Long-read metagenomic sequencing reveals shifts in associations of antibiotic resistance genes with mobile genetic elements from sewage to activated sludge

Background: There is concern that the microbially rich activated sludge environment of wastewater treatment plants (WWTPs) may contribute to the dissemination of antibiotic resistance genes (ARGs). We applied long-read (nanopore) sequencing to profile ARGs and their neighboring genes to illuminate their fate in the activated sludge treatment by comparing their abundance, genetic locations, mobility potential, and bacterial hosts within activated sludge relative to those in influent sewage across five WWTPs from three continents. Results: The abundances (gene copies per Gb of reads, aka gc/Gb) of all ARGs and those carried by putative pathogens decreased 75-90% from influent sewage (192-605 gc/Gb) to activated sludge (31-62 gc/Gb) at all five WWTPs. Long reads enabled quantification of the percent abundance of ARGs with mobility potential (i.e., located on plasmids or co-located with other mobile genetic elements (MGEs)). The abundance of plasmid-associated ARGs decreased at four of five WWTPs (from 40-73 to 31-68%), and ARGs co-located with transposable, integrative, and conjugative element hallmark genes showed similar trends. Most ARG-associated elements decreased 0.35-13.52% while integrative and transposable elements displayed slight increases at two WWTPs (1.4-2.4%). While resistome and taxonomic compositions both shifted significantly, host phyla for chromosomal ARG classes remained relatively consistent, indicating vertical gene transfer via active biomass growth in activated sludge as the key pathway of chromosomal ARG dissemination. Conclusions: Overall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention.