An Analysis of the Epidemic of Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae: Convergence of Two Evolutionary Mechanisms Creates the "Perfect Storm"

Abstract

Background: Carbapenem resistance is a critical healthcare challenge worldwide. Particularly concerning is the widespread dissemination of Klebsiella pneumoniae carbapenemase (KPC). Klebsiella pneumoniae harboring blaKPC (KPC-Kpn) is endemic in many areas including the United States, where the epidemic was primarily mediated by the clonal dissemination of Kpn ST258. We postulated that the spread of blaKPC in other regions occurs by different and more complex mechanisms. To test this, we investigated the evolution and dynamics of spread of KPC-Kpn in Colombia, where KPC became rapidly endemic after emerging in 2005.

Methods: We sequenced the genomes of 133 clinical isolates recovered from 24 tertiary care hospitals located in 10 cities throughout Colombia, between 2002 (before the emergence of KPC-Kpn) and 2014. Phylogenetic reconstructions and evolutionary mapping were performed to determine temporal and genetic associations between the isolates.

Results: Our results indicate that the start of the epidemic was driven by horizontal dissemination of mobile genetic elements carrying blaKPC-2, followed by the introduction and subsequent spread of clonal group 258 (CG258) isolates containing blaKPC-3.

Conclusions: The combination of 2 evolutionary mechanisms of KPC-Kpn within a challenged health system of a developing country created the "perfect storm" for sustained endemicity of these multidrug-resistant organisms in Colombia.

Keywords: Colombia; KPC; Klebsiella pneumoniae.

Figure 1.

Map of Colombia showing the locations where isolates were collected. The 10 cities included in the study are located in the most densely populated areas of the country (as indicated by darker hues). The number of hospitals per city is indicated by colored circles.

Figure 2.

Resistance determinants by antibiotic class and plasmid replicons identified in the 133 Klebsiella pneumoniae isolates included in the study. Isolates are chronologically organized by year of isolation (2002 to 2014). Klebsiella pneumoniae isolates belonging to ST258 are identified by a red triangle; isolates belonging to ST512 are identified by a black triangle; first bla_KPC-2 harboring isolates recovered in 2005 are denoted by black circles. For all classes of resistance determinants, white means absence and any color means presence. Gray scale indicates the number of copies of the same gene, regardless of the variant found (light, 1 copy; medium, 2 copies; dark, 3 copies). β-lactamases of special interest are highlighted in color: bla_CTX-M-12 (green), bla_CTX-M-15 (orange), bla_KPC-2 (red), and bla_KPC-3 (blue). Resistance determinants detected in the isolates include: narrow spectrum β-lactamases (TEM-1, SHV-1 SHV-11, OKP-1, LEN-1/12/16); extended spectrum β-lactamases (SHV-5/11/12/25/27/31/33/101/108/129, CTX-M 2/12/15/96, OXA-1/2/9/47), carbapenemases (KPC-2/3, VIM-24); aminoglycoside-modifying enzymes [aminoglycoside N-acetyltransferases (AAC), aminoglycoside O-nucleotidyltranferases (ANT), aminoglycoside O-phosphotransferases (APH)]; 16S rRNA-modifying enzymes [methyltransferases (RMT)]; quinolone conferring resistance enzymes [plasmid-mediated quinolone-resistance (QNR), OqxA-B efflux pump, and N-acetyltransferase AAC(6′)-Ib-cr]; fosfomycin resistance proteins (FosA, FosB); macrolides, lincosamides, and streptogramins (MLS) resistance determinants [ErmB rRNA methylase (ERM) and macrolide phosphorylases (MphA/B)]; chloramphenicol resistance determinants [chloramphenicol acetyltransferase (CatA1) and chloramphenicol resistance efflux protein, CmlA]; rifampin ADP-ribosyltransferase (Arr); trimethoprim dihydrofolate reductase (DfrA); tetracycline efflux pump (TetA-C); sul1, sul2, and sul3 genes encoding dihydropteroate synthetase (DHPS). For plasmid replicons, an 80% similarity cutoff in PlasmidFinder was used; gray indicates presence, and color indicates plasmid types of interest: IncFIB(pQIL), cyan; IncI2, purple; IncP6, magenta.

Figure 3.

Transposable element carrying bla_KPC, virulence factors and capsular diversity as revealed by wzi typing in the bla_KPC harboring K. pneumoniae isolates, identified by year of isolation and ST. Indicated in red are isolates corresponding to the first bla_KPC-harboring isolates reported in Colombia; in blue is the oldest bla_KPC-harboring CG258 isolate found in our collection; in green is the index isolate from the “Israeli clone”. Genes encoding for the main virulence factors described in K. pneumoniae are grouped (type 3 fimbriae, yerisiniabactin, iron transporter, colibactin); a dot indicates presence of at least one gene from the group, blank indicates absence. Each wzi type is indicated by a different color. For the transposable element, ND indicates not determined.

Figure 4.

Circular representation of the transformed phylogenetic tree (ignoring branch lengths) generated by RAxML and drawn using iTOL version 3.2.4, showing the genetic relationships among 133 Colombian Klebsiella pneumoniae isolates. Isolates in gray correspond to K. pneumoniae isolates from different parts of the world added as reference. Isolates in red correspond to the first 2 Colombian K. pneumoniae isolates described, carrying bla_KPC-2. Isolate in purple corresponds to the oldest ST258 isolate in our collection. Isolate in blue corresponds to the oldest ST258 KPC-Kpn isolate in our collection. Isolate in green corresponds to the index isolate from the “Israeli outbreak” in Medellin. ^aFirst KPC-Kpn reported in the world (North Carolina).

Figure 5.

Circular representation of the transformed phylogenetic tree generated by RAxML and drawn using iTOL version 3.2.4, showing the genetic relationships among 331 CG258 Klebsiella pneumoniae isolates. Isolates shown in bold correspond to Colombian isolates collected in this study, all other isolates were obtained from GenBank and are named with the respective country where they were collected followed by 2 digits indicating the year of isolation. Outer ring indicates the sequence type (ST); inner ring indicates the KPC variant carried by each isolate. Isolates highlighted in pink indicate the cluster including the “Israeli outbreak”-related Colombian isolates (ST512), as well as other worldwide isolates found on GenBank. Isolates highlighted in blue indicate the cluster including the oldest ST258 isolate in our collection (KPC^48a) as well as most of the other ST258 Colombian isolates reported in this study.