Benchmarking of Methods for Genomic Taxonomy

Larsen, Mette Voldby; Cosentino, Salvatore; Lukjancenko, Oksana; Saputra, Dhany; Rasmussen, Simon; Hasman, Henrik; Sicheritz-Pontén, Thomas; Aarestrup, Frank Møller; Ussery, David W.; Lund, Ole

doi:10.1128/jcm.02981-13

Cited by 282 publications

(208 citation statements)

References 51 publications

(56 reference statements)

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“…KmerFinder is a novel program/database for rapid species identifica-tion using WGS data (36). Briefly, 1,647 (in a later update 5,029) complete bacterial genomes were downloaded from NCBI, and each k-mer (k ϭ 16) with the prefix ATGAC was saved in a database using an in-house script.…”

Section: Methodsmentioning

confidence: 99%

Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

et al. 2014

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c Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens. Bacterial pathogens still pose a major threat to public health, and in order to limit their spread and prevent infectious disease outbreaks, accurate and rapid diagnostics and classification of isolates are of great importance. In current routine practice, isolation and identification are mostly performed at clinical microbiological laboratories, and verification and further characterization are performed for a few selected pathogens at national, or regional, reference laboratories, using a variety of species-specific methods. Typing and surveillance of bacterial pathogens rely mainly on well-established, standardized phenotypic and molecular typing methods, such as serotyping and pulsed-field gel electrophoresis (PFGE) (1, 2). However, to obtain sufficient discrimination between isolates, it is typically necessary to combine typing results from several different typing techniques, both phenotypic and genotypic. As a result, it is laborious, time-consuming, and expensive to perform proper typing for surveillance and outbreak detection.As the cost of whole-genome sequencing (WGS) has decreased and benchtop sequencing machines enable fast turnaround, it has become increasingly attractive...

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Section: Methodsmentioning

confidence: 99%

Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

et al. 2014

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“…The challenge now at stake for genomic Microbial taxonomy is to examine how the existing genomic databases, bioinformatics tools, and access facilities may be further developed into prototypes to be further tested and discussed. Automated methods such as the ones benchmarked by Larsen et al (2014) will enable the use of WGS for higher resolution and more phylogenetically accurate classifications. It has been noticed that to date, microbial taxonomy has barely taken the wealth of information contained in completed sequenced genomes into account .…”

Section: Statements Arguing In Favor Of a Genomic Microbial Taxonomymentioning

confidence: 99%

Microbial taxonomy in the post-genomic era: Rebuilding from scratch?

et al. 2014

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“…Assembled genomes were uploaded to the online bioinformatics tools KmerFinder v2.0, ResFinder v2.1, and MLST v1.7 (Center for Genomic Epidemiology, Technical University of Denmark, Lingby, Denmark) (32)(33)(34). Species identification was based on the "winner takes it all" scoring method of KmerFinder.…”

Section: Esbl-e Isolatesmentioning

confidence: 99%

Whole-Genome Multilocus Sequence Typing of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae

et al. 2016

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e Molecular typing has become indispensable in the detection of nosocomial transmission of bacterial pathogens and the identification of sources and routes of transmission in outbreak settings, but current methods are labor-intensive, are difficult to standardize, or have limited resolution. Whole-genome multilocus sequence typing (wgMLST) has emerged as a whole-genome sequencing (WGS)-based gene-by-gene typing method that may overcome these limitations and has been applied successfully for several species in outbreak settings. In this study, genus-, genetic-complex-, and species-specific wgMLST schemes were developed for Citrobacter spp., the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae and used to type a national collection of 1,798 extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E) isolates obtained from patients in Dutch hospitals. Genus-, genetic-complex-, and species-specific thresholds for genetic distance that accurately distinguish between epidemiologically related and unrelated isolates were defined for Citrobacter spp., the E. cloacae complex, E. coli, and K. pneumoniae. wgMLST was shown to have higher discriminatory power and typeability than in silico MLST. In conclusion, the wgMLST schemes developed in this study facilitate high-resolution WGS-based typing of the most prevalent ESBL-producing species in clinical practice and may contribute to further elucidation of the complex epidemiology of antimicrobial-resistant Enterobacteriaceae. wgMLST opens up possibilities for the creation of a Web-accessible database for the global surveillance of ESBL-producing bacterial clones. The continuing global spread of extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E) constitutes a major public health threat in both health care and community settings (1-4). Extended-spectrum beta-lactamases (ESBL) confer resistance to the majority of beta-lactam antibiotics, including third-generation cephalosporins, which limits the options for antimicrobial therapy and results in increased morbidity and mortality and health care costs (5, 6). Infection control guidelines recommend prevention of the spread of ESBL-E in health care settings (7,8). Molecular typing has become an important tool in infection control, as it enables the detection of nosocomial transmission of bacterial pathogens and the identification of sources and routes of transmission in outbreak settings. Different molecular typing methods have been used, ranging from fingerprintbased methods, like pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP), to sequencebased methods, like multilocus sequence typing (MLST) (9-11). Although widely adopted, PFGE is labor-intensive and difficult to standardize and has limited interlaboratory reproducibility (12)(13)(14). AFLP is less time-consuming than PFGE but may have lower discriminatory power for typing of Enterobacteriaceae (13, 15). MLST targets 7 or 8 housekeeping genes, depending on the Ent...

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Benchmarking of Methods for Genomic Taxonomy

Cited by 282 publications

References 51 publications

Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

Microbial taxonomy in the post-genomic era: Rebuilding from scratch?

Whole-Genome Multilocus Sequence Typing of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae

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