Carbapenem-Resistant, Virulence Plasmid-Harboring Klebsiella pneumoniae, United States

Abstract

Carbapenem-resistant and virulence plasmid-harboring Klebsiella pneumoniae (pVir-CRKP) has emerged and spread globally, yet clinical investigations from the United States remain limited. We conducted a genomic analysis of 884 unique carbapenem-resistant K. pneumoniae isolates from a multicenter US cohort and identified 6 pVir-CRKP isolates, including 2 sequence type (ST) 23, 2 ST893, and 2 ST11 isolates. Patients infected with pVir-CRKP experienced high Pitt bacteremia scores and a 33% 30-day mortality rate. The pVir-CRKP isolates exhibited significant sequence variation in virulence genes and plasmids, along with differences in mucoviscosity, capsule production, survival in normal human serum, resistance to killing by human polymorphonuclear neutrophils, and in vivo pathogenicity. Phylogenetic analyses showed that most pVir-CRKP isolates were genetically similar to strains reported from other global regions. The emergence of pVir-CRKP with higher virulence potential and carbapenem resistance in the United States than the predominant carbapenem-resistant K. pneumoniae clone underscores the need for active global surveillance.

Keywords: Klebsiella pneumoniae; United States; antimicrobial resistance; bacteria; carbapenem resistance; clinical and genomic characterization; pVir-CRKP; virulence; virulence plasmid.

Figure 1

Information of 884 isolates of carbapenem-resistant Klebsiella pneumoniae isolates, United States. A) Unadjusted distribution of desirability of outcome ranking outcomes. B) Heatmap of infections, virulence genes, antimicrobial resistance genes, and porin mutations in STs with >2 isolates. *MgrB or PmrB loss-of-function mutations. #Mutations in the quinolone resistance–determining regions of GyrA and ParC. C) Distribution of drug classes by genome for which antimicrobial resistance elements were detected. Bars colors indicate the presence of virulence genes. D, E) Distributions of virulence genes (D) and virulence gene combination (E) per genomes. Bars are colored based on STs. ESBL, extended-spectrum β-lactamase; ST, sequence type.

Figure 2

Linear alignment of virulence plasmids in isolates of carbapenem-resistant Klebsiella pneumoniae and hypervirulent K. pneumoniae NTUH-K2044, United States. Arrowheads represent genes; colors indicate gene function classification. Light blue shading represents regions of homology. IS, insertion sequence; KL, capsular locus; ST, sequence type.

Figure 3

Capsule heterogeneity in 6 clinical isolates and reference isolates in study of carbapenem-resistant, virulence plasmid–harboring Klebsiella pneumoniae isolates, United States. A) Hypermucoid index scores; B) capsule production measured by uronic acid content of isolates displaying different mucoidity. The hypermucoid index and uronic acid production of the 6 isolates were compared to those of ATCC 43816 and NJST 258–2 using t-tests. Asterisk (*) indicates p<0.05 vs. ATCC43816; hash mark (#) indicates p<0.05 vs. NJST258_2. Results represent the mean (bars) + SDs (error bars) of 3 samples for each isolate. KL, capsular locus; OD₆₀₀, optical density at 600 nm; ST, sequence type.

Figure 4

Serum and PMN bactericidal activity in 6 clinical isolates in study of carbapenem-resistant, virulence plasmid–harboring Klebsiella pneumoniae isolates, United States. A–B) The indicated clinical isolates were cultured with 0%, 5%, 25%, or 83% NHS for 1 hour, and recovered viable bacteria were enumerated as both percentages (A) and absolute values (B). Asterisk (*) indicates p<0.05 vs. 0% serum by using a repeated-measures analysis of variance and Dunnett posttest; hash mark (#) indicates p<0.05 vs. ARLG-3254 in 25% or 83% NHS by using a t-test. C–D) Survival of clinical isolates determined following synchronized PMN phagocytosis assays at 30 or 60 min as described. Data are expressed as percentage survival (C) or colony-forming units (D). Results represent the mean (dots) + SD (error bars) of 3 separate experiments. Asterisk (*) indicates p<0.05 as determined by using a repeated-measures analysis of variance and Bonferroni posttest (selected pairs of data) or Kruskal-Wallis test and Dunn posttest for nonparametric analysis of ARLG-4744; hash mark (#) indicates p<0.05 vs. ARLG-3254 for 30 or 60 min in PMN by using a t-test. We conducted statistical analyses by using CFUs. NHS, normal human serum; PMN, polymorphonuclear neutrophils; ST, sequence type.

Figure 5

Virulence comparison of 3 selected isolates in study of carbapenem-resistant, virulence plasmid–harboring Klebsiella pneumoniae isolates, United States. Virulence phenotypes were determined in a murine model of K. pneumoniae pneumonia. A) Changes in mouse weight relative to the baseline at 0 h during the course of infection (n = 5). Results represent the mean (dots) + standard deviation (t bars) of the indicated number of samples. Asterisks (*) indicate p<0.05 vs. 24 h; hash marks (#) indicate p<0.05 vs. ATCC43816. B–D) Bacteria recovered from mouse lungs at 2 h (B, n = 3) and 48 h (C, n = 5) postinfection and from spleens at 48 h postinfection (D, n = 5). Individual CFU values (dots) and mean values (horizontal lines) are shown. We used t-tests for all the comparisons. Dashed lines indicate limits of detection. KL, capsular locus; ST, sequence type.