Abstract Description

Wastewater treatment plants (WWTPs) are crucial for mitigating antimicrobial resistance (AMR) but can also facilitate its propagation and spread into natural ecosystems. Mobile antimicrobial resistance genes (ARGs) are of particular concern in these environments, as they mediate AMR spread across taxa and compartments. However, the ecological factors shaping ARG persistence and mobility in aquatic systems remain poorly understood, especially in understudied African settings. We investigated the chemical and environmental factors shaping ARG diversity and mobility across nine WWTP-river systems in South Africa. Shotgun metagenomic sequencing was performed on both intracellular and extracellular DNA (exDNA) fractions, alongside physicochemical profiling of nutrient and heavy metal concentrations. While WWTPs significantly reduced total ARG richness (p ≤ 0.05), removal was incomplete. High-risk and mobile ARGs conferring resistance to critical antibiotics, including colistin, tigecycline, carbapenems, cephalosporins, aminoglycosides and macrolides, were enriched in WWTP effluents and downstream rivers, particularly in sediments. Notably, exDNA emerged as a distinct and ecologically relevant ARG reservoir in effluents and river systems, mainly dominated by plasmid-borne genes from Pseudomonadota and Bacteroidota. Metagenome-assembled genomes showed that antibiotic-resistant bacteria (ARB) and virulence gene carriers were more prevalent in water samples than sediments, reflecting compartment-specific ecological niches and transmission dynamics. Environmental parameters, including pH, temperature, salinity, nutrients and heavy metals, were significantly correlated with ARG abundance and distribution, indicating strong selective pressures. Resistance determinants also exhibited niche partitioning: macrolide, beta-lactam and aminoglycoside ARGs were widespread across all sample types, whereas ARGs for colistin, quinolones and nitroimidazoles were mostly confined to the water column. To our knowledge, this is the first study of exDNA-associated resistomes across an African WWTP-river network, demonstrating that this fraction contributes to AMR persistence by acting as a stable reservoir for mobile ARGs. Importantly, the association of ARG abundance with physicochemical factors suggests that climate- and pollutant-driven co-selection, rather than antibiotic pressure alone, may be a key mechanism sustaining AMR across environmental compartments. Our findings call for ecologically integrated, compartment-specific AMR surveillance strategies that incorporate exDNA and environmental drivers to more accurately track and mitigate resistance in WTTPs and freshwater systems.