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Journal ArticleDOI

Whole-genome typing and characterization of blaVIM19-harbouring ST383 Klebsiella pneumoniae by PFGE, whole-genome mapping and WGS

01 Jun 2016-Journal of Antimicrobial Chemotherapy (Oxford University Press)-Vol. 71, Iss: 6, pp 1501-1509

TL;DR: This study demonstrates the application of WGM to understanding the epidemiology of hospital-associated K. pneumoniae and presents the first longitudinal genomic characterization of the highly dynamic carbapenem-resistant ST383 K.neumoniae clone that is rapidly gaining importance in Europe.

AbstractObjectives We utilized whole-genome mapping (WGM) and WGS to characterize 12 clinical carbapenem-resistant Klebsiella pneumoniae strains (TGH1-TGH12). Methods All strains were screened for carbapenemase genes by PCR, and typed by MLST, PFGE (XbaI) and WGM (AflII) (OpGen, USA). WGS (Illumina) was performed on TGH8 and TGH10. Reads were de novo assembled and annotated [SPAdes, Rapid Annotation Subsystem Technology (RAST)]. Contigs were aligned directly, and after in silico AflII restriction, with corresponding WGMs (MapSolver, OpGen; BioNumerics, Applied Maths). Results All 12 strains were ST383. Of the 12 strains, 11 were carbapenem resistant, 7 harboured blaKPC-2 and 11 harboured blaVIM-19. Varying the parameters for assigning WGM clusters showed that these were comparable to STs and to the eight PFGE types or subtypes (difference of three or more bands). A 95% similarity coefficient assigned all 12 WGMs to a single cluster, whereas a 99% similarity coefficient (or ≥10 unmatched-fragment difference) assigned the 12 WGMs to eight (sub)clusters. Based on a difference of three or more bands between PFGE profiles, the Simpson's diversity indices (SDIs) of WGM (0.94, Jackknife pseudo-values CI: 0.883-0.996) and PFGE (0.93, Jackknife pseudo-values CI: 0.828-1.000) were similar (P = 0.649). However, the discriminatory power of WGM was significantly higher (SDI: 0.94, Jackknife pseudo-values CI: 0.883-0.996) than that of PFGE profiles typed on a difference of seven or more bands (SDI: 0.53, Jackknife pseudo-values CI: 0.212-0.849) (P = 0.007). Conclusions This study demonstrates the application of WGM to understanding the epidemiology of hospital-associated K. pneumoniae. Utilizing a combination of WGM and WGS, we also present here the first longitudinal genomic characterization of the highly dynamic carbapenem-resistant ST383 K. pneumoniae clone that is rapidly gaining importance in Europe.

Summary (1 min read)

Introduction

  • These can be utilized to develop in 70 silico restriction maps using the same enzymes as used for experimentally-generated WGMs, 71 allowing comparison of restriction patterns and eventual identification of the genetic content of the 72 variable regions in the strains of interest.
  • 5 73 In this study, the authors demonstrate the utility of WGM in conjunction with WGS for typing, 74 characterizing, and dissecting the genomic features of carbapenem-resistant K. pneumoniae 75 isolated at the Tzaneio General (TG) hospital, Piraeus, Greece.

Strain collection 84

  • Twelve K. pneumoniae (TGH1-TGH12), harbouring blaVIM and/or blaKPC, and isolated from 85 patients admitted to the intensive care unit (n=8) or surgical ward (n=4) at the TG hospital during 86 2010-2013 were studied.
  • Clinical data and strain characteristics are outlined in Table 1. 87.

Antimicrobial resistance profiling 88

  • All 12 strains were screened for resistance to 17 antibiotics, including β-lactams with and without 89 β-lactamase inhibitors, by disk diffusion (Table 1).
  • MICs of carbapenems (ertapenem, imipenem 90 and meropenem) were determined by Etest (bioMérieux Inc., Durham, NC), and results 91 interpreted according to CLSI cut-offs.
  • 11 Strains were screened for presence of extended-spectrum 92 β-lactamase (ESBL) and carbapenemase genes by PCR and Sanger sequencing as described 93 previously.

MLST and PFGE 95

  • MLST was performed as described previously for seven marker genes (gapA, infB, mdh, pgi, 96 phoE, rpoB, tonB), 15 and sequence types were assigned using the Institute Pasteur database 97 (www.pasteur.fr/mlst).
  • Both parameters showed 186 similar (sub) clustering of WGMs, identifying two clusters, C1 and C2, and two singletons, C3 187 and C4 , with C1 and C2 each divided into two sub clusters and a singleton .

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This item is the archived peer-reviewed author-version of:
Whole-genome typing and characterization of -harbouring ST383
Klebsiella pneumoniae by PFGE, whole-genome mapping and WGS
Reference:
Sabirova Julia, Xavier Basil Britto, Coppens Jasmine, Zarkotou Olympia, Lammens Christine, Janssens Lore, Burggrave
Ronald, Wagner Trevor, Goossens Herman, Malhotra Surbhi.- Whole-genome typing and characterization of -
harbouring ST383 Klebsiella pneumoniae by PFGE, whole-genome mapping and WGS
The journal of antimicrobial chemotherapy - ISSN 1460-2091 - (2016), p. 1-9
Full text (Publishers DOI): http://dx.doi.org/doi:10.1093/JAC/DKW003
To cite this reference: http://hdl.handle.net/10067/1318080151162165141
Institutional repository IRUA

Whole genome typing and characterisation of bla
VIM19
-harbouring
1
ST383 Klebsiella pneumoniae by PFGE, whole genome mapping and
2
sequencing
3
4
5
Julia S. Sabirova
a*
, Basil Britto Xavier
a*
, Jasmine Coppens
a
, Olympia Zarkotou
b
, Christine
6
Lammens
a
, Lore Janssens
a
, Ronald Burggrave
c
, Trevor Wagner
c
, Herman Goossens
a
, Surbhi
7
Malhotra-Kumar
a#
8
a
Department of Medical Microbiology, Vaccine & Infectious Disease Institute, Universiteit Antwerpen,
9
Antwerp, Belgium
a
; Department of Microbiology, Tzaneio General Hospital, Piraeus, Greece
b
; OpGen,
10
Inc., Gaithersburg, Maryland, USA
c
.
11
12
Running Title: bla
VIM19
-harbouring ST383 K. pneumoniae
13
14
Key words: Whole genome sequencing, class 1 integron, integrase, ICEPm1, ST383, optical
15
mapping, whole genome maps, PFGE
16
Text word count: 3749
17
Synopsis word count: 248
18
19
*Equal contribution authors
20
#
Corresponding author mailing address: Department of Medical Microbiology, Campus Drie
21
Eiken, University of Antwerp, S6, Universiteitsplein 1, B-2610 Wilrijk, Belgium. Phone: 32-3-
22
265-27-52. Fax: 32-3-265-26-63. E-mail: surbhi.malhotra@uantwerpen.be
23

Synopsis
24
Objectives: We utilized whole-genome mapping (WGM) and WGS to characterize 12 clinical
25
carbapenem-resistant Klebsiella pneumoniae (TGH1-TGH12).
26
Methods: All strains were screened for carbapenemase genes by PCR, and typed by MLST, PFGE
27
(XbaI), and WGM (AflII) (Opgen, USA). WGS (Illumina) was performed on TGH8 and TGH10.
28
Reads were denovo assembled and annotated (SPAdes, RAST). Contigs were aligned directly, and
29
after in silico AflII restriction, with corresponding WGMs (MapSolver, OpGen; BioNumerics,
30
Applied Maths).
31
Results: All 12 strains were ST383. Eleven of the 12 strains were carbapenem-resistant and 7
32
harboured bla
KPC-2,
and 11, bla
VIM-19 .
Varying the parameters for assigning WGM clusters showed
33
that these were comparable to ST types, and to the 8 PFGE (sub)types (≥ 3-band difference). A
34
95% similarity coefficient assigned all 12 WGMs to a single cluster while a 99% similarity
35
coefficient (or ≥ 10 unmatched-fragment difference) assigned the 12 WGMs to 8 (sub)clusters.
36
Based on a ≥ 3-band difference between PFGE profiles, the Simpson’s diversity index (SDI) of
37
WGM (0.94, Jackknife pseudo-values CI: 0.883-0.996) and PFGE (0.93, CI: 0.828-1.000) were
38
similar (p=0.649). However, discriminatory power of WGM was significantly higher (SDI: 0.94,
39
CI: 0.883-0.996) than PFGE profiles typed on a ≥7-band difference (SDI: 0.53, CI: 0.212-0.849)
40
(p=0.007).
41
Conclusions: This study demonstrates the application of whole-genome mapping to understanding
42
the epidemiology of hospital-associated K. pneumoniae. Utilizing a combination of WGM and
43
WGS, we also present here the first longitudinal genomic characterization of the highly dynamic
44
carbapenem-resistant ST383 K. pneumoniae clone that is rapidly gaining importance in Europe.
45

INTRODUCTION
46
The spread of multidrug-resistant K. pneumoniae strains in hospitals constitutes a pressing global
47
health problem. The increasing prevalence of plasmid-encoded carbapenem-hydrolysing enzymes
48
in K. pneumoniae is of particular concern due to their ability to hydrolyse almost all β-lactam
49
antibiotics, as well as their genetic association with transferable multidrug resistance.
1-3
Infections
50
due to carbapenem-resistant K. pneumoniae are not only difficult to treat due to limited therapeutic
51
options, but such clones could potentially cause hospital epidemics if not promptly detected and
52
contained.
4
53
Molecular typing techniques are effective surveillance tools to monitor the dynamics of multidrug-
54
resistant clones circulating in hospitals during non-outbreak situations and to detect early signs of
55
an outbreak. Currently used techniques are based on amplification of marker genes followed by
56
sequencing (MLST), or targeting entire genomes by PFGE or by whole genome mapping (WGM),
57
with the restriction fragments analysed on a gel or in a microfluidic device, respectively. Of the
58
three techniques, MLST is most commonly utilized for bacteria strain typing. While it is highly
59
reproducible and relatively inexpensive, the resolution achieved by clustering strains based on
60
sequenced segments of seven or more housekeeping genes is not high enough to study inter- and
61
intra-clonal genetic diversity. Compared to PFGE, WGM has the advantage of being less labour
62
intensive and, importantly, allowing inter-lab reproducibility and comparison of results that has
63
been a major shortcoming of PFGE typing. WGM is also far less technologically challenging than
64
whole genome sequencing (WGS) and does not require bioinformatics expertise. Furthermore,
65
results can be generated on the same day with WGM that offers a distinct advantage for
66
microbiology laboratories undertaking outbreak investigations. Notwithstanding instrument costs,
67
the cost of running a single strain for PFGE < WGM ≤ WGS. Also with WGM, the genetic
68
content of the detected variable genome regions can be extracted and identified by utilizing the
69
vast number of publicly available whole genome sequences. These can be utilized to develop in
70

silico restriction maps using the same enzymes as used for experimentally-generated WGMs,
71
allowing comparison of restriction patterns and eventual identification of the genetic content of the
72
variable regions in the strains of interest.
5
73
In this study, we demonstrate the utility of WGM in conjunction with WGS for typing,
74
characterizing, and dissecting the genomic features of carbapenem-resistant K. pneumoniae
75
isolated at the Tzaneio General (TG) hospital, Piraeus, Greece. Carbapenem-resistant K.
76
pneumoniae producing VIM-type metallo-β-lactamases have been endemic in Greek hospitals
77
since the early 2000s.
6
Many strains with VIM-1producing K. pneumoniae have also been
78
described.
7
From 2007, KPC-type carbapenemases became prevalent and even caused outbreaks,
8,
79
9
followed by emergence of strains coproducing KPC and VIM.
10
We studied VIM- and KPC-
80
+VIM-producing K. pneumoniae isolated from colonized or infected patients during 2010-2013 at
81
the TG hospital.
82

Citations
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In this paper, a whole-genome typing and characterization of harboring ST383 Klebsiella pneumoniae by PFGE, wholegenome mapping and WGS is presented.