Longitudinal metagenomic analysis on antibiotic resistome, mobilome, and microbiome of river ecosystems in a sub-tropical metropolitan city

Rivers play an important role as reservoirs and sinks for antibiotic resistance genes (ARGs). However, it remains underexplored for the resistome and associated mobilome in river ecosystems, and hosts of riverine ARGs particularly the pathogenic ones are rarely studied. This study for the first time conducted a longitudinal metagenomic analysis to unveil the resistome, mobilome, and microbiome in river water, by collecting samples from 16 rivers in Hong Kong over a three-year period and using both short-read and long-read sequencing. Results revealed that aminoglycoside, bacitracin, β-lactam, macrolide lincosamide-streptogramin, and sulfonamide were the predominant ARG types in the river water samples. Riverine ARGs exhibited high spatial variations in abundance and diversity. Environmental factors such as fecal coliform count, Escherichia coli count, 5-day biochemical oxygen demand (BOD₅), dissolved oxygen (DO), and total organic carbon (TOC) had a significant correlation to the absolute concentrations of ARGs. Nanopore sequencing was used to reveal the physical genetic linkage of mobile genetic elements (MGEs) with ARGs in river water samples. The results showed that qacEdelta, transposase, integrase, and Tn916 had a high prevalence in ARG-carrying long reads. Host tracking using ARG-carrying reads identified 23 pathogenic bacteria species that harbored ARGs. Some ARGs were shared by different bacterial groups. This study presented a nuanced insight of resistome in river water by a longitudinal metagenomic analysis and deepened our understanding of common and divergent riverine antimicrobial resistant risk across the regional patterns.