The Genomic Characterization of KPC-Producing Klebsiella pneumoniae from the ICU of a Teaching Hospital in Shanghai, China

Abstract

Purpose: This study retrospectively analyzed the genome characteristics of blaKPC-2 in multidrug-resistant Klebsiella pneumoniae collected from the ICU of a teaching hospital in Shanghai, China.

Methods: From February 2018 to December 2019, 36 strains of multidrug-resistant Klebsiella pneumoniae were collected from the bronchoalveolar lavage fluid of critically ill patients. The genome of all isolates was obtained through the Illumina sequence, and single nucleotide polymorphisms of the blaKPC-2 gene were analyzed to explore blaKPC-2's evolutionary characteristics. Different strains' genetic relationships and homology were studied by constructing an evolutionary tree on a single copy orthologue. Pacbio combined Illumina sequence was conducted to evaluate the structure and potential mobility of drug-resistant plasmids of the strain KP-s26.

Results: The distribution of resistance and virulence genes had little difference, but most strains had significant differences in the plasmid-encoded region. Most strains (31/36) carried the carbapenemase gene blaKPC-2, with no single nucleotide polymorphism in different strains. Extended-spectrum β-lactamase resistance genes, such as blaCTX-M and blaSHV, were found in the isolates, but no metallo-β-lactamases were detected. All strains with blaKPC-2 coexisted with chromosomal-associated fosfomycin resistance genes fosA6, and the coexistence of blaKPC-2 and blaCTX variants (blaCTX-M-15, blaCTX-M-65, and blaCTX-M-27) was also detected in 29/31 strains. The isolate KP-s26 carried five circular plasmids. pA and pB were conjugate plasmids, as they carried drug resistance genes and contained a complete IV secretion system.

Conclusion: The blaKPC-2 carbapenemase gene is relatively conservative in the process of evolution; drug-resistant plasmids containing conjugated transfer elements contribute to the spreading of drug resistance. The coexistence of blaKPC-2 with fosA6 or blaCTX-M variants was associated with increased fosfomycin resistance and broad-spectrum β-lactam resistance, respectively.

Clinical trials registration: Clinical Trials.gov Identifier: NCT03950544.

Keywords: Klebsiella pneumoniae; blaKPC-2; drug-resistant plasmids; single nucleotide polymorphism; whole-genome sequencing.

Conflict of interest statement

The authors report no conflicts of interest in this work.

© 2022 Du et al.

Figures

Figure 1

Drug susceptibility heat map of 33 KPC-producing Klebsiella pneumoniae. The minimum inhibitory concentrations (MICs) of 33 KPC-producing Klebsiella pneumoniae strains were homogenized against 17 antibiotics, and the colors in the legend indicate the change in susceptibility of the strains to the antibiotics,kP-s18, KP-s29 and KP-s39 without carbapenemase resistance genes were indicated in gray.

Figure 2

Comparative genomic circle map of 36 strains of Klebsiella pneumoniae. Taking KP-s26 as the reference genome, the remaining 35 Klebsiella pneumoniae were compared with KP-s26 for circle map.In the legend different colors represent different strains, black is GC content, green is GC offset of the leading chain, and purple is GC offset of lagging chain.Mark the missing genes on the circle map.

Figure 3

Phylogenetic tree of 36 Klebsiella pneumoniae. Construction Phylogenetic tree of 36 Klebsiella pneumoniae strains.In the legend different colors were used to represent the ST typing of strains, the expression of blaKPC and blaCTX in the second and third layers of the figure, respectively.

Figure 4

KPC gene sequence comparison of 33 KPC-producing Klebsiella pneumoniae. The figure shows the results of KPC gene sequence comparison among 33 KPC-producing Klebsiella pneumoniae strains. Different colors indicated the degree of sequence inconsistency, and black indicated that the sequence similarity was 100%. The KPC-24 gene carried by KP-s42 and KP-f04 is located at the edge of the scaffold, and the prediction is incomplete.

Figure 5

Continued.

Figure 5

Plasmid map. Plasmid pA were compared with pKP18069-CTX, pNMBU-W07E18_01, pC2601-2, pC2972_2, pC2974_2, p1_015093, and plasmid pB uses MK191023.1 as the reference genome. In the outermost layer of the map, the drug resistance gene, movable element and type IV secretion system carried by the plasmid were marked. The missing portion indicates no expression or less than 50% genomic similarity in the strain genome compared to the reference genome.