Minim typing - A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae

Patiyan Andersson, Steven Tong, Jan Bell, J Turnidge, Philip Giffard

    Research output: Contribution to journalArticleResearchpeer-review

    3 Downloads (Pure)

    Abstract

    Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.
    Original languageEnglish
    Article numbere33530
    Pages (from-to)1-7
    Number of pages7
    JournalPLoS One
    Volume7
    Issue number3
    DOIs
    Publication statusPublished - 2012

    Fingerprint

    Multilocus Sequence Typing
    Klebsiella pneumoniae
    melting
    Melting
    Freezing
    Costs and Cost Analysis
    Costs
    Single Nucleotide Polymorphism
    Base Composition
    Assays
    alleles
    Streptococcus pyogenes
    Alleles
    loci
    Enterococcus faecium
    Optical resolving power
    assays
    multilocus sequence typing
    Polymorphism
    antibiotic resistance

    Cite this

    Andersson, Patiyan ; Tong, Steven ; Bell, Jan ; Turnidge, J ; Giffard, Philip. / Minim typing - A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae. In: PLoS One. 2012 ; Vol. 7, No. 3. pp. 1-7.
    @article{a1fb3498ccb249f9a6308fe6167c42d7,
    title = "Minim typing - A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae",
    abstract = "Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.",
    keywords = "bacterial DNA, DNA fragment, primer DNA, allele, analytical parameters, antibiotic resistance, article, bacterial gene, bacterium isolate, controlled study, cost control, GC rich sequence, gene amplification, gene locus, genetic variability, genotype, high resolution melting analysis, high throughput sequencing, infB gene, Klebsiella pneumoniae, mdh gene, multilocus sequence typing, nonhuman, nucleotide sequence, phoE gene, prediction, real time polymerase chain reaction, rpoB gene, sequence analysis, Simpsons Index of Diversity, single nucleotide polymorphism, TonB gene, Australia, bacterium identification, biology, chromosome map, DNA base composition, genetics, methodology, phylogeny, transition temperature, Enterococcus faecium, Staphylococcus aureus, Streptococcus pyogenes, Bacterial Typing Techniques, Base Composition, Chromosome Mapping, Computational Biology, DNA Primers, Genotype, Multilocus Sequence Typing, Phylogeny, Polymorphism, Single Nucleotide, Real-Time Polymerase Chain Reaction, Transition Temperature",
    author = "Patiyan Andersson and Steven Tong and Jan Bell and J Turnidge and Philip Giffard",
    year = "2012",
    doi = "10.1371/journal.pone.0033530",
    language = "English",
    volume = "7",
    pages = "1--7",
    journal = "PLoS One",
    issn = "1932-6203",
    publisher = "Public Library of Science (PLoS)",
    number = "3",

    }

    Minim typing - A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae. / Andersson, Patiyan; Tong, Steven; Bell, Jan; Turnidge, J; Giffard, Philip.

    In: PLoS One, Vol. 7, No. 3, e33530, 2012, p. 1-7.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - Minim typing - A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae

    AU - Andersson, Patiyan

    AU - Tong, Steven

    AU - Bell, Jan

    AU - Turnidge, J

    AU - Giffard, Philip

    PY - 2012

    Y1 - 2012

    N2 - Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.

    AB - Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.

    KW - bacterial DNA

    KW - DNA fragment

    KW - primer DNA

    KW - allele

    KW - analytical parameters

    KW - antibiotic resistance

    KW - article

    KW - bacterial gene

    KW - bacterium isolate

    KW - controlled study

    KW - cost control

    KW - GC rich sequence

    KW - gene amplification

    KW - gene locus

    KW - genetic variability

    KW - genotype

    KW - high resolution melting analysis

    KW - high throughput sequencing

    KW - infB gene

    KW - Klebsiella pneumoniae

    KW - mdh gene

    KW - multilocus sequence typing

    KW - nonhuman

    KW - nucleotide sequence

    KW - phoE gene

    KW - prediction

    KW - real time polymerase chain reaction

    KW - rpoB gene

    KW - sequence analysis

    KW - Simpsons Index of Diversity

    KW - single nucleotide polymorphism

    KW - TonB gene

    KW - Australia

    KW - bacterium identification

    KW - biology

    KW - chromosome map

    KW - DNA base composition

    KW - genetics

    KW - methodology

    KW - phylogeny

    KW - transition temperature

    KW - Enterococcus faecium

    KW - Staphylococcus aureus

    KW - Streptococcus pyogenes

    KW - Bacterial Typing Techniques

    KW - Base Composition

    KW - Chromosome Mapping

    KW - Computational Biology

    KW - DNA Primers

    KW - Genotype

    KW - Multilocus Sequence Typing

    KW - Phylogeny

    KW - Polymorphism, Single Nucleotide

    KW - Real-Time Polymerase Chain Reaction

    KW - Transition Temperature

    UR - http://www.scopus.com/inward/record.url?scp=84858054024&partnerID=8YFLogxK

    U2 - 10.1371/journal.pone.0033530

    DO - 10.1371/journal.pone.0033530

    M3 - Article

    VL - 7

    SP - 1

    EP - 7

    JO - PLoS One

    JF - PLoS One

    SN - 1932-6203

    IS - 3

    M1 - e33530

    ER -