Targeted Enrichment Sequencing for Wastewater Resistome Surveillance: A Proof-of-Concept Study Across Treatment Stages and Seasons.

The detection of antibiotic resistance genes (ARGs) in wastewater is commonly based on targeted PCR assays, limiting analysis to predefined genes. Consequently, the structure of wastewater resistomes often remains poorly characterised. Targeted enrichment sequencing offers an alternative approach for comprehensive resistome profiling. A panel targeting 2786 ARGs was applied to eight influent-effluent wastewater samples collected over one year from a large wastewater treatment plant in central Italy. Sequencing data were analysed at both the ARG and Antimicrobial Agent Categories (AACs) levels, and seasonal variation was explored using a ratio-based clustering approach. Overall, 743 ARGs were detected in both influents and effluents, with 84% of them. The resistome showed a strongly uneven structure, with a limited number of highly abundant ARGs accounting for ~60% of total abundance, while more than 500 genes were detected at ultra-low abundance. Thirty-five AACs were represented. Seasonal resistome profiles were generally stable, although winter samples showed a distinct enrichment pattern involving multiple ARGs, particularly tetracycline-associated ARGs. This structure was evident in influents becoming less defined in effluents. Targeted enrichment sequencing provided a broader view of the wastewater resistome, revealing a large pool of ARGs and seasonal patterns that would likely remain undetected using conventional PCR-based approaches.