Resolving Resistome and Mobilome Dynamics in Wastewater Treatment Plants Using Long—Read Metagenomics

Wastewater treatment plants (WWTPs) are key reservoirs for antibiotic resistance genes (ARGs), particularly when linked to mobile genetic elements (MGEs) and specific microbial hosts. We applied Oxford Nanopore long-read sequencing using complementary contig- and read-based approaches to profile the resistome, mobilome, and host taxonomy in influent and effluent samples from WWTPs in Slovakia and Taiwan. Multidrug resistance was the dominant class in all samples, reaching 40.29–60.06% in Taiwanese and 20.00–35.56% in Slovak WWTPs, followed by MLS and tetracycline resistance. These differences reflect country-specific inputs: Taiwanese WWTPs receiving hospital effluent showed higher multidrug resistance, while Slovak WWTPs, fed by municipal and agricultural wastewater, were dominated by tetracycline resistance and Aliarcobacter cryaerophilus. In Taiwan, Acinetobacter baumannii carried multiple ARGs, including msrE and the regulatory gene ompR, co-localized with MGEs on plasmid- and chromosome-associated contigs. Clinically important Enterococcus faecium (Taiwan) and Staphylococcus pseudintermedius (Slovakia), both WHO-priority pathogens, were identified as hosts for MLS and multidrug resistance genes co-localized with MGEs. These findings suggest that integrating contig- and read-based long-read analyses improves ARG compartmentalization, MGE co-localization, and host assignment in wastewater environments beyond either approach alone.