Genomic surveillance for multidrug-resistant or hypervirulent Klebsiella pneumoniae among United States bloodstream isolates

Abstract

Background: Klebsiella pneumoniae strains have been divided into two major categories: classical K. pneumoniae, which are frequently multidrug-resistant and cause hospital-acquired infections in patients with impaired defenses, and hypervirulent K. pneumoniae, which cause severe community-acquired and disseminated infections in normal hosts. Both types of infections may lead to bacteremia and are associated with significant morbidity and mortality. The relative burden of these two types of K. pneumoniae among bloodstream isolates within the United States is not well understood.

Methods: We evaluated consecutive K. pneumoniae isolates cultured from the blood of hospitalized patients at Northwestern Memorial Hospital (NMH) in Chicago, Illinois between April 2015 and April 2017. Bloodstream isolates underwent whole genome sequencing, and sequence types (STs), capsule loci (KLs), virulence genes, and antimicrobial resistance genes were identified in the genomes using the bioinformatic tools Kleborate and Kaptive. Patient demographic, comorbidity, and infection information, as well as the phenotypic antimicrobial resistance of the isolates were extracted from the electronic health record. Candidate hypervirulent isolates were tested in a murine model of pneumonia, and their plasmids were characterized using long-read sequencing. We also extracted STs, KLs, and virulence and antimicrobial resistance genes from the genomes of bloodstream isolates submitted from 33 United States institutions between 2007 and 2021 to the National Center for Biotechnology Information (NCBI) database.

Results: Consecutive K. pneumoniae bloodstream isolates (n = 104, one per patient) from NMH consisted of 75 distinct STs and 51 unique capsule loci. The majority of these isolates (n = 58, 55.8%) were susceptible to all tested antibiotics except ampicillin, but 17 (16.3%) were multidrug-resistant. A total of 32 (30.8%) of these isolates were STs of known high-risk clones, including ST258 and ST45. In particular, 18 (17.3%) were resistant to ceftriaxone (of which 17 harbored extended-spectrum beta-lactamase genes) and 9 (8.7%) were resistant to meropenem (all of which harbored a carbapenemase genes). Four (3.8%) of the 104 isolates were hypervirulent K. pneumoniae, as evidenced by hypermucoviscous phenotypes, high levels of virulence in a murine model of pneumonia, and the presence of large plasmids similar to characterized hypervirulence plasmids. These isolates were cultured from patients who had not recently traveled to Asia. Two of these hypervirulent isolates belonged to the well characterized ST23 lineage and one to the re-emerging ST66 lineage. Of particular concern, two of these isolates contained plasmids with tra conjugation loci suggesting the potential for transmission. We also analyzed 963 publicly available genomes of K. pneumoniae bloodstream isolates from locations within the United States. Of these, 465 (48.3%) and 760 (78.9%) contained extended-spectrum beta-lactamase genes or carbapenemase genes, respectively, suggesting a bias towards submission of antibiotic-resistant isolates. The known multidrug-resistant high-risk clones ST258 and ST307 were the predominant sequence types. A total of 32 (3.3%) of these isolates contained aerobactin biosynthesis genes and 26 (2.7%) contained at least two genetic features of hvKP strains, suggesting elevated levels of virulence. We identified 6 (0.6%) isolates that were STs associated with hvKP: ST23 (n = 4), ST380 (n = 1), and ST65 (n = 1).

Conclusions: Examination of consecutive isolates from a single center demonstrated that multidrug-resistant high-risk clones are indeed common, but a small number of hypervirulent K. pneumoniae isolates were also observed in patients with no recent travel history to Asia, suggesting that these isolates are undergoing community spread in the United States. A larger collection of publicly available bloodstream isolate genomes also suggested that hypervirulent K. pneumoniae strains are present but rare in the USA; however, this collection appears to be heavily biased towards highly antibiotic-resistant isolates (and correspondingly away from hypervirulent isolates).

Keywords: Bacteremia; Hypervirulent Klebsiella; Klebsiella pneumoniae; Pathogenesis; Whole-genome sequencing.

Fig. 1

Virulence loci associated with hypervirulence in K. pneumoniae. PAL-1 and PAL-2 contents and organization are depicted for pK2044, KP52.145pII, pTK421_2, and ICEKp1 (A). Genomic elements and plasmid architecture are depicted for pK2044 (B), KP52.145pII (C) and pTK421 (D) (drawings not to scale)

Fig. 2

Core genome phylogenetic tree, genomic content, and antibiotic resistance of 104 K. pneumoniae bloodstream isolates. A Maximum likelihood phylogenetic tree was generated from core genome SNP loci in 104 K. pneumoniae bloodstream isolates. Sequence types, the presence of virulence genes, hypermucoviscosity, and antibiotic resistances are indicated. ybt = yersiniabactin biosynthesis loci, clb = colibactin biosynthesis loci, rmpADC = mucoid regulator operon, rmpA2 = regulator of mucoid phenotype 2, iuc = aerobactin biosynthesis genes, iro = salmochelin biosynthesis genes, Amp = ampicillin, TMP/SMX = trimethoprim-sulfamethoxazole. Strains were considered hypermucoviscous if they were positive by string test. The complete genome of NTUH-K2044 was used as the reference genome

Fig. 3

Prediction and analysis of antimicrobial-resistant plasmids in ceftriaxone-resistant K. pneumoniae isolates. MOB-suite was used to infer the presence and sequence of plasmids containing ESBL and carbapenemase genes in ceftriaxone-resistant isolates. Inferred plasmids were then assigned to plasmid groups (represented by different colors) based on replicon family, relaxase type, and matepair formation and transferability (A). Clustering of inferred plasmids was performed using Mash. Each node represents a plasmid, and two plasmids are connected by an edge if their Jaccard index is ≥ 0.95 (B). Networks were graphed using Cytoscape

Fig. 4

The bloodstream isolates KPN115 and KPN8 harbor pK2044-like plasmids. pKPN115_1 and pKPN8_1 sequences were aligned to the pK2044 sequence (NC_006625.1) using blast ring image generator (BRIG) (A). The indicated plasmids sequences were aligned to KP52.145pII using BRIG (B). A sequence identity threshold of 85% was used. Aerobactin biosynthesis genes are indicated with green arrows, salmochelin with orange arrows, rmpA with a blue arrow, and rmpA2 with a red arrow

Fig. 5

The bloodstream isolates KPN49 and KPN165 harbor KP52.145pII-like plasmids. De novo clustering of plasmids using Mash (A). Individual nodes represent a sequenced plasmid. The thickness of the edge connecting plasmids is determined by Jaccard index as indicated. Networks were graphed using Cytoscape. The indicated plasmid sequences were aligned to pKPN49_1 using BRIG (B). A sequence identity threshold of 85% was used. The ~ 40 kb tra locus insertion is labeled. Aerobactin biosynthesis genes are indicated with green arrows, salmochelin with orange arrows, and rmpA with a blue arrow. Plasmid accession numbers for previously published plasmids are listed in Table S7

Fig. 6

Virulence of representative NMH K. pneumoniae isolates containing hvKP pathogenicity loci in a murine model of pneumonia. C57BL/6 mice were infected by nasal aspiration with the indicated doses of hvKP5 (A), CRE-229 (B), CRE-163 (C), KPN49 (D), KPN8 (E), KPN23 (F), KPN80 (G), and TK421 (H). The estimated log₁₀ LD₅₀ values are listed in blue. The number of mice used for each dose are listed in Additional file 20: Table S8

Fig. 7

Phylogenetic tree of United States K. pneumoniae bloodstream isolates. Maximum likelihood phylogenetic tree generated from core genome SNP loci in 1067 K. pneumoniae bloodstream isolates (NMH and NCBI). Sequence types, capsule loci, and the presence of virulence and antibiotic resistance genes are indicated. iuc = aerobactin biosynthesis genes, CR = contains a carbapenemase, ESBL = contains an extended-spectrum beta-lactamase gene, NMH = isolate was collected from Northwestern Memorial Hospital. The complete genome of NTUH-K2044 was used as the reference genome. Branch lengths are not to scale