Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

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Diagnostic Microbiology and Infectious Disease

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Whole genome mapping of the first reported case of KPC-2–positiveKlebsiella pneumoniae ST258 in Nebraska

Randal C. Fowler, Caitlyn R. Scharn, Mir A. Ali, Stephen J. Cavalieri, Richard V. Goering, Nancy D. Hanson⁎Center for Research in Anti-Infectives and Biotechnology, Department ofMedical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178

a b s t r a c ta r t i c l e i n f o

⁎ Corresponding author. Tel.: +1-402-280-5837; fax:E-mail address: ndhanson@creighton.edu (N.D. Hans

http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.0230732-8893/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Fowler RC, et al, Win Nebraska, Diagn Microbiol Infect Dis (20

Article history:Received 17 December 2013Received in revised form 18 March 2014Accepted 31 March 2014Available online xxxx

Keywords:blaKPC-2PFGEWhole genome mappingMLST

Three ertapenem-resistant Klebsiella pneumoniae carrying blaKPC-2 were isolated from a single patient inNebraska over a span of 5 months. A comparative analysis of the genetic relatedness of these isolates wasinvestigated using pulsed-field gel electrophoresis, multilocus sequence typing, and whole genome mapping.

+1-402-280-1875.on).

hole genome mapping of the first reported c14), http://dx.doi.org/10.1016/j.diagmicrobio

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

Carbapenem-resistant Enterobacteriaceae (CREs), particularlyKPC-producing Klebsiella pneumoniae, have emerged as multidrugresistant pathogens that pose an immediate threat to public health(Davies et al., 2011; Kitchel et al., 2009; Roth and Hanson, 2013;Tzouvelekis et al., 2012). The spread of KPC-producing K. pneumoniaestrains is a complex global epidemic that is highly associated with thedissemination of clone ST258within the United States andworldwide.(Davies et al., 2011; Kallen and Srinivasan, 2010; Kitchel et al., 2009;Won et al., 2011). Infections caused by KPC-producing pathogenshave led to higher incidence of morbidity andmortality due to limitedantibiotic options for therapy (Bratu et al., 2005; Gasink et al., 2009;Tzouvelekis et al, 2012; Won et al., 2011; Zarkotou et al., 2011).

K. pneumoniae harboring blaKPC have been primarily describedin hospital settings and metropolitan areas worldwide (Munoz-Priceet al., 2013), whereas less populated rural communities representregions where little is known about the prevalence of microorganismsharboring antibiotic resistance mechanisms (Pope et al., 2006).Patients in less populated rural communities may act as reservoirsfor CREs. It has been reported that most rural hospitals do notroutinely use methods beyond standard antibiotic susceptibilitytesting for the detection of antibiotic resistance mechanisms(Stevenson et al., 2003). In addition, when performed, these routinesusceptibility tests are not always adequate to identify KPC-producingEnterobacteriaceae (Bratu et al., 2005; Tenover et al., 2006).Therefore, implementation of antibiotic resistance surveillance pro-grams in these less populated areas may offer valuable information

that could be useful for containing the spread of KPC-producingmicroorganisms and guiding antibiotic therapies for patients.

Between February and August of 2013, 3 ertapenem-resistantK. pneumoniae were cultured from the urine of a 57-year-old man livingin rural, central Nebraska, 95 miles from a metropolitan area with apopulation of N200,000.Hismedical history consisted of recurrent urinarytract infections (UTIs), cystolithiasis, and thoracic spine osteomyelitis. InNovember 2012, thepatientwas transferred froma long-termcare facilityinNebraska to ahospital inMinneapolis,MN, for a cystoithotripsy. A rectalswab from the patient tested positive for CREs; therefore, the patient wasmoved to isolation, but no records regarding treatment were available. InDecember 2012, the patient was transferred back to Nebraska, and inMarch, urine cultures tested positive for possible ESBL-producing K.pneumoniae (KpVA2a); records indicated the patient was not treated andwas discharged in May. One month later, the patient presented withanother UTI resulting from an ertapenem-resistant K. pneumoniae(KpVA2b), and the patient was treated empirically with ciprofloxacinfor 10 days. In July, the patient was admitted to a hospital in Omaha, NE,for a UTI, and a K. pneumoniae (KpVA2c) was isolated; the patient wastreated with ertapenem. Ertapenem treatment was stopped due to thepatient being asymptomatic. Travel history beyond the hospitalization inMinnesota was not available.

MICs were determined by the Vitek-2. In addition to ertapenemresistance, all 3 isolates were resistant to ceftriaxone, tobramycin,amikacin, and ciprofloxacin while remaining susceptible to gentamicin.Interestingly, using the Vitek-2, KpVA2a and KpVA2b were resistant toimipenembut susceptible to cefepime,while KpVA2cwas susceptible toimipenem but resistant to cefepime (Table 1). However, whenevaluated by Etest®, no difference was observed for the imipenemand cefepimeMICs (Table 1). To identify themechanismassociatedwithertapenem resistance in these isolates, a KPC real-time PCR/High

ase of KPC-2–positive Klebsiella pneumoniae ST258.2014.03.023

1 2 3 4

~320 Kb

~220 Kb

Fig. 1. PFGE analysis of XbaI chromosomal digests of K. pneumoniae isolates KpVA2a(lane 1), KpVA2b (lane 2), KpVA2c (lane 3), and size standard Salmonella braenderupH9812 (lane 4).

Fig. 2.Whole genomemapping analysis of AflII chromosomal digests of K. pneumoniae carryinKpVA2b, and KpVA2c.

Table 1MICs determined by Vitek2 and Etest for K. pneumoniae isolates.

Antibiotic MICs (μg/mL)

KpVA2a KpVA2b KpVA2c

Vitek2 Etest Vitek2 Etest Vitek2 Etest

Ertapenem 4 4 4 8 N4 8Imipenem 2 4 8 8 1 8Ceftriaxone 16 16 16 16 N16 16Cefepime 2 8 2 8 N16 8Gentamicin ≤1 1 ≤1 1 ≤4 1.5Amikacin ≥64 ND 32 ND N32 ND

Tobramycin ≥16 32 ≥16 32 N8 32Ciprofloxacin ≥4 ND ≥4 ND N2 ND

ND = not determined.

2 R.C. Fowler et al. / Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

Please cite this article as: Fowler RC, et al, Whole genome mapping of thin Nebraska, Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.101

Resolution Melting (HRM) assay and ScreenClust analysis wereperformed to detect the presence of blaKPC (Roth and Hanson, 2013).HRMdetected a KPC-2–like gene in each isolate whichwas identified asblaKPC-2 by sequence analysis. All 3 isolates were negative for blaNDM,blaVIM, blaIMP, blaGIM, and blaSPM.

Pulsed-field gel electrophoresis (PFGE) and multilocus sequencetyping (MLST) analyses showed these isolates were highly related toeach other and showed that KpVA2b and KpVA2c were of ST258, andKpVA2a was ST258-like (Diancourt et al., 2005; Goering, 2004). ThePFGE profiles were indistinguishable between KpVA2a and KpVA2c,while a single band shift of N100 Kb was observed in KpVA2b (Fig. 1).PFGE is considered the gold standard for strain typing of microorgan-isms, but Whole Genome Mapping (WGM) has the ability to identifyregions of genetic diversity between highly related isolates that wouldotherwise be missed by traditional typing methods such as PFGE andMLST. Therefore,WGMusing the restriction enzymeAflIIwasperformedby OpGen, Inc. (Gaithersburg, MD, USA) to locate the position in thegenome associated with the N100 Kb band shift observed using PFGE ofthese isolates. As shown in Fig. 2, ordered restrictionmaps of the 3 KPC-positive K. pneumoniae isolates were indistinguishable except for a 140-Kb deletion in KpVA2b (Fig. 2). To determine the location of the 140-Kbdeletion found in the chromosomeofKpVA2b,WGMswere compared toa mock AflII restriction digest on K. pneumoniae strain HS11286(GenBankaccession#CP003200.1) using BioNumerics software (AppliedMaths, Sint-Martens-Latem, Belgium). This deletion in KpVA2b (relativeto KpVA2a and KpVA2c) was specifically localized to a P4 integraseextending from 3,433,717 to 3,434,979 bp (protein ID: KPHS_34600 inCP003200). Thus, the deletion likely relates to the loss of an insertionsequence, a transposon, or another mobile genetic element. WGM canalso be used to detect plasmids; however, the detection limit is N150 Kb.K. pneumoniae strains can carry plasmids that range in size from small(≤3 Kb) to large (N150 Kb). Therefore, plasmid profiles were completedon the 3 isolates to determine the number and approximate sizes of theplasmids isolated (Kurpiel and Hanson, 2011). Five identical plasmidprofiles were observed among KpVA2a and KpVA2b ranging from ~8 toN165 Kb (data not shown). However, due to the production of a nucleaseand smearing of the DNA from isolate KpVA2c, only 3 of the 5 smallerplasmids could be visualized. Taken together, WGM supports the PFGEandMLSTdata inproviding ahigh-resolution comparisonof thegenomesfor these isolates. Thedata strongly suggest that these3 isolates representsubtypes of a single KPC-2–producingK. pneumoniae strain in this patientwhile demonstrating the genetic variation that may occur through thegain or loss of genetic material over a relatively short period of time.

To our knowledge, this is the first report of K. pneumoniae of ST258harboring blaKPC-2 in Nebraska. WGM was able to provide additionalinformation about the genetic variation among the 3 isolates evaluated.However,WGMof these isolates supported the PFGE andMLST data, andthus it may better serve as a supplementary method for strain typing ofmicrobial pathogens in outbreak situations or other epidemiologicalinvestigations. This report demonstrates that multidrug-resistant Gram-

g blaKPC. Alignment and assembly of chromosomal DNA collected from isolates KpVA2a,

e first reported case of KPC-2–positive Klebsiella pneumoniae ST2586/j.diagmicrobio.2014.03.023

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negative bacteria can be found outside of densely populatedmetropolitancenters and underscores the need formolecular surveillance of importantantimicrobial resistancemechanisms for isolates collected from these lesspopulated areas so that infection control measures can be implementedto limit patient-to-patient spread.

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e first reported case of KPC-2–positive Klebsiella pneumoniae ST2586/j.diagmicrobio.2014.03.023