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

Abstract

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.

Globally distributed mining-impacted environments are underexplored hotspots of multidrug resistance genes

“…MEGARes 2.0 integrated BacMet database [ 39 ] to a well-organized hierarchical classification ontology. Using the UBLAST algorithm, genes were screened with minimum query coverage of 40%, identity of 70% and e value below 1 × 10 −5 and top hits were classified according to the MRG hierarchy [ 40 ].…”

“…Reference sequences from ISfinder (accessed on 18 September 2019) [ 41 ], INTEGRALL (20 September 2019) [ 42 ] and the Transposon Registry (11 October 2019) [ 43 ] were integrated and de-replicated using CD-HIT at a criterion of 95% identity over 90% of the shorter gene length to construct a database of mobile genetic elements (MGEs, Table S5 ), which included 10,829 transposases, 2615 integrases containing 88 class 1 integron integrase ( intI1 ) sequences, 848 resolvases, and 526 recombinases. Genes were aligned against the self-constructed MGEs database using blastx implemented in DIAMOND v. 2.0.9 [ 44 ] at a criterion of minimum query coverage of 40%, minimal identity of 25%, and e-value below 1 × 10 −5 , and top hits were annotated as corresponding MGEs [ 40 , 45 ]. The PlasFlow software [ 46 ] with default setting was used to predict plasmid sequences for all ARG-carrying contigs.…”

Occurrence and removal of antibiotics, antibiotic resistance genes, and bacterial communities in hospital wastewater

“…bla OXA-1 is frequently detected (57.9%) in Escherichia coli resistant to cefotaxime in hospital wastewater (Adegoke et al 2020). Mobile genetic elements or plasmid-associated OXA β-lactamase genes were found to be significantly enriched in the plasmid reads of secondary wastewater samples (Barlow and Hall 2002;Martin et al 2020). To provide a real picture of ARG pollution in wastewater, the ARG pollution in this study and data from other parts of the world is summarized in Table A.9.…”

Antibiotic resistance: Time of synthesis in a post-genomic age

“…The application of sequencing technologies allowing long reads seems to be a better approach to solve this problem [219] . While PacBio [220] is accurate enough to distinguish among alleles, Nanopore [221] , and associated analytic tools as NanoGalaxy, which can be extremely useful to analyse arranges of ARGs, mobile elements [222] , [223] , or the presence of acquired ARGs in a given microorganism (see below), are not accurate enough for the analysis of alleles presenting few differences in their sequences. Nevertheless, the portability and the fast generation of results of Nanopore devices make this technology an excellent choice for in situ metagenomic studies [224] .…”