Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Klebsiella pneumoniae.

Chen Liu,Hongyu Ren,Wenbing Liu,Haijian Zhou,Tian Qin

doi:10.3389/fmicb.2017.00371

Abstract

At present, the most used methods for Klebsiella pneumoniae subtyping are multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). However, the discriminatory power of MLST could not meet the need for distinguishing outbreak and non-outbreak isolates and the PFGE is time-consuming and labor-intensive. A core genome multilocus sequence typing (cgMLST) scheme for whole-genome sequence-based typing of K. pneumoniae was developed for solving the disadvantages of these traditional molecular subtyping methods. Firstly, we used the complete genome of K. pneumoniae strain HKUOPLC as the reference genome and 907 genomes of K. pneumoniae download from NCBI database as original genome dataset to determine cgMLST target genes. A total of 1,143 genes were retained as cgMLST target genes. Secondly, we used 26 K. pneumoniae strains from a nosocomial infection outbreak to evaluate the cgMLST scheme. cgMLST enabled clustering of outbreak strains with <10 alleles difference and unambiguous separation from unrelated outgroup strains. Moreover, cgMLST revealed that there may be several sub-clones of epidemic ST11 clone. In conclusion, the novel cgMLST scheme not only showed higher discriminatory power compared with PFGE and MLST in outbreak investigations but also showed ability to reveal more population structure characteristics than MLST.

Highlights

Klebsiella pneumoniae is associated with a wide range of diseases, including urinary tract infections, respiratory tract infections, septicemia, pyogenic liver abscess, primary osteomyelitis and so on (Podschun and Ullmann, 1998; Brisse et al, 2009; Prokesch et al, 2016)
In this study we developed a core genome multilocus sequence typing (cgMLST) typing scheme for K. pneumoniae and we evaluated this scheme for ability in outbreak investigations using isolates from one outbreak and several sporadic cases
Using K. pneumoniae strain HKUOPLC (GenBank assembly accession number GCA_001280925.1) as the reference genome (4,764 genes), after basic filter based on minimum length, start codon, stop codon, homologous gene and gene overlap, 4,543 genes were retained

Summary

INTRODUCTION

Klebsiella pneumoniae is associated with a wide range of diseases, including urinary tract infections, respiratory tract infections, septicemia, pyogenic liver abscess, primary osteomyelitis and so on (Podschun and Ullmann, 1998; Brisse et al, 2009; Prokesch et al, 2016). A large number of subtyping techniques have been used to identify clusters and outbreaks, trace the source and determine the spreading chain in the investigation of K. pneumonia infections or outbreak and to study the population structure and pathogen evolution of K. pneumonia (Podschun and Ullmann, 1998) Among these techniques, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are the two most frequently used methods to investigate outbreaks of K. pneumoniae infection (Jin et al, 2015; Mshana et al, 2015; Zhu et al, 2016). A possible solution to this problem has been suggested by extending the MLST concept to the genome level based on whole-genome sequence data and comparison to a set of loci (e.g., the genes of the core genome) and allele variants indexed (Jolley and Maiden, 2010; Maiden et al, 2013) This method is known as core genome multilocus sequence typing (cgMLST). In this study we developed a cgMLST typing scheme for K. pneumoniae and we evaluated this scheme for ability in outbreak investigations using isolates from one outbreak and several sporadic cases

MATERIALS AND METHODS

RESULTS

DISCUSSION