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(R) 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/482492v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1a5eedcorg.highwire.dtl.DTLVardef@e4da78org.highwire.dtl.DTLVardef@c324bforg.highwire.dtl.DTLVardef@1a18138_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA DNA database from an AGS reactor was constructed to study the system resistome, mobilome, and microbiome. C_LIO_LIThe genera Pseudomonas and Rhodoferax were the predominant ARG carriers in the system. C_LIO_LIMGEs and ARGs often co-localize on contigs recovered from the exDNA of the effluent. C_LIO_LIAGS plants are efficient at reducing ARB. C_LIO_LIThe exDNA is an underestimated DNA fraction containing ARGs in the effluent. C_LI