Antimicrobial resistance dissemination via horizontal gene transfer is constrained in stratified waters.

Aquatic ecosystems are major reservoirs of antibiotic resistance genes (ARGs) and hubs for microbial interactions that can facilitate their spread through horizontal gene transfer (HGT). While mobile genetic elements (MGEs), including plasmids and viruses, are recognized as important drivers of ARG mobility, the extent to which water column stratification constrains their vertical dissemination remains unresolved. Here, we analysed depth-resolved metagenomic data from stratified freshwater and marine systems to assess the role of HGT in ARG spread. We found that ARG diversity is consistently lower in marine than freshwater environments and that only a small fraction of ARGs is mobilized by plasmids and viruses. Importantly, we detected no evidence for recent HGT-mediated dissemination of ARGs across depth layers, despite genetic compatibility among co-occurring bacteria. Instead, ARGs appear largely confined to lineage-specific inheritance and within-layer persistence. These findings suggest that stratification acts as a barrier, limiting vertical ARG transfer while promoting within-layer accumulation. Given projections of intensified and prolonged stratification under climate change, our results imply reduced vertical connectivity of ARGs in aquatic environments, with potential consequences of further mitigation in its dynamics by water stratification.