Water reuse within a circular economy: Long-read metagenomics reveals distinct residual resistome profiles across full-scale wastewater-reclamation systems in Murcia, Spain.

Water reuse is a key pillar of circular economy strategies, particularly in water-scarce regions, by preserving freshwater resources and supporting agricultural production. Current regulations promote the use of reclaimed water for irrigation, placing wastewater treatment plants (WWTPs) at the center of this transition. However, emerging risks related to antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) challenge the safety of reuse as these determinants may persist after treatment. This study evaluated the effectiveness of ozonation, sodium hypochlorite, UV-C irradiation, and membrane bioreactor (MBR) systems in reducing ARGs in effluents from the Region of Murcia (Spain) across five sampling campaigns between 2023 and 2024. Metagenomic sequencing identified ARGs associated with 17 antibiotic classes, with macrolide-, β-lactam-, and tetracycline-ARGs dominating. Total ARG abundance was lower in treated effluents than in influents, with the greatest percentage reduction in MBR system, although based on only two effluent samples. Selected macrolide-, sulfonamide-, tetracycline- and β-lactam-ARGs nevertheless remained detectable after treatment, with distinct gene-specific profiles among the wastewater-reclamation systems. Effluent samples differed in the taxonomic composition of ARG-carrying reads and in the detection of ARGs putatively co-localized on mobilome associated contigs. These findings show that total ARG reduction alone does not fully characterize wastewater-reclamation performance because substantial decreases in total ARG burden were accompanied by different residual resistome profiles. Combining ARG abundance, identity, potential hosts, and genomic context enables a fuller assessment of reclaimed-water treatment. These metagenomic associations indicate potential persistence and mobility but do not constitute direct evidence of horizontal gene transfer or quantitative environmental risk.