Hypervirulent Klebsiella pneumoniae Causing Neonatal Bloodstream Infections: Emergence of NDM-1-Producing Hypervirulent ST11-K2 and ST15-K54 Strains Possessing pLVPK-Associated Markers

Abstract

Klebsiella pneumoniae is a major cause of neonatal sepsis. Hypervirulent Klebsiella pneumoniae (hvKP) that cause invasive infections and/or carbapenem-resistant hvKP (CR-hvKP) limit therapeutic options. Such strains causing neonatal sepsis have rarely been studied. Characterization of neonatal septicemic hvKP/CR-hvKP strains in terms of resistance and virulence was carried out. Antibiotic susceptibility, molecular characterization, evaluation of clonality, in vitro virulence, and transmissibility of carbapenemase genes were evaluated. Whole-genome sequencing (WGS) and mouse lethality assays were performed on strains harboring pLVPK-associated markers. About one-fourth (26%, 28/107) of the studied strains, leading to mortality in 39% (11/28) of the infected neonates, were categorized as hvKP. hvKP-K2 was the prevalent pathotype (64.2%, 18/28), but K54 and K57 were also identified. Most strains were clonally diverse belonging to 12 sequence types, of which ST14 was most common. Majority of hvKPs possessed virulence determinants, strong biofilm-forming, and high serum resistance ability. Nine hvKPs were carbapenem-resistant, harboring bla_NDM-1/bla_NDM-5 on conjugative plasmids of different replicon types. Two NDM-1-producing high-risk clones, ST11 and ST15, had pLVPK-associated markers (rmpA, rmpA2, iroBCDEN, iucABCDiutA, and peg-344), of which one co-transferred the markers along with bla_NDM-1. The 2 strains revealed high inter-genomic resemblance with the other hvKP reference genomes, and were lethal in mouse model. To the best of our knowledge, this study is the first to report on the NDM-1-producing hvKP ST11-K2 and ST15-K54 strains causing fatal neonatal sepsis. The presence of pLVPK-associated markers and bla_NDM-1 in high-risk clones, and the co-transmission of these genes via conjugation calls for surveillance of these strains. IMPORTANCE Klebsiella pneumoniae is a leading cause of sepsis in newborns and adults. Among the 2 major pathotypes of K. pneumoniae, classical (cKP) and hypervirulent (hvKP), hvKP causes community-acquired severe fatal invasive infections in even healthy individuals, as it possesses several virulence factors. The lack of comprehensive studies on neonatal septicemic hvKPs prompted this work. Nearly 26% diverse hvKP strains were recovered possessing several resistance and virulence determinants. The majority of them exhibited strong biofilm-forming and high serum resistance ability. Nine of these strains were also carbapenem (last-resort antibiotic)-resistant, of which 2 high-risk clones (ST11-K2 and ST15-K54) harbored markers (pLVPK) noted for their virulence, and were lethal in the mouse model. Genome-level characterization of the high-risk clones showed resemblance with the other hvKP reference genomes. The presence of transmissible carbapenem-resistant gene, bla_NDM, along with pLVPK-markers calls for vigilance, as most clinical microbiology laboratories do not test for them.

Keywords: India; Klebsiella pneumoniae; antibiotic resistance; carbapenem resistance; hypervirulence; neonatal sepsis.

FIG 1

PFGE profiles along with distribution of different antimicrobial resistance (both phenotypic and genotypic) and virulence determinants of the neonatal septicemic, carbapenem-resistant (a) and carbapenem-susceptible (b) hvKP strains. Dark blue filled-up boxes indicate the presence of different antimicrobial resistance and virulence determinants.

FIG 2

Comparative BLASTN analysis of NDM-1-producing neonatal septicemic CR-hvKP strains EN5180 and EN5289 and 11 other hvKP reference strains. The circular map was generated using the CGView^BETA comparison tool. From the center to the periphery: Ring 1 and 2 show the GC content and GC skew, respectively; Ring 3 and ring 4 display CR-hvKP strains EN5289 (accession: JAELUW000000000) and EN5180 (accession: JAELUV000000000), respectively; Ring 4: strain 1084 (accession: CP003785.1); Ring 5: strain 1158 (accession: CP006722.1); Ring 6: strain CG43 (accession: CP006648.1); Ring 7: strain ED2 (accession: CP016813.1); Ring 8: strain ED23 (accession: CP016814.1); Ring 9: strain EN5275 (accession: VINI00000000); Ring 10: strain KCTC-2242 (accession: CP002910.1); Ring 11: strain NTUH-2044 (accession: NC_012731.1); Ring 12: strain NUHL30457 (accession: CP026586.1); Ring 13: strain RJF293 (accession: CP014008.1); Ring 14: strain RJF999 (accession: CP014010.1). The sequence comparison revealed that the 13 hvKP strains were shown to have a high degree of similarity.

FIG 3

Virulence plasmid sequence alignment analysis among the bla_NDM-1 and hypervirulence determinants co-encoding hybrid plasmid, pvirEN5289, and the previously reported virulence plasmid, pLVPK (GenBank accession AY378100) (a), sequence alignment analysis among the bla_CTX-M-15 and hypervirulent markers co-harboring hybrid plasmid, pvirEN5180 and pLVPK (b). Although, both the genomes harbored all the hypervirulent biomarkers, some of the molecular markers were not portrayed in the given virulence plasmid maps due to their low sequence identity against the available sequence databases.

FIG 4

Kaplan–Meier survival curves for neonatal septicemic, carbapenem-resistant hypervirulent K. pneumoniae EN5180- and EN5289-infected mice. Female BALB/c mice (4 to 6-weeks-old, 5 mice/strain) were intraperitoneally challenged with 1 × 10⁵ CFU of the different K. pneumoniae strains (EN5180, EN5289, hypervirulent control strain SB42, and low virulence control strain ATCC 700603). Saline was administered as vehicle control. Mice were monitored every 24 h up to 7 days, and scored for death. During the 7-day period, no mice in the ATCC 700603 or saline groups died.