Persistence of high-risk antimicrobial resistance genes in extracellular DNA along an urban wastewater-river continuum

Inadequate wastewater treatment can drive the spread of antimicrobial resistance (AMR), threatening ecosystems and human health. Extracellular DNA (exDNA) stabilizes antimicrobial resistance genes (ARGs) in the environment and facilitates horizontal gene transfer, yet its taxonomic structure and influence on AMR ecology remain poorly understood, especially in African aquatic systems. We profile exDNA-associated resistomes across nine South African wastewater treatment plants and receiving rivers, comparing single-stage activated sludge process (ASP-only) and combined ASP-biofilter systems. exDNA harbors high-risk mobile ARGs conferring resistance to last-resort antibiotics, with enrichment in effluents and downstream rivers. Surprisingly, upstream river samples also carry abundant ARGs, indicating cumulative inputs from multiple environmental reservoirs. ARGs are mainly associated with Pseudomonadota and Bacteroidota, suggesting that exDNA constitutes an ecologically distinct AMR reservoir dominated by key taxa. These findings underscore the need to integrate exDNA into AMR surveillance and highlight its broader role in microbial adaptation within freshwater environments.