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.
Highlights
Multilocus sequence typing (MLST) is a widely used method for determining bacterial population structures, and assigning isolates to lineages [1]
Method design In order to design a K. pneumoniae Minim procedure, the Minimum single nucleotide polymorphism (SNP) software was used to identify a set of six SNPs that provided as high as possible D value with respect to the MLST database
To maximise that probability of the SNPs forming the basis of a robust method, the choice of SNPs was confined to: i) G/ C«A/T SNPs, because in these cases, the alleles differ in their numbers of hydrogen bonds, and are more likely to be resolved by highresolution melting (HRM) analysis, and ii) SNPs that are close to conserved regions that may be used for the design of PCR primer sets that will amplify fragments approximately 50–150 bp in size, which is appropriate for the Minim approach [10]
Summary
Multilocus sequence typing (MLST) is a widely used method for determining bacterial population structures, and assigning isolates to lineages [1]. MLST is based on sequence data from standardised fragments of housekeeping genes. Unique sequences at each locus are assigned an allele number. The seven allele numbers comprise a profile that defines the sequence type (ST). Alleles and sequence types are numbered in accordance with the chronology of their discovery, and are accessible online (http:// www.mlst.net/, http://www.pasteur.fr/). While MLST is very effective, it typically requires 14 separate sequencing reactions per isolate. The time and cost of this and the associated data analysis has limited the use of MLST in large-scale studies, and in the near future, MLST may not be competitive with whole genome sequencing using generation technologies
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