatment plants pose challenges to public health

Wastewater treatment plants (WWTPs) are essential for controlling antimicrobial resistance, but also serve as hotspots for resistance gene persistence and dissemination. In this study, a novel combinatory approach using genome-resolved metagenomics and metatranscriptomics was employed to examine the resistome of a full-scale municipal WWTP across treatment stages and seasons. Results reveal that although human-associated, potentially pathogenic, antibiotic resistance gene (ARG)-harbouring metagenome-assembled genomes (ARB_MAGs) declined in abundance during treatment, many ARGs remained transcriptionally active, particularly efflux, beta-lactam, and macrolide-lincosamide-streptogramin genes conferring resistance against fluoroquinolones, cephalosporins, and macrolides. Environment and treatment-adapted microbes become increasingly dominant, with plasmids identified as major vectors of mobile resistance. Dominant potential pathogenic human-associated ARB_MAGs were abundant during spring and summer, whereas environmental ARB_MAGs predominated in colder seasons. Fluoroquinolone resistance genes displayed varying expression levels across ARB_MAG types, with the lowest levels observed during the anaerobic phase of treatment. Although clinically relevant ARGs were detected at low relative abundance and expression levels, one of the carriers was Citrobacter freundii ARB_MAG, a human-associated potential pathogen. These findings underscore the value of integrating genomic and transcriptomic data to assess site-specific and ARB_MAG-type-specific resistance and to strengthen antibiotic resistance monitoring in engineered systems.