Metagenomic profiling of resistome and mobilome dynamics in diverse freshwater aquaculture modes

The widespread presence of antibiotic resistance genes (ARGs) in aquaculture environments poses a growing threat to public health. However, comprehensive understanding of ARG distribution and transmission potential across different freshwater aquaculture modes remains limited. This study employed integrated short- and long-read metagenomic sequencing to characterize the resistome, mobilome, and associated microbial communities across three predominant freshwater aquaculture modes (grass carp, crayfish, and crab ponds), using water, sediment, and intestinal samples analyzed at both contig and metagenome-assembled genome (MAG) levels. The results revealed that aquaculture modes and environmental media jointly shaped microbial and ARG compositions. At the contig level, the crayfish system harbored the highest relative abundance of both ARGs and mobile genetic elements (MGEs), with gut samples consistently emerging as the dominant reservoir across all modes. A significant positive correlation between ARG and MGE alpha diversity indicated that the gut microbiome, particularly in crayfish, provides a selective environment that co-enriches resistance genes and their mobile carriers. High-risk core ARGs (Rank I) were at least 19 times more abundant in the crayfish gut than in any other compartment, underscoring the intestinal microbiome as a hotspot for clinically relevant resistance accumulation. At the MAG level, over half of the recovered MAGs met near-complete or high-quality thresholds, and approximately 38% of ARG-carrying MAGs were classified as multidrug-resistant (MDR). MDR MAG abundance was significantly higher in gut than in sediment and water samples, with the crayfish gut as the most enriched compartment. Critically, several crayfish-associated MDR MAGs affiliated with Klebsiella aerogenes carried virulence factor genes (VFGs) and exhibited ARG-MGE-VFG co-localization within prophage sequences, suggesting phage-mediated co-dissemination of resistance and virulence traits. These findings highlight the intestinal microbiome of aquaculture species as a critical hotspot for resistance dissemination and provide a scientific basis for evaluating freshwater aquaculture-associated ARG risks under the One Health framework.