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Epidemiology and Surveillance

Genome Sequencing of an Extended Series of NDM-Producing Klebsiella pneumoniae Isolates from Neonatal Infections in a Nepali Hospital Characterizes the Extent of Community- versus Hospital-Associated Transmission in an Endemic Setting

N. Stoesser, A. Giess, E. M. Batty, A. E. Sheppard, A. S. Walker, D. J. Wilson, X. Didelot, A. Bashir, R. Sebra, A. Kasarskis, B. Sthapit, M. Shakya, D. Kelly, A. J. Pollard, T. E. A. Peto, D. W. Crook, P. Donnelly, S. Thorson, P. Amatya, S. Joshi
N. Stoesser
aNuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
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A. Giess
aNuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
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E. M. Batty
bWellcome Trust Center for Human Genetics, Oxford, United Kingdom
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A. E. Sheppard
aNuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
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A. S. Walker
cNIHR Biomedical Research Center, University of Oxford/Oxford University Hospitals NHS Trust, Oxford, United Kingdom
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D. J. Wilson
aNuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
bWellcome Trust Center for Human Genetics, Oxford, United Kingdom
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X. Didelot
dSchool of Public Health, Imperial College London, London, United Kingdom
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A. Bashir
eIcahn School of Medicine at Mount Sinai, New York, New York, USA
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R. Sebra
eIcahn School of Medicine at Mount Sinai, New York, New York, USA
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A. Kasarskis
eIcahn School of Medicine at Mount Sinai, New York, New York, USA
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B. Sthapit
fDepartment of Pediatrics, Patan Hospital, Kathmandu, Nepal
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M. Shakya
fDepartment of Pediatrics, Patan Hospital, Kathmandu, Nepal
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D. Kelly
cNIHR Biomedical Research Center, University of Oxford/Oxford University Hospitals NHS Trust, Oxford, United Kingdom
gOxford Vaccine Group, Center for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford, United Kingdom
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A. J. Pollard
cNIHR Biomedical Research Center, University of Oxford/Oxford University Hospitals NHS Trust, Oxford, United Kingdom
gOxford Vaccine Group, Center for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford, United Kingdom
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T. E. A. Peto
aNuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
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D. W. Crook
aNuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
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P. Donnelly
bWellcome Trust Center for Human Genetics, Oxford, United Kingdom
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S. Thorson
fDepartment of Pediatrics, Patan Hospital, Kathmandu, Nepal
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P. Amatya
fDepartment of Pediatrics, Patan Hospital, Kathmandu, Nepal
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S. Joshi
fDepartment of Pediatrics, Patan Hospital, Kathmandu, Nepal
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DOI: 10.1128/AAC.03900-14
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  • FIG 1
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    FIG 1

    Timeline of Klebsiella pneumoniae cases, including individuals who were both part of epidemiologically defined case clusters and had genetically linked outbreak strains. H2 was found to be genetically unrelated to the other outbreak isolates; H30 was thought to share a relatively recent common ancestor but was not involved in the immediate transmission network. There were no clinical details available for H1460, which is therefore not shown.

  • FIG 2
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    FIG 2

    Maximum-likelihood phylogeny of all sequenced Klebsiella pneumoniae study isolates (deduplicated by individual). Isolates in red represent those originally considered part of case clusters on the basis of clinical suspicion and susceptibility testing. Additional closely genetically related clusters of nonoutbreak strains are indicated with curly brackets. “CA” denotes community-associated strains, and “HA” hospital-associated strains; the time interval listed is that spanning the isolation dates of clustered isolates. Symbols indicating bootstrap support at nodes are defined in the key.

  • FIG 3
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    FIG 3

    Resistance genotypes and susceptibility phenotypes as determined by the Phoenix automated system are shown for all sequenced Klebsiella pneumoniae isolates (A) and for all sequenced non-Klebsiella pneumoniae isolates (B). Copy numbers calculated for blaNDM, where present, are also shown. Susceptibility categories were determined by Phoenix in accordance with EUCAST breakpoints. S, susceptible; I, intermediate; R, resistant; NA, no result available; AMP, ampicillin-amoxicillin; AMC, amoxicillin-clavulanate; TZP, piperacillin-tazobactam; CXM, cefuroxime; CRO, ceftriaxone; CAZ, ceftazidime; CIP, ciprofloxacin; ERT, ertapenem; MEM, meropenem; ATM, aztreonam; GEN, gentamicin; AMK, amikacin; SXT, trimethoprim-sulfamethoxazole; CST, colistin.

  • FIG 4
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    FIG 4

    (A) Nucleotide and amino acid level variations in outbreak isolates with respect to the sequences of the PMK1 reference chromosome and pPMK1-B and pPMK1-NDM. del, deletion; NA, unannotated hypothetical protein; -, stop codon. The order of the isolates approximates the outbreak time frame but also accommodates the grouping of isolates that share identical genetic sequences based on mapping to the PMK1 reference chromosome (i.e., 0 SNV differences; shown in boxes with black borders). Brown-shaded chromosomal positions represent noncoding positions, gray-shaded positions represent positions in plasmids. Nonsynonymous, persistent mutations occurred in ppx (an exopolyphosphatase), azlC (an azaleucine resistance protein), adaA (a methyltransferase), gcd (a quinoprotein glucose dehydrogenase), bmR (a transcriptional repressor), oprD (an outer membrane porin), ssuC (an alkane sulfonate transporter subunit), and virB4 (a type IV secretion system). (B) Phylogenetic tree summarizing chromosomal genetic relationships between all outbreak isolates. Colored nodes represent sampled isolates and black nodes unsampled intermediates; node colors are defined in the key. Each solid branch represents a single SNV, with branch colors indicating mutation types as follows: orange, nonsynonymous; green, synonymous; gray, intergenic; gray dashed line, 121,366-bp deletion; black dashed line, 21 single-nucleotide variants (SNVs).

  • FIG 5
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    FIG 5

    Transmission network inferred using Outbreaker. The sizes of the nodes reflect the numbers of secondary cases per infected individual. The colors of nodes reflect the sampling date as indicated in the key at the upper left. Shading of arrows represents the degree of posterior support for the transmission link, as indicated in the key at the bottom left.

  • FIG. 6
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    FIG. 6

    Mapping of coverage of reference plasmids in outbreak and nonoutbreak strains. Reference plasmid coordinates are displayed in kilobases. Heatmap values represent the number of bases mapped, scaled by the number of bases mapped to the whole reference plasmid. A value of zero represents absence. A value of 1 represents presence at the average coverage or greater. Values between zero and 1 represent presence at a lower-than-average coverage.

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Genome Sequencing of an Extended Series of NDM-Producing Klebsiella pneumoniae Isolates from Neonatal Infections in a Nepali Hospital Characterizes the Extent of Community- versus Hospital-Associated Transmission in an Endemic Setting
N. Stoesser, A. Giess, E. M. Batty, A. E. Sheppard, A. S. Walker, D. J. Wilson, X. Didelot, A. Bashir, R. Sebra, A. Kasarskis, B. Sthapit, M. Shakya, D. Kelly, A. J. Pollard, T. E. A. Peto, D. W. Crook, P. Donnelly, S. Thorson, P. Amatya, S. Joshi
Antimicrobial Agents and Chemotherapy Nov 2014, 58 (12) 7347-7357; DOI: 10.1128/AAC.03900-14

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Genome Sequencing of an Extended Series of NDM-Producing Klebsiella pneumoniae Isolates from Neonatal Infections in a Nepali Hospital Characterizes the Extent of Community- versus Hospital-Associated Transmission in an Endemic Setting
N. Stoesser, A. Giess, E. M. Batty, A. E. Sheppard, A. S. Walker, D. J. Wilson, X. Didelot, A. Bashir, R. Sebra, A. Kasarskis, B. Sthapit, M. Shakya, D. Kelly, A. J. Pollard, T. E. A. Peto, D. W. Crook, P. Donnelly, S. Thorson, P. Amatya, S. Joshi
Antimicrobial Agents and Chemotherapy Nov 2014, 58 (12) 7347-7357; DOI: 10.1128/AAC.03900-14
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