Plasmid diversity among genetically related Klebsiella pneumoniae blaKPC-2 and blaKPC-3 isolates collected in the Dutch national surveillance

Abstract

Carbapenemase-producing Klebsiella pneumoniae emerged as a nosocomial pathogen causing morbidity and mortality in patients. For infection prevention it is important to track the spread of K. pneumoniae and its plasmids between patients. Therefore, the major aim was to recapitulate the contents and diversity of the plasmids of genetically related K. pneumoniae strains harboring the beta-lactamase gene bla_KPC-2 or bla_KPC-3 to determine their dissemination in the Netherlands and the former Dutch Caribbean islands from 2014 to 2019. Next-generation sequencing was combined with long-read third-generation sequencing to reconstruct 22 plasmids. wgMLST revealed five genetic clusters comprised of K. pneumoniae bla_KPC-2 isolates and four clusters consisted of bla_KPC-3 isolates. KpnCluster-019 bla_KPC-2 isolates were found both in the Netherlands and the Caribbean islands, while bla_KPC-3 cluster isolates only in the Netherlands. Each K. pneumoniae bla_KPC-2 or bla_KPC-3 cluster was characterized by a distinct resistome and plasmidome. However, the large and medium plasmids contained a variety of antibiotic resistance genes, conjugation machinery, cation transport systems, transposons, toxin/antitoxins, insertion sequences and prophage-related elements. The small plasmids carried genes implicated in virulence. Thus, implementing long-read plasmid sequencing analysis for K. pneumoniae surveillance provided important insights in the transmission of a KpnCluster-019 bla_KPC-2 strain between the Netherlands and the Caribbean.

Figure 1

Minimum spanning tree based on wgMLST of 478 sequenced K. pneumoniae isolates. Circles represent K. pneumoniae isolates, and the sizes of the circles indicate the numbers of isolates. Lines connecting the circles represent the genetic distance in numbers of alleles; the longer the connecting line, the larger the genetic distance. K. pneumoniae bla_KPC-2 isolates were marked blue and K. pneumoniae bla_KPC-3 were marked magenta. K. pneumoniae bla_KPC-2 or bla_KPC-3 cluster isolates that were sequenced with TGS were marked green. Genetic clusters were indicated with either a blue or a magenta halo around the circles, if two or more isolates differ ≤ 20 alleles. A categorical coefficient was used for the clustering. Cluster names are indicated. Inset: genetic distance between the KpnClusters in which the allelic difference is indicated by numbers.

Figure 2

Resistome of K. pneumoniae bla_KPC-2 and bla_KPC-3 cluster isolates. K. pneumoniae bla_KPC-2 and bla_KPC-3 cluster isolates were indicated on the y-axis and AMR genes on the x-axis. All isolates analysed contained the fosA, oqxA and oqxB AMR genes and were not included in this figure. The clustering was based on the presence (squares) and absence of AMR genes. Antibiotic classes are indicated above the AMR genes in different colors. Resistance genes in K. pneumoniae bla_KPC-2 or bla_KPC-3 cluster isolates that were sequenced with TGS were marked with green squares. Genetic relatedness was depicted in an UPGMA tree in which K. pneumoniae bla_KPC-2 isolates were marked with blue branches, and K. pneumoniae bla_KPC-3 were marked magenta. Dutch KpnCluster-019 isolates were marked with an *. A dotted line marks the 85% cut off.

Figure 3

Antimicrobial resistance genes on chromosomes and plasmids. The presence of AMR genes among the 22 plasmids of seven TGS sequenced isolates is indicated with black squares and for the chromosomes using green squares. Chromosomes (cRIVM_C0xxxx) and plasmids (pRIVM_C0xxxx) are depicted on the Y-axis, and AMR genes on the x-axis. Antibiotic classes are indicated above the AMR genes in different colors.

Figure 4

K. pneumoniae plasmid gene content. An UPGMA clustering was performed based on the plasmid DNA sequence for the determination of the genetic relation among the 22 plasmids. Similarity is indicated on the y-axis using a scale from 0 (not similar) to 100% (identical). A similarity of ≥ 85 to 100% is regarded as the same plasmid. The plasmids are indicated on the x-axis. The presence (black squares) and absence is indicated of annotated genes among the 22 plasmids of seven TGS sequenced isolates. If a gene was present twice, blue squares were used and more than 2, red squares were used. Colors indicated different groups of genes with a specific function. In the UPGMA tree, large plasmids are indicated in red, medium plasmids in black and small plasmids in green color.

Figure 5

K. pneumoniae plasmid-localized transposases. The presence (black squares) and absence is indicated of annotated transposases among the 22 plasmids of six TGS sequenced isolates. The plasmids are indicated on the x-axis. If a transposon was present twice, blue squares were used and more than 2, red squares were used. The light grey area indicates specific transposons found in only one plasmid. In the UPGMA tree, large plasmids are indicated in red, medium plasmids in black and small plasmids in green color.