Abstract Description

Wastewater treatment plants (WWTPs) are a critical hotspot for the environmental evolution and dissemination of antimicrobial resistance genes (ARGs). In these environments, mobile genetic elements (MGEs) mediate the horizontal gene transfer (HGT) of ARGs, yet their role in the mobility of the resistome across the WWTP-continuum, particularly under short term temporal fluctuations, remains understudied. In this study, we conducted temporal sampling every six hours over 7 consecutive days across a WWTP-river continuum in Tshwane, South Africa, collecting samples from industrial and domestic influent, effluent and river sites upstream and downstream of the WWTP. ARG-MGE associations were characterized by metagenomic profiling. Our findings showed strong coupling between ARGs and MGEs, with integration and insertion elements being the most abundant. These MGEs were associated with tetracyclines, aminoglycosides, and cephalosporins, which demonstrated their consistent coverage at all sampling times. Phyla such as Proteobacteria, Pseudomonadota hosted these genes, which could be linked to their prevalence, while some genes, such as colistin, quinolone, and rifamycin, were host-restricted, pointing to their lower prevalence. Overall, the findings suggest that short-term temporal dynamics and ecological mixing influence resistome composition and potential HGT events in WWTPs rather than bacterial community composition alone. Further, we highlight the potential for of specific integration elements such as tpnA potential markers for the mobility risk in AMR assessment.