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Research Article

Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307

S. Wesley Long, Randall J. Olsen, Todd N. Eagar, Stephen B. Beres, Picheng Zhao, James J. Davis, Thomas Brettin, Fangfang Xia, James M. Musser
Steven J. Projan, Editor
S. Wesley Long
a Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas, USA
b Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York, USA
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Randall J. Olsen
a Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas, USA
b Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York, USA
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Todd N. Eagar
a Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas, USA
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Stephen B. Beres
a Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas, USA
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Picheng Zhao
a Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas, USA
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James J. Davis
c Computing, Environment and Life Sciences, Argonne National Laboratory, Argonne, Illinois, USA
d Computation Institute, University of Chicago, Chicago, Illinois, USA
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Thomas Brettin
c Computing, Environment and Life Sciences, Argonne National Laboratory, Argonne, Illinois, USA
d Computation Institute, University of Chicago, Chicago, Illinois, USA
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Fangfang Xia
c Computing, Environment and Life Sciences, Argonne National Laboratory, Argonne, Illinois, USA
d Computation Institute, University of Chicago, Chicago, Illinois, USA
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James M. Musser
a Center for Molecular and Translational Human Infectious Diseases Research, Department of Pathology and Genomic Medicine, Houston Methodist Research Institute and Houston Methodist Hospital, Houston, Texas, USA
b Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York, USA
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Steven J. Projan
MedImmune
Roles: Editor
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Michael Dunne
bioMérieux, Inc.
Roles: Solicited external reviewer
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Robert Bonomo
Louis Stokes Veterans Affairs Medical Center
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DOI: 10.1128/mBio.00489-17
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  • FIG 1 
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    FIG 1 

    Estimates of genetic relationships among 1,777 ESBL-producing K. pneumoniae strains recovered from infected patients in a multiple-hospital system in the Houston metropolitan area. Polymorphisms were called against the genome of CG258 K. pneumoniae reference strain NJST258_2 (NCBI GenBank accession no. CP006918.1 ). Colors represent the major clonal groups, including CG258 (red) and CG307 (blue), and the remaining heterogeneous STs (gray). Phylogenetic relationships were defined by the neighbor-joining method in FastTreeMP with double precision. The core genome was defined as the chromosomal sequence with phage sequence regions excluded. To simplify presentation, outliers have been cropped out of frame at 3 and 9 o’clock. The outlier strains, which were identified as K. pneumoniae by matrix-assisted laser desorption ionization mass spectrometry and MLST, may be allied with the genomes of some strains referred to as K. quasipneumoniae and K. variicola.

  • FIG 2 
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    FIG 2 

    Spatial relationships of ESBL-producing K. pneumoniae strains from the multiple hospitals in the Houston Methodist system with clonal groups and associated patient deaths. This circular cladogram displays the first isolate from each patient, color coded by the five hospitals of origin (teal, yellow, red, purple, and gray). The circles surrounding the cladogram, from innermost to outermost, indicate the clonal group of the strain, yersiniabactin locus, colibactin locus, and patient death. In the innermost circle, the CG258 strains are red and the CG307 strains are blue. Other clonal types are indicated with a gray bar. The yersiniabactin circle is color coded to indicate which yersiniabactin locus is present, if any, with the two most common loci, ybt17 and ybt10, in yellow and teal, respectively; all other ybt loci are gray. The colibactin circle is purple to indicate the presence of clb3, the only colibactin synthesis locus detected in our collection. In the outermost circle, in-hospital death is indicated by a black line. For patients with multiple strains, only the first isolate from the patient is represented on the tree.

  • FIG 3 
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    FIG 3 

    ESBL-producing K. pneumoniae strains recovered during the course of this study. The first strain recovered from each patient is graphed (circles, green linear regression). The number of strains classified as CG307 (triangles, blue linear regression) and CG258 (inverted triangles, red linear regression) are also shown. This figure demonstrates that CG258 and CG307 strains were abundant throughout the study period.

  • FIG 4 
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    FIG 4 

    Gene content differences between the reference genomes of CG258 and CG307. CG258 is the globally abundant strain of K. pneumoniae, and CG307 is the locally abundant strain in our study. Bidirectional BLAST was performed by using the PATRIC resource to illustrate the differences in gene content between these two reference genomes. The color indicates the percent identity of the BLAST hit for each gene, with darker shading indicating a bidirectional hit and lighter shading indicating a unidirectional hit (top). A progressive Mauve alignment of the CG258 and CG307 reference genomes is shown. Local colinear blocks are organized by color (bottom). NJST258_2 is the CG258 reference genome, and KPN11 is the CG307 reference assembly.

  • FIG 5 
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    FIG 5 

    Genetic variation in ESBL-producing K. pneumoniae clinical strains in the same patient over time. (A) Clonal group of each strain recovered from 196 patients with multiple strains. (B) Clonal group of each isolate recovered from 10 patients (numbered 1 to 10) with multiple strains spanning the longest period. Each circle represents one isolate. The vertically overlapping circles shown for patient 5 indicate that two different strains were recovered from different specimens on the same day or consecutive days.

  • FIG 6 
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    FIG 6 

    RNA-seq analysis of 10 genomically diverse ESBL-producing K. pneumoniae strains. Strains of CG258 (n = 4, red), CG307 (n = 3, blue), and other STs (n = 3, gray) were analyzed. (A) Growth curves of strains. RNA-seq analysis of duplicate cultures was done (the mean absorbance of each strain pair is shown); the growth curves of all of the strains are similar. Cells were harvested at the mid-exponential phase of growth (OD600 of 0.6; horizontal dashed green line). (B) RNA-seq transcriptome data for chromosomally located genes of each strain were compared to those of CG258 reference strain BK30684. Plotted are expression relationships among the strains based on principal component 1 (PC1) and principal component 2 (PC2), which account for the two largest unrelated variances in the data. The strains are distributed across both axes, showing transcriptome diversity within and between CGs. (C) Number of genes with significantly altered expression relative to reference strain BK30684. Plotted are the numbers of genes with significantly increased (positive y axis) and decreased (negative y axis) expression for each strain. (D) Box plot summarizing data from the 10 strains combined or within CGs.

  • FIG 7 
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    FIG 7 

    Host immune response to ESBL-producing K. pneumoniae in a mouse model of pneumonia. Flow cytometry analysis of leukocytes recovered from infected lungs, including neutrophils (A), monocytes (B), T cells (C), B cells (D), alveolar macrophages (E), CD4 T cells (F), γδ T cells (G), IL-17-producing CD4 T cells (H), and IL-17-producing γδ T cells (I). (J) IL-17-producing γδ T cells were also recovered from the spleen.

  • FIG 8 
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    FIG 8 

    Receiver operating characteristic curves of 16 AdaBoost-based classifiers predicting antimicrobial resistance. Antibiotic agent abbreviations: AMK, amikacin; ATM, aztreonam; FEP, cefepime; FOX, cefoxitin; CIP, ciprofloxacin; ETP, ertapenem; FOF, fosfomycin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; MEM, meropenem; TZP, piperacillin-tazobactam; TET, tetracycline; TGC, tigecycline; TOB, tobramycin; SXT, trimethoprim-sulfamethoxazole. AUC is area under the concentration-time curve. The F1 score, the harmonic mean of precision and recall, is commonly used to compare classification methods. Data are the results of a 10-fold cross validation.

Tables

  • Figures
  • Supplemental Material
  • TABLE 1 

    Summary of clonal groups and specimen sources of 1,777 K. pneumoniae isolates

    Source(s)No. of isolates (% of total)
    CG258CG307Other CGTotal
    Blood34 (1.9)54 (3.0)59 (3.3)147 (8.3)
    Respiratory130 (7.3)111 (6.2)143 (8.0)384 (21.6)
    Urine220 (12.4)374 (21.0)333 (18.7)927 (52.2)
    Othera90 (5.1)105 (5.9)124 (7.0)319 (18.0)
    All474 (26.7)644 (36.2)659 (37.1)1,777 (100)
    • ↵ a Other specimen sources include tissue, wounds, and body fluids.

  • TABLE 2 

    bla NDM-1-containing strains recovered from six patients (A to F)

    StrainPatientSTDate collectedSourceST geographic association(s)
    528AST1412/2012SputumIndia
    537AST1412/2012TracheaIndia
    553ASLVa of ST1412/2012WoundIndia
    603ASLV of ST141/2013UrineIndia
    658AST142/2013BloodIndia
    755BSLV of ST144/2013UrineIndia
    764BST144/2013UrineIndia
    1010BST1410/2013UrineIndia
    1273BSLV of ST141/2014UrineIndia
    1145CST1412/2013UrineIndia
    1402CSLV of ST144/2014BileIndia
    1426DST375/2014UrineIndia/United Kingdom
    1458DST375/2014UrineIndia/United Kingdom
    1481EST9066/2014UrineIsrael
    1844FST30710/2014UrineHouston
    • ↵ a SLV, single-locus variant.

Supplemental Material

  • Figures
  • Tables
  • TABLE S1 

    (A) Summary of the CGs/STs, capsule loci, collection dates, sources, yersiniabactin loci, and colibactin loci of 1,777 K. pneumoniae strains. (B) Summary of selected antimicrobial resistance genes. (C) Summary of the top six plasmid replicons from 1,777 K. pneumoniae strains. Download TABLE S1, XLSX file, 0.3 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S1 

    Antimicrobial agent resistances and plasmid contents of ESBL-producing K. pneumoniae strains. Plasmid replicons and antimicrobial resistance gene contents were identified by using SRST2. Neighbor-joining phylogenetic relationships relative to NJST258_2 are shown in a cladogram for the major clades CG258 (red) and CG307 (blue) and the remaining heterogeneous diverse CGs (gray). The presence of a KPC gene allele is indicated in the first track as KPC-2 (green) or KPC-3 (purple). The six tracks to the right indicate the presence (black) or absence (white) of plasmid replicons in the replicon types listed. To prevent bias from multiple strains collected from the same patient, only the first isolate per patient is shown. Download FIG S1, PDF file, 0.4 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • TABLE S2 

    STs of the strains recovered in this study. Download TABLE S2, PDF file, 0.1 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • TABLE S3 

    Strains sequenced to closure in this study. Download TABLE S3, PDF file, 0.04 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • TABLE S4 

    Annotated genes present in NJST258_2 but not in CG307 strain KPN11. Download TABLE S4, PDF file, 0.1 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • TABLE S5 

    Annotated genes present in KPN11 but not in NJST258_2. Download TABLE S5, PDF file, 0.1 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S2 

    Timeline for recovery of strains with blaNDM-1-positive plasmids from six patients. The patients (A to F) are shown relative to the calendar (below). Each box represents a single episode of care. The number indicates the count of blaNDM-1 -positive strains recovered. ST14 (blue) and ST37 (red) strains are classically associated with NDM-1. CG307 strains (gray) are numerically dominant in Houston but have not been previously associated with blaNDM-1. Download FIG S2, PDF file, 0.04 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • TABLE S6 

    Strains used for RNA-seq analysis. Genome analysis detected the presence (+) or absence (−) of the genes encoding KPC, NDM-1, and OXA-48. Download TABLE S6, PDF file, 0.1 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

  • FIG S3 

    RNA-seq analysis of 10 genomically diverse K. pneumoniae strains. (A) Box plot summarizing data from the 10 strains combined, GC258 strains, CG307 strains, and diverse ST strains. Reads were mapped to the annotated assembly of the chromosome of CG307 strain KPN11. (B) Reads were mapped to the annotated assembly of the chromosome of CG14 strain KPN528. (C, D) RNA-seq transcriptome data for reference strain BK30684 and clinical strains KPN528 (plasmid analysis detected the blaNDM-1 and OXA-48 genes) and KPN1402 (plasmid analysis detected the blaNDM-1 gene). Reads were mapped to the blaNDM-1-containing plasmid pPKPN1 or OXA-48-containing plasmid pKP112. (E) Reads were mapped to KPC-containing plasmid pNJST258C2. (F) Reads were mapped to the ybt-containing chromosome of CG14 strain KPN528. Normalized counts are graphed as reads per kilobase per million (RPKM) mapped reads for each gene. Download FIG S3, TIF file, 1.3 MB.

    Copyright © 2017 Long et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .

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Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307
S. Wesley Long, Randall J. Olsen, Todd N. Eagar, Stephen B. Beres, Picheng Zhao, James J. Davis, Thomas Brettin, Fangfang Xia, James M. Musser
mBio May 2017, 8 (3) e00489-17; DOI: 10.1128/mBio.00489-17

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Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307
S. Wesley Long, Randall J. Olsen, Todd N. Eagar, Stephen B. Beres, Picheng Zhao, James J. Davis, Thomas Brettin, Fangfang Xia, James M. Musser
mBio May 2017, 8 (3) e00489-17; DOI: 10.1128/mBio.00489-17
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KEYWORDS

Genome, Bacterial
Klebsiella Infections
Klebsiella pneumoniae
beta-lactamases

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