Fish gut-water interface as a hotspot for the dissemination of antibiotic resistance genes across natural and aquaculture systems.

Freshwater ecosystems are important reservoirs and transmission pathways for antibiotic resistance genes (ARGs), yet host-mediated microbial selection and anthropogenic pressure on fish gut resistome remain poorly characterised, especially in major South Asian river systems. We performed shotgun metagenomics and genome-resolved binning from 194 fish representing four species, along with host-associated water samples, collected from six geographically distinct sites spanning two major river systems (the Yamuna and the Indus) and two aquaculture farms. The fish gut nurtures distinct microbial communities from the surrounding water, revealing strong host-mediated filtering of environmental microbiota. Across all samples, 1108 ARG subtypes conferring resistance to 14 antibiotic classes were detected, including extended-spectrum β-lactamases (blaTEM and blaCTX-M) and WHO critical-priority carbapenemases (blaIMP and blaOXA). Fish from the Indus River maintained diverse but comparatively stable resistomes dominated by intrinsic chromosomal efflux mechanisms, whereas fish from the urbanized Yamuna River, particularly Labeo boggut, exhibited noticeable enrichment of clinically important ARGs. The prevalence of mobile genetic elements (MGEs) and virulence factors (VFs) were consistently more abundant in the fish gut microbiome than in the host's surrounding water, indicating an increased potential for horizontal gene transfer and microbial persistence. A total of 19 metagenome-assembled genomes (MAGs) carrying multiple ARGs, VFs, and plasmid-associated markers were detected, identifying bacterial populations capable of maintaining and disseminating antimicrobial resistance. These outcomes confirm that fish inhabiting anthropogenically influenced river systems can serve as important reservoirs of clinically relevant resistance determinants, highlighting potential risks for environmental dissemination, aquaculture and human exposure through aquatic ecosystems.