Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups

Abstract

Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected.

Figure 1

Phylogenetic network of the 167 Klebsiella pneumoniae genomes as determined on the basis of the allelic profiles of the 694 core genome multilocus sequence typing (cgMLST) genes. The network was constructed by using the neighbor-net method implemented in SplitsTree v4.13.1 (18). Nodes are colored according to the clonal group (CG). Only the 10 most relevant CGs are highlighted; note that ST35 was subdivided into 2 CGs (CG35-A and CG35-B). Gray shading indicates CG258. Gray dots indicate phylogroups KpII-B and KpIII (K. variicola). Bold indicates reference strains. Scale bar represents 100 allelic mismatches. ATCC, American Type Culture Collection; ST, sequence type.

Figure 2

Phylogenetic tree of the 167 Klebsiella pneumoniae genomes as determined on the basis of core genome multilocus sequence typing (cgMLST) genes and distribution of virulence and resistance features. The tree was inferred from minimum evolution analysis based on aligned cgMLST sequences, with K. variicola and KpII-B sequences as outgroups. Terminal branches corresponding to different taxa from the same clonal group (CG) or sequence type (ST) are shown as triangles of depth proportional to internal diversity. Bootstrap values >50% based on 1,000 gene-by-gene replicates are given at branches. Scale bar represents 0.05% estimated sequence divergence. The virulence and resistance gene content (indicated along the top of the figure) of identified clones is represented by squares, which are colored in black proportionally to the percentage of presence of a gene or cluster among members of a given CG or ST.

Figure 3

Phylogenetic network of the 82 Klebsiella pneumoniae strains belonging to clonal group (CG) 258 as determined on the basis of the allelic profiles of the 694 core genome multilocus sequence typing (cgMLST) genes. The 20 genomes corresponding to isolates from the 2011 K. pneumoniae outbreak at the National Institutes of Health Clinical Center (Bethesda, Maryland, USA) (24) are highlighted by gray shading. Scale bar represents 10 allelic mismatches. ST, sequence type.