Accessory Genomes Drive Independent Spread of Carbapenem-Resistant Klebsiella pneumoniae Clonal Groups 258 and 307 in Houston, TX

Abstract

Carbapenem-resistant Klebsiella pneumoniae (CRKp) is an urgent public health threat. Worldwide dissemination of CRKp has been largely attributed to clonal group (CG) 258. However, recent evidence indicates the global emergence of a CRKp CG307 lineage. Houston, TX, is the first large city in the United States with detected cocirculation of both CRKp CG307 and CG258. We sought to characterize the genomic and clinical factors contributing to the parallel endemic spread of CG258 and CG307. CRKp isolates were collected as part of the prospective, Consortium on Resistance against Carbapenems in Klebsiella and other Enterobacterales 2 (CRACKLE-2) study. Hybrid short-read and long-read genome assemblies were generated from 119 CRKp isolates (95 originated from Houston hospitals). A comprehensive characterization of phylogenies, gene transfer, and plasmid content with pan-genome analysis was performed on all CRKp isolates. Plasmid mating experiments were performed with CG307 and CG258 isolates of interest. Dissection of the accessory genomes suggested independent evolution and limited horizontal gene transfer between CG307 and CG258 lineages. CG307 contained a diverse repertoire of mobile genetic elements, which were shared with other non-CG258 K. pneumoniae isolates. Three unique clades of Houston CG307 isolates clustered distinctly from other global CG307 isolates, indicating potential selective adaptation of particular CG307 lineages to their respective geographical niches. CG307 strains were often isolated from the urine of hospitalized patients, likely serving as important reservoirs for genes encoding carbapenemases and extended-spectrum β-lactamases. Our findings suggest parallel cocirculation of high-risk lineages with potentially divergent evolution. IMPORTANCE The prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKp) infections in nosocomial settings remains a public health challenge. High-risk clones such as clonal group 258 (CG258) are particularly concerning due to their association with bla_KPC carriage, which can severely complicate antimicrobial treatments. There is a recent emergence of clonal group 307 (CG307) worldwide with little understanding of how this successful clone has been able to adapt while cocirculating with CG258. We provide the first evidence of potentially divergent evolution between CG258 and CG307 with limited sharing of adaptive genes. Houston, TX, is home to the largest medical center in the world, with a large influx of domestic and international patients. Thus, our unique geographical setting, where two pandemic strains of CRKp are circulating, provides an indication of how differential accessory genome content can drive stable, endemic populations of CRKp. Pan-genomic analyses such as these can reveal unique signatures of successful CRKp dissemination, such as the CG307-associated plasmid (pCG307_HTX), and provide invaluable insights into the surveillance of local carbapenem-resistant Enterobacterales (CRE) epidemiology.

Keywords: CG258; CG307; carbapenem-resistant Klebsiella pneumoniae; divergent evolution; genomic surveillance; mobile genetic elements.

FIG 1

Core gene pangenomic population structure of 94 CRKp isolates in the Houston, TX, cohort. Maximum-likelihood phylogenetic, midpoint rooted tree demonstrating the two predominant cocirculating clades, CG258 (blue clade) and CG307 (red clade). Internal node bootstrap values of ≥95% are denoted as black circles. Label backgrounds indicate the 6 hierarchical clustering groups identified using Bayesian analysis of population structure. Clonal groups associated with each hierarchical group are as follows: CG258 (blue), CG307 (red), CG147 (green), CG15 (pink), CG20 (purple), and other CGs (yellow). The first column indicates each of the 10 hospital sites in Houston, TX. The second column indicates carbapenemase carriage. The third column indicates if carbapenemase carriage was chromosomal. The final 13 columns indicate the plasmid vectors that were identified to have at least one sample with carbapenemase carriage. Carbapenemase carriage of each plasmid vector is distinguished by carbapenemase positive (filled shape) and carbapenemase negative (empty shape).

FIG 2

Core SNP-inferred Maximum Likelihood (ML) phylogenomic trees of CG258 and CG307 isolates using a midpoint rooted tree. Internal node bootstrap values of ≥95% are denoted as black circles. Isolate label backgrounds indicate hierarchical clustering groups identified using Bayesian analysis of population structure for each respective sublineage. Legend designations are as follows: (1) region from which isolate was collected, (2) capsular synthesis/lipopolysaccharide (LPS) allele type, (3) ICEKp variant with yersiniabactin and colibactin gene cluster lineages listed, (4) carbapenemase carriage status, (5) ESBL carriage (CG258)/bla_CTX-M-15 copy number (CG307). (A) CG258 core SNP phylogeny using a reference-based alignment with a C268 isolate. (B) CG307 core SNP phylogeny using reference-based alignment with a C246 isolate. Stars in the bla_CTX-M-15 copy number column (i.e., column 5) indicate an isolate with one truncated bla_CTX-M-15 copy.

FIG 3

Population structure of previously characterized CG307 isolates with the CG307 Houston CRACKLE II isolates. Maximum-likelihood inferred phylogeny of CG307 isolates (n = 798) using C234 as a reference for core gene alignment with midpoint rooting. The branch label background corresponds to the hierBAPS predicted clade. The outer ring indicates the region where the isolate was collected. Clades I, III, and IV are predominantly made up of Houston isolates. Clade II is disseminated worldwide and shares a paraphyletic relationship with clade I.

FIG 4

Clustering of CRKP isolates with predicted protein functional characterization of the accessory genome. (A and B) t-Distributed stochastic neighbor embedding (t-SNE) two-dimensional (2-D) plot of accessory genome clustering of CRKp isolates. Clonal group is indicated by shape. ‡, Georgia isolate (C682) that clusters with Houston CG307 isolates. (A) Geographical region stratification of isolates. (B) Cluster group prediction (k = 4) using a PAM algorithm to determine cluster assignment. *, Exceptions to CG258 cluster-to-clade correlation. (C) Stacked bar chart of Cluster of Orthologous Genes (COGs) functional category proportions based on annotated genes found in the accessory genome. The n above each group indicates the absolute count of COGs identified in each clonal group per each genome. (D) The proportion of functionally annotated accessory genes differs significantly across pairwise comparisons of clonal groups as demonstrated in the scatterplot, which plots CG258 versus CG307 relative frequency proportions of COG functional groups denoted by the COG group letter (defined in panel C), with significant adjusted P values (Fisher’s exact test) indicated for a greater proportion of CG258 (blue) or CG307 (red) for each group labeled accordingly. These COG function group proportions exclude “S – unknown function” and “undefined homologs.” Nonsignificant COG functional group differences are labeled in gray.

FIG 5

Dendrogram and heatmap of plasmidome mash distances. Legends for row and column labels are listed to the left of each graph. Each dendrogram is constructed through agglomerative hierarchical clustering using an “average” linkage. (A) Plasmidome mash distance matrix by isolate (n = 121). The Legend is labeled as follows: (1) region, (2) clonal group, (3) carbapenemase. There is a noted primary clustering group of CG258 isolates (blue-labeled clade), whereas there are two Houston-based CG307 clustering groups (red-labeled clades) with diffuse clustering occurring with other CGs. (B) Plasmidome mash distance matrix by plasmid type (n = 295) with small, primarily ColE1-like plasmids excluded from analysis. The legend is labeled as follows: (1) region, (2) clonal group, (3) plasmid type, (4) carbapenemase. This analysis shows clustering of primarily CG258 X2-type plasmids, multireplicon FIIK-type plasmids, and pKpQIL plasmids indicated with blue-labeled cluster groups. This is in contrast to one primary CG307 plasmid cluster group which includes R-type plasmids as well the novel pCG307_HTX plasmid associated with the Houston group.