Rapid resistome mapping using nanopore sequencing

Helm, Eric van der; Imamovic, Lejla; Ellabaan, Mostafa Mostafa Hashim; Schaik, Willem van; Koza, Anna; Sommer, Morten Otto Alexander

doi:10.1093/nar/gkw1328

Cited by 57 publications

(49 citation statements)

References 36 publications

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“…Our results demonstrate that Nanopore sequencing can be employed to both accurately and rapidly predict phenotypic AST profiles. Our study builds off previously published proof-of-concept or early insight studies applying Nanopore sequencing for the detection of antimicrobial resistance genes (8,(10)(11)(12)(13)(14)(15)(16). We found an overall agreement of 92% between genotypic results and comprehensive AST results using a Nanopore assembly-based approach.…”

Section: Discussionsupporting

confidence: 68%

Applying Rapid Whole-Genome Sequencing To Predict Phenotypic Antimicrobial Susceptibility Testing Results among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates

Tamma

Fan

Bergman

et al. 2019

Antimicrob Agents Chemother

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Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. Here, we sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole-genome sequencing in predicting AST results. Using a cohort of 40 clinical isolates (21 carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae, 10 non-carbapenemase-producing carbapenem-resistant K. pneumoniae, and 9 carbapenem-susceptible K. pneumoniae isolates), three separate sequencing and analysis pipelines were performed, as follows: (i) a real-time Nanopore analysis approach identifying acquired AMR genes, (ii) an assembly-based Nanopore approach identifying acquired AMR genes and chromosomal mutations, and (iii) an approach using short-read correction of Nanopore assemblies. The short-read correction of Nanopore assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results. With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 h from subcultured isolates. Assemblies sufficient for full resistance gene and single-nucleotide polymorphism annotation were available within 14 h from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40 K. pneumoniae isolates was 77% (range, 30% to 100%) and 92% (range, 80% to 100%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could shorten the median time to effective antibiotic therapy by 20 h and 26 h, respectively, compared to standard AST. Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms and to predict AST results for K. pneumoniae isolates. Bioinformatics improvements enabling real-time alignment, coupled with rapid extraction and library preparation, will further enhance the accuracy and workflow of the Nanopore real-time approach.

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Section: Discussionsupporting

confidence: 68%

Applying Rapid Whole-Genome Sequencing To Predict Phenotypic Antimicrobial Susceptibility Testing Results among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates

Tamma

Fan

Bergman

et al. 2019

Antimicrob Agents Chemother

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“…Subsequently, we mapped sequence of the metagenomic gene catalog to CARD database to predict resistomes [60]. To ensure the accuracy of ARGs, we selected 80% identity cut off as the search criteria [61][62][63].…”

Section: Dna Sequence Assembly and Annotationmentioning

confidence: 99%

Metagenomic analysis reveals the microbiome and resistome in migratory birds

et al. 2020

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Background: Antibiotic-resistant pathogens pose high risks to human and animal health worldwide. In recent years, the role of gut microbiota as a reservoir of antibiotic resistance genes (ARGs) in humans and animals has been increasingly investigated. However, the structure and function of the gut bacterial community, as well as the ARGs they carry in migratory birds remain unknown. Results: Here, we collected samples from migratory bird species and their associated environments and characterized their gut microbiomes and resistomes using shotgun metagenomic sequencing. We found that migratory birds vary greatly in gut bacterial composition but are similar in their microbiome metabolism and function. Birds from the same environment tend to harbor similar bacterial communities. In total, 1030 different ARGs (202 resistance types) conferring resistance to tetracycline, aminoglycoside, β-lactam, sulphonamide, chloramphenicol, macrolide-lincosamide-streptogramin (MLS), and quinolone are identified. Procrustes analysis indicated that microbial community structure is not correlated with the resistome in migratory birds. Moreover, metagenomic assembly-based host tracking revealed that most of the ARG-carrying contigs originate from Proteobacteria. Co-occurrence patterns revealed by network analysis showed that emrD, emrY, ANT(6)-Ia, and tetO, the hubs of ARG type network, are indicators of other co-occurring ARG types. Compared with the microbiomes and resistomes in the environment, migratory birds harbor a lower phylogenetic diversity but have more antibiotic resistance proteins. Interestingly, we found that the mcr-1 resistance gene is widespread among different birds, accounting for 50% of the total samples. Meanwhile, a large number of novel β-lactamase genes are also reconstructed from bird metagenomic assemblies based on fARGene software. Conclusions: Our study provides a comprehensive overview of the diversity and abundance of ARGs in migratory birds and highlights the possible role of migratory birds as ARG disseminators into the environment.

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“…The availability of portable, real-time, long-read sequencing platforms presents new opportunities and challenges for pathogenome and resistome monitoring 30,31 . In this study, we combined extensive shortread shotgun metagenomic sequencing of multiple sites, wards and timepoints (355 samples), with enrichment and nanopore sequencing of antibiotic-resistant mixed cultures (1661 samples), to provide the most extensive genetic characterization of hospital environments to date.…”

Section: Introductionmentioning

confidence: 99%

Cartography of opportunistic pathogens and antibiotic resistance genes in a tertiary hospital environment

Chng

Bertrand

et al. 2019

Preprint

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There is growing attention surrounding hospital acquired infections (HAIs) due to high associated healthcare costs, compounded by the scourge of widespread multi-antibiotic resistance. Although hospital environment disinfection is well acknowledged to be key for infection control, an understanding of colonization patterns and resistome profiles of environment-dwelling microbes is currently lacking. We report the first extensive genomic characterization of microbiomes (355), common HAI-associated microbes (891) and transmissible drug resistance cassettes (1435) in a tertiary hospital environment based on a 2-timepoint sampling of 179 sites from 45 beds. Deep shotgun metagenomic sequencing unveiled two distinct ecological niches of microbes and antibiotic resistance genes characterized by biofilm-forming and human microbiome influenced environments that display corresponding patterns of divergence over space and time. To study common nosocomial pathogens that were typically present at low abundances, a combination of culture enrichment and long-read nanopore sequencing was used to obtain thousands of high contiguity genomes (2347) and closed plasmids (5910), a significant fraction of which (>58%) are not represented in current sequence databases. These high-quality assemblies and metadata enabled a rich characterization of resistance gene combinations, plasmid architectures, and the dynamic nature of hospital environment resistomes and their reservoirs. Phylogenetic analysis identified multidrug resistant clonal strains as being more widely disseminated and stably colonizing across hospital sites. Further genomic comparisons with clinical isolates across multiple species supports the hypothesis that multidrug resistant strains can persist in the hospital environment for extended periods (>8 years) to opportunistically infect patients. These findings highlight the importance of characterizing antibiotic resistance reservoirs in the hospital environment and establishes the feasibility of systematic genomic surveys to help target resources more efficiently for preventing HAIs.

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Rapid resistome mapping using nanopore sequencing

Cited by 57 publications

References 36 publications

Applying Rapid Whole-Genome Sequencing To Predict Phenotypic Antimicrobial Susceptibility Testing Results among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates

Applying Rapid Whole-Genome Sequencing To Predict Phenotypic Antimicrobial Susceptibility Testing Results among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates

Metagenomic analysis reveals the microbiome and resistome in migratory birds

Cartography of opportunistic pathogens and antibiotic resistance genes in a tertiary hospital environment

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