Integrated chromosomal and plasmid sequence analyses reveal diverse modes of carbapenemase gene spread among Klebsiella pneumoniae

Abstract

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of Klebsiella pneumoniae isolates (n = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, bla_OXA-48-like genes have spread primarily via the single epidemic pOXA-48-like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, bla_VIM and bla_NDM genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, bla_KPC genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying bla_KPC Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.

Keywords: Klebsiella pneumoniae; carbapenem resistance; carbapenemase genes; genomics; plasmids.

Fig. 1.

High prevalence of the pOXA-48–like plasmid sequence across bla_OXA-48-like–carrying isolates. (A) The phylogenetic tree includes 248 bla_OXA-48-like–carrying isolates from K. pneumoniae sensu stricto (the single bla_OXA-48-like–carrying isolate from K. quasipneumoniae was excluded). All non–bla_OXA-48-like–carrying isolates, which would be interspersed among the isolates here, were also excluded. Long read-sequenced isolates are marked with a diamond or circle, depending on whether they carry bla_OXA-48-like on a putative plasmid sequence or the chromosome, respectively. The diamond colors represent distinct plasmids that were obtained. The first two columns, from left to right, show the genetic context group of isolates assigned using the short-read assembly contigs (ambiguous isolates not assigned to any group are in gray) and the bla_OXA-48-like variant. Remaining columns show the percentage length of bla_OXA-48-like–carrying plasmid sequences obtained from the hybrid assemblies that are mapped by short reads of the 248 bla_OXA-48-like–carrying isolates (note the nonlinear color gradient). Mapping is shown to single representatives of the IncL/M(pOXA48) (i.e., pOXA-48–like) and ColKP3 plasmids since several highly similar plasmids were obtained. Each plasmid sequence is indicated by a diamond of the same color as that indicating the isolate(s) in the tree from which the plasmid was recovered. Mapping data for two shorter bla_OXA-48-like–carrying putative plasmids (20.3 and 2.5 kb) are not shown. C, circular; NC, noncircular. (B) The tanglegram links phylogenetic trees constructed using SNPs in the core genome (Left) and the pOXA48-like plasmid (Right). Both trees are midpoint rooted and include 207 isolates from K. pneumoniae sensu stricto that had mapping and bases called (A/T/C/G rather than N) at ≥90% of positions in the plasmid reference sequence. These comprise 202 isolates with bla_OXA-48-like genes and 5 with bla_VIM genes, the latter of which are shaded in yellow in the plasmid tree. Lines have been drawn between tips in the trees representing the same isolate, while the tree branches were rotated to minimize the number of overlapping lines required. The lines are colored by the nucleotide sequence variant of the plasmid. Unique plasmid variants are colored black.

Fig. 2.

Plasmids carrying bla_VIM and bla_NDM genes are associated with individual clonal expansions. The phylogenetic trees show 56 bla_VIM-carrying isolates (A) and 79 bla_NDM-carrying isolates (B) from K. pneumoniae sensu stricto. We excluded all non–bla_VIM and non–bla_NDM-carrying isolates, respectively, which would be interspersed among the isolates here. Long read-sequenced isolates are marked next to the tree with a diamond or circle, depending on whether they carry the carbapenemase gene on a putative plasmid sequence or chromosome, respectively. The diamond colors represent distinct carbapenemase-carrying plasmids that were obtained. Columns, from left to right, show the genetic context group of isolates assigned using the short-read assembly contigs (ambiguous isolates not assigned to any group are in gray), the country of isolation, and the gene variant (for bla_VIM genes only as all bla_NDM genes were bla_NDM-1). Remaining columns show the percentage length of putative plasmids carrying bla_VIM (A) and bla_NDM (B) genes obtained from the hybrid assemblies that were mapped by short reads (note the nonlinear color gradient). Each putative plasmid sequence is indicated by a diamond with the same color as that indicating the isolate in the tree from which the plasmid was recovered. Mapping data for one shorter bla_VIM-carrying putative plasmid are not shown (EuSCAPE_GR075—2.9 kb). C, circular; NC, noncircular.

Fig. 3.

Comparison of 24 circularized bla_KPC-carrying plasmids shows dominance of two major IncF backbone types. The phylogenetic tree contains 311 bla_KPC-carrying isolates from K. pneumoniae sensu stricto (the single bla_KPC-carrying isolate from K. variicola is excluded). Twenty-four isolates from which circularized bla_KPC-carrying plasmids were obtained are marked by red circles in the tree. The heat map shows the percentage of bases in each plasmid that could be aligned to each of the other plasmids using NUCmer (the row and column orders are the same). Dotted lines link the 24 long read-sequenced isolates in the phylogenetic tree to their respective plasmids in the heat map.

Fig. 4.

High congruence between pKpQIL-like and IncX3 plasmid phylogenies with the core genome phylogeny of ST258/512 reveals shared evolutionary histories. Each tanglegram comprises a phylogeny of the ST258/512 lineage constructed using all SNPs in the core genome (mapping) alignment and either the pKpQIL-like (A) or IncX3 (B) plasmids. The core genome phylogenies include all 236 ST258/512 isolates and were rooted using an outgroup. Ninety-one pKpQIL-like and 135 IncX3 plasmid sequences from isolates that had bases (A/T/C/G) called at ≥99% positions in the plasmid reference were included in the plasmid phylogenies. Lines are drawn between tips in the two trees representing the same isolate. The solid purple lines indicate isolates that were found to carry bla_KPC on a pKpQIL-like (A) or IncX3 (B) plasmid in the hybrid assemblies. Red lines (in B only) indicate isolates that were found to carry bla_KPC on an alternative plasmid to an IncX3 plasmid in the hybrid assemblies.

Fig. 5.

Movement of bla_KPC genes between plasmids in the ST258/512 lineage. (A) The phylogenetic tree contains 236 ST258/512 isolates and was constructed using SNPs from a core genome (mapping) alignment. Thirty-two long read-sequenced isolates carrying bla_KPC on a putative plasmid sequence are indicated by small circles on the tree tips. Putative plasmid sequences derived from the hybrid genome assemblies with at least one known replicon type and/or containing bla_KPC are depicted next to the tree. These are scaled by size and colored by any replicon types found in the sequence. A star indicates the presence of bla_KPC within these sequences. (B) Metadata columns, from left to right, show the bla_KPC variant, the Tn4401 variant, the 10-bp left and right flanking regions of Tn4401, and presence or absence of eight plasmid replicon types that are associated with bla_KPC in the hybrid assemblies. Tn4401a-N1 to Tn4401a-N7 represent novel SNP variants of the structural variant, Tn4401a. Tn4401-NS1 represents a novel structural variant of Tn4401. NA, not applicable.