Metagenomic analysis of urban wastewater resistome and mobilome: Implications for Reclaimed Water Safety in Agriculture.

The reuse of reclaimed wastewater for agricultural irrigation is a promising solution in regions experiencing water scarcity, yet it raises concerns about the persistence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). This study employed metagenomic analyses to characterize the resistome and mobilome in influent, effluent, and sludge samples from three urban wastewater treatment plants (WWTPs) in Barcelona, Spain. Weekly samples were collected over three weeks and analyzed using high-throughput DNA sequencing and UHPLC-qTOF to quantify ARGs, MGEs, and antibiotic concentrations. The results revealed high levels of ARGs across all WWTPs, with ARG removal efficiencies ranging from 2.34 to 2.43 log reductions and antibiotic removals between 50–99%. Despite treatment, effluents retained moderate levels of ARGs, some of which were associated with human activity, high mobility, and pathogenicity. These findings suggest potential risks related to the use of reclaimed water in agriculture and underscore the need for robust risk assessment and management strategies. Structural equation modelling and source tracking further elucidated factors influencing ARG persistence. This study contributes critical insights for improving water reuse safety and informing policy on wastewater treatment standards.