Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae

Abstract

Infections caused by drug-resistant bacteria are a major problem worldwide. Carbapenem-resistant Klebsiella pneumoniae, most notably isolates classified as multilocus sequence type (ST) 258, have emerged as an important cause of hospital deaths. ST258 isolates are predominantly multidrug resistant, and therefore infections caused by them are difficult to treat. It is not known why the ST258 lineage is the most prevalent cause of multidrug-resistant K. pneumoniae infections in the United States and other countries. Here we tested the hypothesis that carbapenem-resistant ST258 K. pneumoniae is a single genetic clone that has disseminated worldwide. We sequenced to closure the genomes of two ST258 clinical isolates and used these genomes as references for comparative genome sequencing of 83 additional clinical isolates recovered from patients at diverse geographic locations worldwide. Phylogenetic analysis of the SNPs in the core genome of these isolates revealed that ST258 K. pneumoniae organisms are two distinct genetic clades. This unexpected finding disproves the single-clone hypothesis. Notably, genetic differentiation between the two clades results from an ∼ 215-kb region of divergence that includes genes involved in capsule polysaccharide biosynthesis. The region of divergence appears to be a hotspot for DNA recombination events, and we suggest that this region has contributed to the success of ST258 K. pneumoniae. Our findings will accelerate research on novel diagnostic, therapeutic, and vaccine strategies designed to prevent and/or treat infections caused by multidrug resistant K. pneumoniae.

Keywords: Enterobacteriaceae; antibiotic resistance; carbapenemase; plasmid.

Fig. 1.

K. pneumoniae genomes. Selected K. pneumoniae genomes were compared with the two reference ST258 genomes, NJST258_1 and NJST258_2 (red and orange inner circles). Notable features, such as prophages (Phage) and other MGEs or variable regions, are indicated by rectangles. An RD is indicated by the bracket and dotted lines.

Fig. 2.

Plasmids and multidrug resistance. Plasmids present in NJST258_1 (A) and NJST258_2 (B). Antibiotic and heavy metal-resistance elements are indicated for each plasmid.

Fig. 3.

SNP-based phylogenetic analysis of ST258 clinical isolates. (A) Phylogenetic analysis of 86 K. pneumoniae clinical isolates [85 study isolates and KPNIH1, an ST258 isolate originally recovered from a patient at the National Institutes of Health Clinical Center (4)] based on 2,436 unique, concatenated SNP nucleotides present in the core genome (excluding MGEs) of the 85 study isolates (Dataset S1). KPNIH1 was included because of its clinical significance. (B) Phylogenetic analysis of the clinical isolates shown in A but excluding the RD (∼215 kb). NJST258_1 is the fully closed mucoid ST258 reference strain to which all isolates are compared (see Fig. 1). NJST258_2 is the fully closed nonmucoid ST258 strain as presented in Fig. 1.

Fig. 4.

RD. (A) Phylogenetic analysis of 85 ST258 (or single-locus multilocus sequence type variants) K. pneumoniae clinical isolates based upon 664 unique, concatenated SNP nucleotides present in the RD alone. (B) Linear schematic of the RD indicating the location of the genes encoding the cps biosynthesis enzymes and ICE Kp258.2. (C) Comparison of the cps regions in clades 1 and 2.