High-resolution melting genotyping of enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms

Steven Tong, Shirley Xie, Leisha Jade Richardson, Susan Ballard, Farshid Dakh, Elizabeth Grabsch, Lindsay Grayson, Benjamin Howden, Paul Johnson, Philip Giffard

    Research output: Contribution to journalArticleResearchpeer-review

    Abstract

    We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure. � 2011 Tong et al.
    Original languageEnglish
    Article numbere29189
    Pages (from-to)1-8
    Number of pages8
    JournalPLoS One
    Volume6
    Issue number12
    DOIs
    Publication statusPublished - 2011

    Fingerprint

    Multilocus Sequence Typing
    Enterococcus faecium
    Polymorphism
    melting
    Freezing
    genotyping
    single nucleotide polymorphism
    Single Nucleotide Polymorphism
    Melting
    Nucleotides
    Pulsed Field Gel Electrophoresis
    pulsed-field gel electrophoresis
    Electrophoresis
    Gels
    multilocus sequence typing
    Databases
    kinetics
    Polymerase Chain Reaction
    Kinetics
    Base Composition

    Cite this

    Tong, Steven ; Xie, Shirley ; Richardson, Leisha Jade ; Ballard, Susan ; Dakh, Farshid ; Grabsch, Elizabeth ; Grayson, Lindsay ; Howden, Benjamin ; Johnson, Paul ; Giffard, Philip. / High-resolution melting genotyping of enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms. In: PLoS One. 2011 ; Vol. 6, No. 12. pp. 1-8.
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    abstract = "We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 {"}melting types{"} (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure. � 2011 Tong et al.",
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    author = "Steven Tong and Shirley Xie and Richardson, {Leisha Jade} and Susan Ballard and Farshid Dakh and Elizabeth Grabsch and Lindsay Grayson and Benjamin Howden and Paul Johnson and Philip Giffard",
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    Tong, S, Xie, S, Richardson, LJ, Ballard, S, Dakh, F, Grabsch, E, Grayson, L, Howden, B, Johnson, P & Giffard, P 2011, 'High-resolution melting genotyping of enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms', PLoS One, vol. 6, no. 12, e29189, pp. 1-8. https://doi.org/10.1371/journal.pone.0029189

    High-resolution melting genotyping of enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms. / Tong, Steven; Xie, Shirley; Richardson, Leisha Jade; Ballard, Susan; Dakh, Farshid; Grabsch, Elizabeth; Grayson, Lindsay; Howden, Benjamin; Johnson, Paul; Giffard, Philip.

    In: PLoS One, Vol. 6, No. 12, e29189, 2011, p. 1-8.

    Research output: Contribution to journalArticleResearchpeer-review

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    T1 - High-resolution melting genotyping of enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms

    AU - Tong, Steven

    AU - Xie, Shirley

    AU - Richardson, Leisha Jade

    AU - Ballard, Susan

    AU - Dakh, Farshid

    AU - Grabsch, Elizabeth

    AU - Grayson, Lindsay

    AU - Howden, Benjamin

    AU - Johnson, Paul

    AU - Giffard, Philip

    PY - 2011

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    N2 - We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure. � 2011 Tong et al.

    AB - We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure. � 2011 Tong et al.

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    KW - intermethod comparison

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    KW - Electrophoresis, Gel, Pulsed-Field

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