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Mechanisms of Resistance

Whole-Genome Assembly of Klebsiella pneumoniae Coproducing NDM-1 and OXA-232 Carbapenemases Using Single-Molecule, Real-Time Sequencing

Yohei Doi, Tracy H. Hazen, Matthew Boitano, Yu-Chih Tsai, Tyson A. Clark, Jonas Korlach, David A. Rasko
Yohei Doi
aDivision of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
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Tracy H. Hazen
bInstitute for Genome Sciences, University of Maryland, Baltimore, Maryland, USA
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Matthew Boitano
cPacific Biosciences, Menlo Park, California, USA
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Yu-Chih Tsai
cPacific Biosciences, Menlo Park, California, USA
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Tyson A. Clark
cPacific Biosciences, Menlo Park, California, USA
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Jonas Korlach
cPacific Biosciences, Menlo Park, California, USA
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David A. Rasko
bInstitute for Genome Sciences, University of Maryland, Baltimore, Maryland, USA
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DOI: 10.1128/AAC.03180-14
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  • FIG 1
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    FIG 1

    BLAST comparison results for the chromosomes of K. pneumoniae strains PittNDM01 and MGH 78578. A large inversion was identified and verified by PCR in PittNDM01 in comparison with MGH 78578, covering 23.3% of the chromosome.

  • FIG 2
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    FIG 2

    Whole-genome phylogeny of K. pneumoniae PittNDM01 relative to other Klebsiella strains. Bootstrap values of ≥50 are shown. The scale bar indicates the distance of 0.04 nucleotide substitutions per site. The tree demonstrates that the core genome of K. pneumoniae PittNDM01 is similar to that of other K. pneumoniae isolates that have been sequenced.

  • FIG 3
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    FIG 3

    Overview of pPKPN1. The plasmid carries 12 resistance genes, including blaNDM-1, armA, and qnrB1, which are clustered in two regions of the plasmid containing class 1 integrons. pPKPN1 therefore confers resistance to the three key antimicrobial classes (carbapenems, aminoglycosides, and fluoroquinolones). The numbers indicate the positions on the plasmid.

  • FIG 4
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    FIG 4

    Alignment of the blaNDM-1 region of pPKPN1 and representative blaNDM-1-carrying plasmids. The antimicrobial resistance genes and mobile genetic elements are colored in red and brown, respectively. The region surrounding blaNDM-1 differed substantially among the plasmids, with pPKPN1 being especially rich in antimicrobial resistance genes, along with pNDM-CIT.

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  • TABLE 1

    Overall features of the K. pneumoniae PittNDM01 genome

    ParameterChromosomepPKPN01pPKPN02pPKPN03pPKPN04
    Size (bp)5,348,284283,371103,69470,8146,141
    No. of reads391,21819,9361,5051,31599
    G+C %57.5046.4151.0052.9252.19
    No. of predicted open reading frames4,940291101808
    Resistance gene(s)blaSHV-28, blaOXA-1, aac(6′)-Ib-cr, cat, macA/macB, fosAblaNDM-1, blaOXA-1, armA, aac(6′)-Ib-cr, aadA2, aphA6, qnrB1, catB4, mel, mph2, dfrA12, sul1NoneblaCTX-M-15, blaSHV-11, blaTEM-1, aadA1, aacC2, strB, ereC, cmlA5, sul1, sul2, arr-2blaOXA-232
  • TABLE 2

    Detection of methylation in K. pneumoniae PittNDM01

    MotifaModification typeNo. of motifs detectedNo. of motifs in genome% motifs detected
    5′-GATC-3′6mA63,45363,69899.6
    3′-CTAG-5′
    5′-CAACNNNNNCGT-3′6mA75675899.7
    3′-GTTGNNNNNGCA-5′75675899.7
    5′-GATGNNNNNNTTG-3′6mA1,0401,04399.7
    3′-CTACNNNNNNAAC-5′1,0371,04399.4
    5′-GTCANNNNNNNNTGT-3′6mA32232599.1
    3′-CAGTNNNNNNNNACA-5′32432599.7
    5′-CCWGG-3′5mC7,29626,52427.5
    3′-GGWCC-5′
    • ↵a The methylated positions in the methyltransferase recognition sequence motifs are in bold type.

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Whole-Genome Assembly of Klebsiella pneumoniae Coproducing NDM-1 and OXA-232 Carbapenemases Using Single-Molecule, Real-Time Sequencing
Yohei Doi, Tracy H. Hazen, Matthew Boitano, Yu-Chih Tsai, Tyson A. Clark, Jonas Korlach, David A. Rasko
Antimicrobial Agents and Chemotherapy Sep 2014, 58 (10) 5947-5953; DOI: 10.1128/AAC.03180-14

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Whole-Genome Assembly of Klebsiella pneumoniae Coproducing NDM-1 and OXA-232 Carbapenemases Using Single-Molecule, Real-Time Sequencing
Yohei Doi, Tracy H. Hazen, Matthew Boitano, Yu-Chih Tsai, Tyson A. Clark, Jonas Korlach, David A. Rasko
Antimicrobial Agents and Chemotherapy Sep 2014, 58 (10) 5947-5953; DOI: 10.1128/AAC.03180-14
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