Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome in wastewater.

In-depth studies of the microbiome and mobile resistome profile of different environments is central to understanding the role of the environment in antimicrobial resistance (AMR), which is one of the urgent threats to global public health. In this study, we demonstrated the use of a rapid (and easily portable) sequencing approach coupled with user-friendly bioinformatics tools, the MinION (Oxford Nanopore Technologies), on the evaluation of the microbial as well as mobile metal and antibiotic resistome profile of semi-rural wastewater. A total of 20 unique phyla, 43 classes, 227 genera, and 469 species were identified in samples collected from the Amherst Wastewater Treatment Plant, both from primary and secondary treated wastewater. Alpha diversity indices indicated that primary samples were significantly richer and more microbially diverse than secondary samples. A total of 1041 ARGs, 68 MRGs, and 17 MGEs were detected in this study. There were more classes of AMR genes in primary than secondary wastewater, but in both cases multidrug, beta-lactam and peptide AMR predominated. Of note, OXA β-lactamases, some of which are also carbapenemases, were enriched in secondary samples. Metal resistance genes against arsenic, copper, zinc and molybdenum were the dominant MRGs in the majority of the samples. A larger proportion of resistome genes were located in chromosome-derived sequences except for mobilome genes, which were predominantly located in plasmid-derived sequences. Genetic elements related to transposase were the most common MGEs in all samples. Mobile or MGE/plasmid-associated resistome genes that confer resistance to last resort antimicrobials such as carbapenems and colistin were detected in most samples. Worryingly, several of these potentially transferable genes were found to be carried by clinically-relevant hosts including pathogenic bacterial species in the orders Aeromonadales, Clostridiales, Enterobacterales and Pseudomonadales. This study demonstrated that the MinION can be used as a metagenomics approach to evaluate the microbiome, resistome, and mobilome profile of primary and secondary wastewater.