Genome analysis of clinical multilocus sequence Type 11 Klebsiella pneumoniae from China

Abstract

The increasing prevalence of KPC-producing Klebsiella pneumoniae strains in clinical settings has been largely attributed to dissemination of organisms of specific multilocus sequence types, such as ST258 and ST11. Compared with the ST258 clone, which is prevalent in North America and Europe, ST11 is common in China but information regarding its genetic features remains scarce. In this study, we performed detailed genetic characterization of ST11 K. pneumoniae strains by analyzing whole-genome sequences of 58 clinical strains collected from diverse geographic locations in China. The ST11 genomes were found to be highly heterogeneous and clustered into at least three major lineages based on the patterns of single-nucleotide polymorphisms. Exhibiting five different capsular types, these ST11 strains were found to harbor multiple resistance and virulence determinants such as the blaKPC-2 gene, which encodes carbapenemase, and the yersiniabactin-associated virulence genes irp, ybt and fyu. Moreover, genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA) were detectable in six genomes, whereas genes encoding salmochelin were found in three genomes. In conclusion, our data indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings. Such findings provide insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.

Keywords: Klebsiella pneumonia; ST11; ST258; antimicrobial resistance; cps loci; genome analysis; virulence.

Fig. 1.

Alignment of the K. pneumoniae genomes. A total of ten K. pneumoniae genomes were compared using the chromosome sequence of strain GD4 (outermost circle) as a reference. Prophages (prophages 11.1–11.8), integrated conjugative elements (ICEKpnHS11286-1 and ICEKpnHS11286-2) and the capsule polysaccharide region are indicated by rectangles. Antimicrobial resistance genes are indicated.

Fig. 2.

Approximately maximum-likelihood phylogeny estimated with the parsnp software [28] based on a total of 6749 unique concatenated SNPs in the core genome of 58 clinical ST11 K. pneumoniae strains from China. (a) Unrooted phylogenetic analysis of 58 K. pneumoniae clinical isolates. GD4, HS11286 and JM45 are ST11 strains with completed genome sequences. Bar, 0.01 substitutions per nucleotide position. (b) Circular phylogenetic tree of the 58 strains. The lengths of the branches are not proportional to the evolutionary distances in the cladogram. Different background colors underneath each strain name indicate the K locus type of the corresponding strain detected by Kaptive [32].

Fig. 3.

Phylogenetic tree and SNPs of representative K. pneumoniae strains from CG258. Pink lines indicate SNPs identified with the harvest suite [28]. Regions of divergence were labelled with double side arrows.

Fig. 4.

Comparison of the cps gene clusters from K. pneumoniae strains of CG258. The order of the pairwise comparisons is defined by the phylogenetic relationships. Locus types KL47 and KL64 correspond to serotypes K47 and K64, respectively. K types KL103, KL125 and KL105 are locus types which have not been phenotypically defined and are defined from DNA sequence data on the basis of gene content [32]. Sequence origins, accession numbers and STs for the respective strains are indicated. Arrows indicate the direction, relative length and function of ORFs. The cps loci of K. pneumoniae strains ATCC BAA-2146 and BO4 were downloaded from the NCBI database and used as references for the comparison. ORFs encoding transposases are colored in red, while those encoding non-initial glycosyltransferases are colored in blue. HP, CL and PLSP are short for hypothetical protein, carbohydrate lyase and pectate lyase superfamily protein, respectively. Homologous regions are connected by areas of different colors reflecting the degree of nucleotide identity (from 64 to 100 %).

Fig. 5.

Heatmap of the antimicrobial resistance genes harbored by clinical ST11 K. pneumoniae strains from China. The presence of resistance genes in a specific genome is represented by a green box and the absence of resistance genes is represented by a purple box.

Fig. 6.

Circular map of plasmid pKPGD4 harbored by K. pneumoniae strain GD4. Resistance determinants are indicated in the outermost circle. Plasmids pCT-KPC (accession number: KT185451.1) and SWU01 unnamed plasmid (accession number: CP018455.1) with similar sequence organizations were aligned against pKPGD4.

Fig. 7.

Heatmap of virulence determinants harbored by clinical ST11 K. pneumoniae strains from China. The presence of virulence genes in a specific genome is represented by the blue box and the absence of virulence genes is represented by a black box.