Liam Brown, Anna Marizzi, Carles M Borrego, Giulia Gionchetta, Zhao Zhengzheng, Rodrigo B. Carneiro, Pablo Gago-Ferrero, Víctor Matamoros, Jessica Subirats
High Resolution Image Download MS PowerPoint Slide The new European (EU) regulation on water reuse explicitly incorporates antimicrobial resistance (AMR) into routine monitoring and risk management, creating an urgent need to define target antibiotic resistance genes (ARGs) for reclaimed irrigation water and agricultural sludge. However, existing global data largely focus on secondary effluents, providing little actionable evidence for reuse-oriented systems. Here, we present the first integrated framework combining targeted antibiotic residue analysis with shotgun metagenomics of the resistome, mobilome, and microbiome across full-scale reuse-oriented wastewater treatment plants (WWTPs) in Southern Europe to identify sentinel antibiotic resistance families for monitoring. Reclaimed effluents exhibited lower AMR exposure levels than those typically reported for secondary effluents (<0.5 ARGs/cell), while mobile genetic element (MGE) abundances were comparable to secondary effluents (1–2 MGEs/cell). Effluent communities differed by WWTP configuration: membrane bioreactor combined with ultrafiltration favored nutrient-removal/oxidative-stress taxa and reduced transferable MGEs, whereas plants relying on physical separation (sand filtration or reverse osmosis) retained fecal-associated taxa and MGEs. Specific clinically relevant ARGs persisted after treatments, including aadA and aph(3′’)-Ibs (resistance to aminoglycosides), ermB and mphA (resistance to macrolides), and bla OXA-129 (resistance to beta-lactams), which we identify as sentinel markers for monitoring reclaimed water and sludge. We advance a generalizable two-step framework, metagenomic discovery to identify sentinel markers, followed by targeted assays for streamlined surveillance, that provides the first operational blueprint for integrating AMR into water reuse management under the EU regulation.