Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/73135
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Type: Journal article
Title: Minim typing - a rapid and low cost MLST based typing tool for Klebsiella pneumoniae
Author: Andersson, P.
Tong, S.
Bell, J.
Turnidge, J.
Giffard, P.
Citation: PLoS One, 2012; 7(3):1-7
Publisher: Public Library of Science
Issue Date: 2012
ISSN: 1932-6203
1932-6203
Statement of
Responsibility: 
Patiyan Andersson, Steven Y.C. Tong, Jan M. Bell, John D. Turnidge and Philip M. Giffard
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: Klebsiella pneumoniae
DNA Primers
Bacterial Typing Techniques
Chromosome Mapping
Computational Biology
Phylogeny
Base Composition
Genotype
Polymorphism, Single Nucleotide
Transition Temperature
Australia
Multilocus Sequence Typing
Real-Time Polymerase Chain Reaction
Rights: Copyright: © 2012 Andersson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
DOI: 10.1371/journal.pone.0033530
Appears in Collections:Aurora harvest 5
Molecular and Biomedical Science publications

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