The Wastewater Resistome: A Shotgun Metagenomics Analysis of Urban Treatment Plants in Sicily

Background/Objectives: Antimicrobial resistance (AMR) in wastewater represents a valuable reservoir of information for wastewater-based epidemiology (WBE) and a major environmental and public health concern, as wastewater treatment plants (WWTPs) are recognized hotspots for the accumulation and dissemination of antimicrobial resistance genes (ARGs). Within the One Health framework, and to better understand the contribution to AMR spread and the potential of metagenomic surveillance, this study aimed to characterize the taxonomic, functional, and resistome profiles of three WWTPs in Sicily, specifically those located in Catania, Giarre, and Syracuse. Methods: Sixty-nine composite influent samples were collected between February 2022 and December 2023. Shotgun metagenomic sequencing was performed on the Illumina NovaSeq platform. Bioinformatic analyses were conducted to assess microbial community composition, functional pathways, and ARG prevalence across sites. Results: Dominant genera included Aliarcobacter, Bacteroides, and Acinetobacter. Site-specific taxonomic variations reflected differences in local microbial ecology. Functional profiling revealed enrichment in membrane-associated, ribosomal, and energy metabolism pathways, consistent with the expected functional redundancy of wastewater microbiomes. Resistome analysis detected a diverse and ubiquitous array of ARGs, dominated by β-lactam and macrolide resistance genes, followed by aminoglycoside, sulphonamide, and tetracycline classes. Conclusions: These findings highlight urban wastewater as a relevant reservoir and dissemination route for AMR and support the integration of metagenomic approaches into wastewater surveillance programs. By providing region-specific, integrated taxonomic, functional, and resistome data from Sicilian WWTPs, this study contributes to the growing body of evidence supporting WBE as a valuable tool for AMR monitoring and One Health-oriented risk assessment.