High-Throughput Profiling of Antibiotic Resistance Genes and Mobile Genetic Elements in Wastewater and Sludge from a Municipal Wastewater Treatment Plant

Antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) are widespread in wastewater treatment plants (WWTPs), but their occurrence in high-solids matrices remains less characterized. This study used high-throughput quantitative PCR (HT-qPCR) to investigate ARG and MGE diversity, relative abundance, resistance mechanisms, and co-occurrence patterns in raw influent (IN), activated sludge (BR), and anaerobic digestate (AD) from a full-scale municipal WWTP. Among the 306 assays targeting ARGs, 197 were detected in at least one of the investigated matrices, with the highest number of detected targets observed in IN (189), followed by BR (132) and AD (120). Aminoglycoside, multidrug resistance (MDR), macrolide–lincosamide–streptogramin B (MLSB), tetracycline, and β-lactam resistance genes were the most diverse groups. Antibiotic deactivation was the dominant resistance mechanism (47.4%), followed by efflux (26.5%) and cellular protection (20.8%). Among 48 MGE assays and four integron-associated targets, 29 MGEs and all integron markers were detected. Co-occurrence analysis identified 56 connected ARGs/MGEs nodes distributed across nine modules. Overall, the results revealed distinct ARG and MGE profiles across the investigated wastewater treatment matrices, with lower numbers of detectable ARG targets in activated sludge and anaerobic digestate than in raw influent. Nevertheless, numerous resistance-associated genetic determinants remained detectable in both sludge-associated matrices, highlighting the importance of including these compartments in wastewater resistome surveillance.