High-Throughput Multilocus Sequence Typing: Bringing Molecular Typing to the Next Level

Boers, Stefan A.; Reijden, Wil A. van der; Jansen, Ruud

doi:10.1371/journal.pone.0039630

Cited by 94 publications

(63 citation statements)

References 32 publications

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“…Though AFLP and MLST are useful techniques for taxonomy and population genetics, they have limited applicability in an epidemiological investigation because of the relatively high cost of multilocus sequencing (29). Furthermore, the results of AFLP fingerprints are difficult to objectively interpret due to minor band variations which cause problems with long-term reproducibility and interlaboratory comparisons (24).…”

Section: Discussionmentioning

confidence: 99%

Identification and Typing of Isolates of Cyphellophora and Relatives by Use of Amplified Fragment Length Polymorphism and Rolling Circle Amplification

et al. 2013

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hThe species diversity and identification of black fungi belonging to Cyphellophora and Phialophora, which colonize and infect human skin and nails, were studied using amplified fragment length polymorphism (AFLP). A total of 76 Cyphellophora and Phialophora isolates were evaluated, and their delimitation was compared to earlier studies using multilocus sequencing. The results of the AFLP analysis and sequencing were in complete agreement with each other. Seven species-specific padlock probes for the most prevalent species were designed on the basis of the ribosomal DNA internal transcribed spacer region, and identification of the respective species could easily be achieved with the aid of rolling circle amplification.

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Section: Discussionmentioning

confidence: 99%

Identification and Typing of Isolates of Cyphellophora and Relatives by Use of Amplified Fragment Length Polymorphism and Rolling Circle Amplification

et al. 2013

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“…For MLST typing, the hiMLST method of Boers et al (2012) using a 454 Junior sequencer (Roche) was employed to sequence the 10 housekeeping genes glp, pntA, pyrC, mdh, gyrB, metG, purM, dtdS, lysA and tnaA as described by Broza et al (2012). The oligonucleotides used for PCR amplification reported in the standardized MLST scheme by Broza et al (2012) were equipped with universal tails for the hiMLST protocol.…”

Section: Bacteriology: Typing and (Molecular) Identificationmentioning

confidence: 99%

“…These strains were compared with the isolated human strain (Dijkstra et al 2009) from the eel farmer suffering from necrotic fasciitis. Strains were identified as V. vulnificus by 16S rRNA gene sequence analysis, and typed by biochemical tests (biotyping) (Tison et al 1982, Bisharat et al 1999, REP-PCR (Diversilab ® ) typing and highthroughput multilocus sequence typing (hiMLST; Sanjuán et al 2011, Boers et al 2012). …”

Section: Introductionmentioning

confidence: 99%

Vibrio vulnificus outbreaks in Dutch eel farms since 1996: strain diversity and impact

Haenen

Zanten²,

Jansen³

et al. 2014

Dis. Aquat. Org.

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Vibrio vulnificus is a potentially zoonotic bacterial pathogen of fish, which can infect humans (causing necrotic fasciitis). We analysed 24 V. vulnificus isolates (from 23 severe eel disease outbreaks in 8 Dutch eel farms during 1996 to 2009, and 1 clinical strain from an eel farmer) for genetic correlation and zoonotic potential. Strains were typed using biotyping and molecular typing by high-throughput multilocus sequence typing (hiMLST) and REP-PCR (Diversilab ® ). We identified 19 strains of biotype 1 and 5 of biotype 2 (4 from eels, 1 from the eel farmer), that were subdivided into 8 MLST types (ST) according to the international standard method. This is the first report of V. vulnificus biotype 1 outbreaks in Dutch eel farms. Seven of the 8 STs, of unknown zoonotic potential, were newly identified and were deposited in the MLST database. The REP-PCR and the MLST were highly concordant, indicating that the REP-PCR is a useful alternative for MLST. The strains isolated from the farmer and his eels were ST 112, a known potential zoonotic strain. Antimicrobial resistance to cefoxitin was found in most of the V. vulnificus strains, and an increasing resistance to quinolones, trimethoprim + sulphonamide and tetracycline was found over time in strain ST 140. Virulence testing of isolates from diseased eels is recommended, and medical practitioners should be informed about the potential risk of zoonotic infections by V. vulnificus from eels for the prevention of infection especially among high-risk individuals. Additional use of molecular typing methods such as hiMLST and Diversilab ® is recommended for epidemiological purposes during V. vulnificus outbreaks.

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“…Furthermore, the flexibility of the method allows the modification and addition of extra markers in case they prove to be relevant for specific populations. This advantage may become very important, as progress in the generation of sequence data (44) can substantially increase the number of target polymorphisms useful for B. cenocepacia genotyping. An online SNP platform associated with the former and successful MLST database may complement and facilitate the analysis of the genotyping data available for this species.…”

Section: Discussionmentioning

confidence: 99%

SNaPBcen : a Novel and Practical Tool for Genotyping Burkholderia cenocepacia

et al. 2013

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c Burkholderia cenocepacia is the most prevalent and feared member of the Burkholderia cepacia complex in lung infections of cystic fibrosis (CF). Genotyping and monitoring of long-term colonization are critical at clinical units; however, the differentiation of specific lineages performed by multilocus sequence typing (MLST) is still limited to a small number of isolates due to the high cost and time-consuming procedure. The aim of this study was to optimize a protocol (the SNaPBcen assay) for extensive bacterial population studies. The strategy used for the SNaPBcen assay is based on targeting single nucleotide polymorphisms (SNPs) located in MLST genes instead of sequencing full MLST sequences. Nonpolymorphic and redundant MLST positions were eliminated, and a set of 24 polymorphisms included in the SNaPBcen assay ensures a high-resolution genomic characterization. These polymorphisms were identified based on the comparative analysis of 137 B. cenocepacia MLST profiles available online (http://pubmlst.org/bcc/). The group of 81 clinical isolates of B. cenocepacia examined in this study using the SNaPBcen assay revealed 51 distinct profiles, and a final discriminatory power of 0.9997 compared with MLST was determined. The SNaPBcen assay was able to reveal isolates with microvariations and the presence of multiple clonal variants in patients chronically colonized with B. cenocepacia. Main phylogenetic subgroups IIIA, IIIB, and IIIC of B. cenocepacia could be separated by the Gl94R polymorphism included in the panel. The SNaPBcen assay proved to be a rapid and robust alternative to the standard MLST for B. cenocepacia, allowing the simultaneous analysis of multiple polymorphisms following amplification and mini-sequencing reactions.

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High-Throughput Multilocus Sequence Typing: Bringing Molecular Typing to the Next Level

Cited by 94 publications

References 32 publications

Identification and Typing of Isolates of Cyphellophora and Relatives by Use of Amplified Fragment Length Polymorphism and Rolling Circle Amplification

Identification and Typing of Isolates of Cyphellophora and Relatives by Use of Amplified Fragment Length Polymorphism and Rolling Circle Amplification

Vibrio vulnificus outbreaks in Dutch eel farms since 1996: strain diversity and impact

SNaPBcen : a Novel and Practical Tool for Genotyping Burkholderia cenocepacia

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