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

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    15
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    Author
    Andersson, P; Tong, SYC; Bell, JM; Turnidge, JD; Giffard, PM
    Date
    2012-03-12
    Source Title
    PLoS One
    Publisher
    PUBLIC LIBRARY SCIENCE
    University of Melbourne Author/s
    Tong, Steven; Andersson, Patiyan
    Affiliation
    Doherty Institute
    Microbiology and Immunology
    Metadata
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    Document Type
    Journal Article
    Citations
    Andersson, P., Tong, S. Y. C., Bell, J. M., Turnidge, J. D. & Giffard, P. M. (2012). Minim Typing - A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae. PLOS ONE, 7 (3), https://doi.org/10.1371/journal.pone.0033530.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/256495
    DOI
    10.1371/journal.pone.0033530
    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.

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