ESKAPE pathogens contribute largely to antibiotic resistance spread via horizontal gene transfer in aquatic...

The overuse of antibiotics in human healthcare, livestock, and aquaculture has led to the accumulation of antibiotic residues in aquatic environments. It promotes the dissemination of antibiotic-resistant bacteria (ARB) that pose a threat to public health. However, the mechanisms that shape antibiotic resistance gene (ARG) profiles in different water types remain poorly understood. In this study, three water types, including hospital wastewater, breeding wastewater, and natural waters, were employed. Using a combination of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), 16S rRNA gene sequencing, and metagenomic analysis, we found that ofloxacin in hospital wastewater posed the highest ecological risk, whereas norfloxacin and tetracycline in natural waters posed elevated health risks. Among 101 detected ARG subtypes, hospital effluents carried the highest abundances of high-risk ARGs and their host bacteria compared to breeding wastewater and natural waters. Interestingly, mobile genetic elements (MGEs) were the primary direct driver of ARG enrichment (PLS-PM path coefficient = 0.725), in contrast to the negligible contributions from typical antibiotic residues, physicochemical parameters, and microbial community structure. Furthermore, genera associated with ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) dominated the co-hosts of both ARGs and MGEs across all water types. Among these, Enterobacter spp. and Klebsiella pneumoniae were found to co-harbor the most diverse MGEs and multidrug-resistant ARGs. Consequently, this study underscores the critical role of ESKAPE pathogens in the environmental dissemination of ARGs and provides a scientific foundation for targeted antibiotic resistance control and sustainable water resource management.