Metagenomic profiling and transfer dynamics of antibiotic resistance determinants in a full-scale granular sludge wastewater treatment plant

In the One Health context, wastewater treatment plants (WWTPs) are central to safeguard water resources. Nonetheless, many questions remain about their effectiveness to prevent the dissemination of antimicrobial resistance (AMR). Most surveillance studies monitor the levels and removal of selected antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in intracellular DNA (iDNA) extracted from WWTP influents and effluents. The role of extracellular free DNA (exDNA) in wastewater is mostly overlooked. In this study, we analyzed the transfer of ARGs and MGEs in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge. We tracked the composition and fate of the iDNA and exDNA pools of influent, sludge, and effluent samples. Metagenomics was used to profile the microbiome, resistome, and mobilome signatures of iDNA and exDNA extracts. Selected ARGs and MGEs were analyzed by qPCR. From 2,840 ARGs identified, the genes arr-3 (2%) , tetC (1.6%) , sul1 (1.5%) , oqxB (1.2%), and aph(3”)-Ib (1.2%) were the most abundant among all sampling points and bioaggregates. Pseudomonas , Acinetobacter , Aeromonas , Acidovorax , Rhodoferax, and Streptomyces populations were the main hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% from exDNA potentially released by cell lysis during aeration in the reactor. MGEs and multiple ARGs were co-localized on the same extracellular genetic contigs. These can pose a risk for AMR dissemination by transformation into microorganisms of receiving water bodies. Total intracellular ARGs decreased 3-42% as a result of wastewater treatment. However, the ermB and sul1 genes increased by 2 and 1 log gene copies mL-1, respectively, in exDNA from influent to effluent. The exDNA fractions need to be considered in AMR surveillance, risk assessment, and mitigation. ![Figure][1] Highlights ### Competing Interest Statement The authors have declared no competing interest. * aac(3) : Ib Aminoglycoside 3’-N-acetyltransferase resistance gene AMR : Antimicrobial resistance aadA6 : Aminoglycoside (3’’) adenyltransferase resistance gene aadA11 : Aminoglycoside (3’’) (9) adenyltransferase resistance gene AGS : Aerobic granular sludge aph(3”)-Ib : Aminoglycoside 3’-phosphotransferase resistance gene aph(6)-Id : Aminoglycoside O-phosphotransferase resistance gene ARB : Antibiotic-resistant bacteria ARG : Antibiotic resistance gene arr-3 : Plasmid-encoded ribosyltransferase resistance gene BGC : Biosynthetic gene clusters COG : Clusters of Orthologous Genes exARG : Free-floating extracellular antibiotic resistance gene exDNA : Free-floating extracellular DNA HGT : Horizontal gene transfer iARG : Intracellular antibiotic resistance gene iDNA : Intracellular DNA MAG : Metagenome-assembled genome MGE : Mobile genetic element oqxB : Fluoroquinolone efflux pump membrane transporter gene qPCR : Quantitative polymerase chain reaction SBR : Sequential batch reactor sul1 : Sulfonamide resistant dihydropteroate synthase of Gram-bacteria, linked to class 1 integrons tetC : Tetracycline efflux pump gene WWT : Wastewater treatment WWTP : Wastewater treatment plant [1]: pending:yes