Fig 1.
Genetic diversity of healthcare-associated K. pneumoniae.
A maximum parsimony phylogeny based on 49,094 core genome SNPs in 165 K. pneumoniae isolates and the reference genome MGH 78578 illustrate the diversity of K. pneumoniae pathogens. The consistency index of the phylogeny is 0.34, reflecting a high number of homoplasious SNPs and indicative of high levels of homologous recombination. (Non-homologous DNA is not analyzed, as it is not part of the core genome.) The main branches of the groups within ST258 are collapsed. In CG258, branches are colored by sequence type. Outer membrane protein sequence was matched by BLAST to a Genbank accession number, except in the case of OmpK36 where matches of high similarity were not always found, in which case the sample name was used as the identifier. The cps loci of all strains were characterized by the wzc and wzi sequences [76, 77] and full-length characterization where genome assemblies allowed.
Table 1.
Results of mapping Illumina sequencing reads from K. pneumoniae isolates to relevant reference genomes.
For further definition of the calculations see Materials and Methods.
Fig 2.
Phylogenies of CG258 and ST258 with large recombined regions removed.
(A) A maximum parsimony phylogeny based on 1,440 core genome SNPs in 138 CG258 isolates using NJST258_1 with the 1.06 Mbp region of recombination [20] masked as a reference reduces the genomic distance between ST258 and the rest of CG258. The consistency index of the maximum parsimony phylogeny is 0.95, indicating most SNPs in the core are vertically transferred. (B) A maximum parsimony phylogeny based on 1,425 core genome SNPs in 102 ST258 isolates, using NJST258_1 with the 215 kb region of recombination [15] masked as a reference illustrate the clonal nature and evolutionary history of ST258. The consistency index is 0.96 for the ST258 maximum parsimony phylogeny, indicating most SNPs in the core are vertically transferred. Analysis of antibiotic resistance genes, Tn4401, capsule type by wzc sequence, and plasmid incompatibility groups lend insight into the vertical versus horizontal transfer of these genetic elements. Complete SRST2 [65] and PlasmidFinder [71] results are in S2 Table.
Fig 3.
Projecting the evolutionary history of ST258.
BEAST analysis based on 1,425 core genome SNPs in 101 ST258 isolates with NJST258_1 reference genome, with the 215 kb region of recombination [15] masked, gives temporal context to the emergence of ST258, with key events and initial reports of KPC-producing K. pneumoniae in different countries plotted. Blue font indicates reports of KPC-producing K. pneumoniae, brown font is ST258. Green shading on the phylogeny shows lines of iterations of Bayesian analyses. The mean mutation rate of K. pneumoniae ST258 is 1.03 x 10−6 (95% HPD 8.09 x 10−7 to 1.24 x 10−6). The TMRCA for the ST258 clade is approximately 20 years ago, around 1995. The plot inset is a root-to-tip analysis of SNP accumulations for each isolate since the MRCA of ST258. The slope of the fit line is 4.66, which is close to the mutation rate calculated by BEAST ((1.03 x 10−6 substitutions per site per year) x (3.8 Mbp core genome size) = 3.9 SNPs per year).
Table 2.
SNP mutations and real-time PCR assays to detect CG258 and ST258.
Lowercase letters in the probes indicate the targeted SNP state.
Fig 4.
Characterization of three cps loci found in ST258 isolates.
Regions of identity are shaded and GenBank BLAST matches labeled. Putative glycosyltransferases are in green and hypothetical proteins are in blue. The IS5-like element disrupting the 5’ region of the US-FL-2011 cps locus is in red and yellow.
Fig 5.
OmpK35 alignment of all alleles found in the 167 isolates.
Sequences are labeled by Genbank accession number when they’re an exact match. WP_004141771 was the most frequently found complete protein in our isolates, and was used as the reference in the alignment. Dots are conserved sites, dashes are sites downstream of a premature stop codon. * US-OR-2010 represents all ST258 isolates in the study. ** These variants are in the divergent isolates US-PA-2001 and US-GA-2009b, not shown in Fig 1.
Fig 6.
OmpK36 alignment of all alleles found in the 167 isolates.
100% identity BLAST matches were not found for several sequences; sample names are used for these sequences. WP_002913005 was the most frequently found protein so was used as the reference. Sequence in green represents the extracellular loop regions of the protein. Dots are conserved sites, dashes are gaps or represent sites downstream of a premature stop codon. * This variant is not shown in Fig 1; it occurs in ST258 isolate US-GA-2007. ** These variants are in the divergent isolates US-PA-2001 and US-GA-2009b, not shown in Fig 1.
Fig 7.
OmpK37 alignment of all alleles found in the 167 isolates.
Sequence in green represents the extracellular loop regions of the protein assumed from the structure of OmpF by Doménech-Sánchez et al. [59]. Dots are conserved sites, dashes are gaps. ** These variants are in the divergent isolates US-PA-2001 and US-GA-2009b, not shown in Fig 1.