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
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sequencing
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Julia S. Sabirova
a*
, Basil Britto Xavier
a*
, Jasmine Coppens
a
, Olympia Zarkotou
b
, Christine
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Lammens
a
, Lore Janssens
a
, Ronald Burggrave
c
, Trevor Wagner
c
, Herman Goossens
a
, Surbhi
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Malhotra-Kumar
a#
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a
Department of Medical Microbiology, Vaccine & Infectious Disease Institute, Universiteit Antwerpen,
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Antwerp, Belgium
a
; Department of Microbiology, Tzaneio General Hospital, Piraeus, Greece
b
; OpGen,
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Inc., Gaithersburg, Maryland, USA
c
.
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Running Title: bla
VIM19
-harbouring ST383 K. pneumoniae
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Key words: Whole genome sequencing, class 1 integron, integrase, ICEPm1, ST383, optical
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mapping, whole genome maps, PFGE
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Text word count: 3749
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Synopsis word count: 248
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*Equal contribution authors
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#
Corresponding author mailing address: Department of Medical Microbiology, Campus Drie
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Eiken, University of Antwerp, S6, Universiteitsplein 1, B-2610 Wilrijk, Belgium. Phone: 32-3-
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265-27-52. Fax: 32-3-265-26-63. E-mail: surbhi.malhotra@uantwerpen.be
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Synopsis
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Objectives: We utilized whole-genome mapping (WGM) and WGS to characterize 12 clinical
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carbapenem-resistant Klebsiella pneumoniae (TGH1-TGH12).
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Methods: All strains were screened for carbapenemase genes by PCR, and typed by MLST, PFGE
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(XbaI), and WGM (AflII) (Opgen, USA). WGS (Illumina) was performed on TGH8 and TGH10.
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Reads were denovo assembled and annotated (SPAdes, RAST). Contigs were aligned directly, and
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after in silico AflII restriction, with corresponding WGMs (MapSolver, OpGen; BioNumerics,
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Applied Maths).
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Results: All 12 strains were ST383. Eleven of the 12 strains were carbapenem-resistant and 7
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harboured bla
KPC-2,
and 11, bla
VIM-19 .
Varying the parameters for assigning WGM clusters showed
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that these were comparable to ST types, and to the 8 PFGE (sub)types (≥ 3-band difference). A
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95% similarity coefficient assigned all 12 WGMs to a single cluster while a 99% similarity
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coefficient (or ≥ 10 unmatched-fragment difference) assigned the 12 WGMs to 8 (sub)clusters.
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Based on a ≥ 3-band difference between PFGE profiles, the Simpson’s diversity index (SDI) of
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WGM (0.94, Jackknife pseudo-values CI: 0.883-0.996) and PFGE (0.93, CI: 0.828-1.000) were
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similar (p=0.649). However, discriminatory power of WGM was significantly higher (SDI: 0.94,
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CI: 0.883-0.996) than PFGE profiles typed on a ≥7-band difference (SDI: 0.53, CI: 0.212-0.849)
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(p=0.007).
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Conclusions: This study demonstrates the application of whole-genome mapping to understanding
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the epidemiology of hospital-associated K. pneumoniae. Utilizing a combination of WGM and
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WGS, we also present here the first longitudinal genomic characterization of the highly dynamic
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carbapenem-resistant ST383 K. pneumoniae clone that is rapidly gaining importance in Europe.
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INTRODUCTION
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The spread of multidrug-resistant K. pneumoniae strains in hospitals constitutes a pressing global
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health problem. The increasing prevalence of plasmid-encoded carbapenem-hydrolysing enzymes
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in K. pneumoniae is of particular concern due to their ability to hydrolyse almost all β-lactam
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antibiotics, as well as their genetic association with transferable multidrug resistance.
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Infections
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due to carbapenem-resistant K. pneumoniae are not only difficult to treat due to limited therapeutic
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options, but such clones could potentially cause hospital epidemics if not promptly detected and
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contained.
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Molecular typing techniques are effective surveillance tools to monitor the dynamics of multidrug-
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resistant clones circulating in hospitals during non-outbreak situations and to detect early signs of
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an outbreak. Currently used techniques are based on amplification of marker genes followed by
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sequencing (MLST), or targeting entire genomes by PFGE or by whole genome mapping (WGM),
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with the restriction fragments analysed on a gel or in a microfluidic device, respectively. Of the
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three techniques, MLST is most commonly utilized for bacteria strain typing. While it is highly
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reproducible and relatively inexpensive, the resolution achieved by clustering strains based on
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sequenced segments of seven or more housekeeping genes is not high enough to study inter- and
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intra-clonal genetic diversity. Compared to PFGE, WGM has the advantage of being less labour
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intensive and, importantly, allowing inter-lab reproducibility and comparison of results that has
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been a major shortcoming of PFGE typing. WGM is also far less technologically challenging than
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whole genome sequencing (WGS) and does not require bioinformatics expertise. Furthermore,
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results can be generated on the same day with WGM that offers a distinct advantage for
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microbiology laboratories undertaking outbreak investigations. Notwithstanding instrument costs,
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the cost of running a single strain for PFGE < WGM ≤ WGS. Also with WGM, the genetic
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content of the detected variable genome regions can be extracted and identified by utilizing the
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vast number of publicly available whole genome sequences. These can be utilized to develop in
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silico restriction maps using the same enzymes as used for experimentally-generated WGMs,
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allowing comparison of restriction patterns and eventual identification of the genetic content of the
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variable regions in the strains of interest.
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In this study, we demonstrate the utility of WGM in conjunction with WGS for typing,
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characterizing, and dissecting the genomic features of carbapenem-resistant K. pneumoniae
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isolated at the Tzaneio General (TG) hospital, Piraeus, Greece. Carbapenem-resistant K.
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pneumoniae producing VIM-type metallo-β-lactamases have been endemic in Greek hospitals
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since the early 2000s.
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Many strains with VIM-1–producing K. pneumoniae have also been
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described.
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From 2007, KPC-type carbapenemases became prevalent and even caused outbreaks,
8,
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followed by emergence of strains coproducing KPC and VIM.
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We studied VIM- and KPC-
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+VIM-producing K. pneumoniae isolated from colonized or infected patients during 2010-2013 at
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the TG hospital.
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