# Metagenomic Assessment of Full-Scale Wastewater Treatment Plants Identifies Sentinel Antibiotic Resistance Gene Families for Monitoring Reclaimed Wastewater and Treated Sludge

## TL;DR

This study identifies key antibiotic resistance genes in wastewater treatment plants to help monitor water and sludge under new EU regulations.

## Contribution

The paper introduces a framework combining metagenomics and targeted analysis to identify sentinel antibiotic resistance genes for monitoring reclaimed water and sludge.

## Key findings

- Reclaimed effluents had lower AMR exposure levels (<0.5 ARGs/cell) compared to secondary effluents.
- Membrane bioreactor systems reduced transferable mobile genetic elements compared to physical separation methods.
- Clinically relevant ARGs like aadA, ermB, and bla_OXA‑129 were identified as sentinel markers for monitoring.

## Abstract

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.