Horizontal gene transfer plays a crucial role in the development of antibiotic resistance in an antibiotic-free shrimp farming system.

Antibiotic selective pressure in aquaculture systems often results in the antibiotic resistance genes (ARGs) proliferation. Nonetheless, a paucity of data exists concerning the mechanisms of ARGs development in aquaculture systems without the influences of antibiotics. This study utilized metagenomic approaches to elucidate the dynamics and transfer mechanisms of ARGs throughout the aquaculture of Pacific white shrimp. A marked change in the resistome was observed throughout the aquaculture without antibiotics. The total ARGs relative abundance increased from 0.05 to 0.33 by day 90 of cultivation, with even higher in mixed wastewater (0.44). Both bacterial communities and mobile genetic elements play pivotal roles in the development of ARGs. Metagenome-assembled genomes showed enrichment of environmentally intrinsic ARGs on chromosomes including macB and mdtK. The plasmid-mediated horizontal transfer was recognized as a principal factor contributing to the rise of ARGs, particularly for tetG and floR, and this led to an escalation of resistance risk, peaking at a risks core of 35.43 on day 90. This study demonstrates that horizontal gene transfer plays a crucial role in ARGs development without antibiotic pressure, which can provide a theoretical foundation for controlling ARGs proliferation in aquaculture systems.