Deciphering the mobility, pathogenic hosts, and co-selection of antibiotic resistance genes in untreated wastewater from three different hospitals.

Objective: Antibiotic resistance genes (ARGs) in hospital wastewater pose significant environmental and public health risks, yet the co-selection mechanisms involving metal/biocide resistance genes (MRGs/BRGs) and the role of mobile genetic elements (MGEs) remain poorly characterized. This study aimed to comprehensively assess the abundance, mobility, pathogenic hosts, and co-selection patterns of ARGs, MRGs, and BRGs in untreated wastewater from three types of hospitals.

Methods: Untreated wastewater samples from nine sources across three hospital types (general, traditional Chinese medicine, and dental) were analyzed using metagenomic sequencing and assembly. ARGs, MRGs, and BRGs were identified via the SARG and BacMet databases. ARG hosts, mobility, and MGE co-occurrence were analyzed using PlasFlow and MOB-suite, with risk levels evaluated alongside pathogenic bacteria databases.

Results: A total of 1911 ARGs (222 subtypes), 1662 MRGs (167 subtypes), and 916 BRGs (139 subtypes) were detected. Tetracycline, multidrug, and β-lactam resistance genes were predominant, with 46.43 % of ARGs being plasmid-associated. Key pathogens including Klebsiella pneumoniae and Enterococcus spp. harbored high-risk ARGs such as KPC-2 and NDM-1. Notably, 76.2 % of ARGs in traditional Chinese medicine hospital wastewater were classified as high-risk. Significant co-occurrence of ARGs with MGEs (e.g., DDE recombinases) and MRGs/BRGs was observed, underscoring the role of horizontal gene transfer and co-selection.

Conclusion: Untreated hospital wastewater represents a significant reservoir of ARGs, with risks exacerbated by pathogenic hosts, MGE-mediated HGT, and metal/biocide co-selection. These findings underscore the urgent need for optimized wastewater treatment strategies to curb the spread of antibiotic resistance and inform future intervention efforts.