Genomic and phenotypic characterization of ST2012 clinical Klebsiella quasipneumoniae subsp. similipneumoniae harboring blaNDM-1 in China

Abstract

Background: Klebsiella pneumoniae has emerged as a significant pathogen that causes community and hospital infections due to its high resistance rate and transmissibility. Klebsiella pneumoniae complex is classified into three phylogroups: Klebsiella pneumoniae (KpI), Klebsiella quasipneumoniae (KpII), and Klebsiella variicola (KpIII) in classical taxonomy, but KpII and KpIII are infrequently isolated clinically. Although pathogenic KpII has been reported worldwide, the understanding of this pathogen remains limited.

Methods: Whole-genome sequencing (WGS) of K. quasipneumoniae subsp. similipneumoniae ACESH00366hy was performed. Plasmid characterization was demonstrated using S1-PFGE, Southern blotting, and conjugation assays. Antimicrobial susceptibility testing was performed and interpreted according to CLSI, EUCAST, and FDA standards. The virulence of the strain was assessed using the Galleria mellonella infection assay, serum-killing assay, and biofilm formation assay. A phylogenetic tree was constructed to explore its biological evolution.

Results: The K. quasipneumoniae subsp. similipneumoniae isolate ACESH00366hy, belonging to ST2012 and KL139, contains several resistance genes including bla_NDM-1, bla_SHV-12, bla_OKP-B-2, and oqxAB, as well as various virulence genes including iroE, iutA, mrkABCDFHIJ, entABCDEFS, and fepABCDG. The bla_NDM-1 and bla_SHV-12 genes were present on the 53,624 bp IncX3 plasmid. Virulence assays showed that the virulence of ACESH00366hy was greater than that of the low-toxicity strain ATCC 700,603. Phylogenetic analysis demonstrated the emergence of ST2012 KpII-B in Asia and revealed the spread of K. quasipneumoniae subsp. similipneumoniae in humans, animals, and the environment.

Conclusion: This study highlights the emergence of clinical ST2012 KpII strains harbouring bla_NDM-1 via the IncX3 plasmid in China. We evaluated its resistance and virulence characteristics and performed phylogenetic analysis, thereby enhancing our understanding of its resistance and pathogenicity and contributing to the clinical surveillance of K. quasipneumoniae.

Keywords: Klebsiella pneumoniae; Klebsiella quasipneumoniae; bla NDM−1; KpII.

Fig. 1

Plasmid characteristic of KpII-B ACESH00366hy. (A) Sequence comparison analysis of pACESH00366hy-NDM with pNDM1_090351 (accession no: CP046892), pAR8538_3 (accession no: CP081829), pA575-NDM (accession no: MH917283) and pF3517-NDM (accession no: CP137176). In the outermost circle, insertion elements are indicated in green, resistance genes in blue, and other genes in red. (B) The genetic environment of bla_NDM−1 in IncX3-type plasmid. Comparison of genes surrounding bla_NDM−1 on E. coli pNDM-1-IncX3 (accession no. CP050161). ORFs are shown as arrows and indicated according to their putative functions. Green indicates gene bla_NDM−1, blue indicates gene bla_SHV−12, and pink represents other functional genes. Regions with a high degree of homology are indicated by grey shading

Fig. 2

Plasmid size determination by S1-PFGE and southern blotting hybridization with bla_NDM−1-specific probe, with the marker Salmonella enterica serotype Braenderup H9812. Plasmids pACESH00366hy-4 and pACESH00366hy-5 are too small, so they are not shown in the electropherogram

Fig. 3

(A) Galleria mellonella infection assay. The survival observation endpoint for ACESH00366hy and ATCC 700,603 was 162 h. The Kaplan-Meier method was used to plot a survival curve for G. mellonella. The survival is shown in percentage. **P < 0.01 (unpaired t-test). (B) Biofilm formation ability of KpII-B ACESH00366hy and ATCC 700,603. Bar in blue and red represent strains FAHZZU2447 and ATCC 700,603. The values shown are the mean ± SD of three independent experiments. **P < 0.01. (C) Serum-killing assay. Survival in a serum-killing assay of ACESH00366hy and ATCC 700,603. The survival rate is denoted in percentage. The bars denote the means and standard errors of the mean. **P < 0.01 (unpaired t-test)

Fig. 4

Phylogenetic tree of KpII-B strain ACESH00366hy. 107 KpII-B strains deposited in the NCBI database and a KpII-B strain in our collection were included in this phylogenetic tree. Circle 1 shows the clonal complex (CC) of the strains (“NA” stands for not assigned to clonal complex classification). Circle 2 denotes the ST type of these strains. Circle 3 depicts the locations of the strains. Circle 4 denotes the strain hosts. Circle 5 denotes the KL type of these strains. Circle 6 depicted the carbapenem-resistant genes harbored by each strain. The pink shade indicates ST2012 (CC13) strains