Research Article Clonality and Resistome Analysis of KPC ...

Research ArticleClonality and Resistome Analysis of KPC-ProducingKlebsiella pneumoniae Strain Isolated in Korea Using WholeGenome Sequencing

Yangsoon Lee1 Bong-Soo Kim2 Jongsik Chun23 Ji Hyun Yong4

Yeong Seon Lee5 Jung Sik Yoo5 Dongeun Yong1 Seong Geun Hong6 Roshan DrsquoSouza1

Kenneth S Thomson7 Kyungwon Lee1 and Yunsop Chong1

1 Department of Laboratory Medicine Research Institute of Bacterial Resistance Yonsei University College of Medicine50 Yonsei-ro Seodaemun-gu Seoul 120-752 Republic of Korea

2 Chunlab Inc Seoul National University Republic of Korea3 School of Biological Sciences Seoul National University Republic of Korea4Department of Science University of British Columbia Vancouver Canada5Division of Antimicrobial Resistance Korea National Institute of Health Osong Republic of Korea6Department of Laboratory Medicine CHA Bundang Medical Center CHA University Seongnam Republic of Korea7Department of Medical Microbiology and Immunology Center for Research in Anti-Infectives and BiotechnologyCreighton University School of Medicine Omaha NE USA

Correspondence should be addressed to Dongeun Yong deyongyuhsac

Received 2 April 2014 Revised 3 June 2014 Accepted 12 June 2014 Published 3 July 2014

Academic Editor Florencio Pazos

Copyright copy 2014 Yangsoon Lee et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We analyzed the whole genome sequence and resistome of the outbreak Klebsiella pneumoniae strain MP14 and comparedit with those of K pneumoniae carbapenemase- (KPC-) producing isolates that showed high similarity in the NCBI genomedatabase A KPC-2-producing multidrug-resistant (MDR) K pneumoniae clinical isolate was obtained from a patient admitted toa Korean hospital in 2011The strainMP14 was resistant to all tested 120573-lactams including monobactam amikacin levofloxacin andcotrimoxazole but susceptible to tigecycline and colistin Resistome analysis showed the presence of 120573-lactamase genes including119887119897119886KPC-2 119887119897119886SHV-11 119887119897119886TEM-169 and 119887119897119886OXA-9 MP14 also possessed aac(61015840-)Ib aadA2 and aph(31015840-)Ia as aminoglycoside resistance-encoding genes mph(A) for macrolides oqxA and oqxB for quinolone catA1 for phenicol sul1 for sulfonamide and dfrA12 fortrimethoprim Both SNP tree and cgMLST analysis showed the close relatedness with the KPC producers (KPNIH strains) isolatedfrom an outbreak in the USA and colistin-resistant strains isolated in ItalyThe plasmid-scaffold genes in plasmids pKpQil pKpQil-IT pKPN3 or pKPN-IT were identified in MP14 KPNIH and Italian strains The KPC-2-producing MDR K pneumoniae ST258stain isolated in Korea was highly clonally related with MDR K pneumoniae strains from the USA and Italy Global spread ofKPC-producing K pneumoniae is a worrying phenomenon

1 Introduction

Klebsiella pneumoniae carbapenemase- (KPC-) producing Kpneumoniae has spread worldwide after the initial reportin the USA [1] and has become a serious problem innosocomial infections due to the associated high mortalitywhich can be as high as 50 [2ndash4] KPC-2 is one of themost common carbapenemases in Enterobacteriaceae in the

USA Multilocus sequence typing (MLST) sequence type(ST) 258 is a common type among KPC-2-producing Kpneumoniae in various parts of the world [5ndash8] In KoreaKPC-2-producing K pneumoniae ST11 first was detected in2010 and a second case of KPC-2-producing K pneumoniaeST258 was also reported [9] Subsequently an outbreak ofthree cases of KPC-2-producing K pneumoniae was detectedin a teaching hospital in Korea [10]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 352862 6 pageshttpdxdoiorg1011552014352862

2 BioMed Research International

Pulsed-field gel electrophoresis (PFGE) and MLST havebeen widely used to understand bacterial disseminationHowever to control the further spread of KPC-2-producingK pneumoniae a better understanding of their mode oftransmission is required With rapid technological advanceswhole genome sequencing (WGS) using a massive parallelsequencer is now becoming a standard protocol in bacterialtyping

Here we analyzed the whole genome sequence and resis-tome of the outbreak strainMP14 and compared it with thoseof KPC-2-producing isolates in the NCBI genome database(httpwwwncbinlmnihgovgenome) that showed highsimilarity to obtain insight into their mode of transfer

2 Materials and Methods

21 Bacterial Isolates and Antimicrobial Susceptibility TestingThe strain MP14 was isolated from the sputum sample ofa 72-year-old man with pneumonia in a Korean hospitalin 2011 He had no recent travel history The species wasidentified by conventional methods and the VITEK 32 GNsystem (bioMerieux Marcy lrsquoEtoile France) Antimicrobialsusceptibility testing was performed using the VITEK II N211system (bioMerieux)

22 Whole Genome Sequencing Genome sequence of MP14was obtained using a combination of Illumina Miseq (150 bppaired end) and Roche 454 (08 kb insert paired end)sequencing systems A total of 3445050 paired reads wereobtained from Miseq run (Q30 gt 78) and 199522 readswere obtained from 454 sequencing systems The sequencesobtained fromMiseq were assembled using the CLC genomicworkbench (CLC Bio Denmark) and the sequences fromthe 454 sequencing systems were assembled using GS DeNovo Assembler 23 (Roche Diagnostics Branford CT)The length of minimum contig was 500 bp and mismatchcost (2) insertion cost (3) deletion cost (3) length fraction(05) and similarity fraction (08) were used for assembly ofIllumina reads inCLCgenomicworkbench For assembly 454sequence reads the length of minimum overlap was 40 bpand the identity of minimum overlap was 90 Alignmentidentity score (2) and alignment difference score (minus3) wereused in GS De Novo Assembler Hybrid assembly of strainMP14 sequences from Miseq and 454 was conducted usingCodonCode Aligner (CodonCode Co MA) The genes wereidentified with Glimmer (maximum overlap length was 50minimum gene length was 110 and threshold score forcalling gene was 30) [11] and annotations were provided byhomology search against COGand SEEDdatabases (databaseobtained at 2012-1-28) [12 13] The whole genomes of thestrains sequenced in this study were compared with thereported genome sequences of K pneumoniae isolates in theNCBI genome database

23 Identification of Resistomes and Plasmids Antimicro-bial resistance genes and plasmid types were analyzedusing ResFinder and PlasmidFinder respectively and usingresources from the Center for Genomic Epidemiology

(httpwwwgenomicepidemiologyorg) ResFinder thresh-old of ID = 98 and PlasmidFinder threshold of ID = 95were selected

24 PFGE and Southern Blotting Whole genomic DNA ofisolates was digested with S1 nuclease (Invitrogen AbingdonUK) and PFGE was performed using a CHEF-DRII device(Bio-Rad Hercules CA) as described previously [14] Gelswith PFGE-separated fragments of DNA were blotted ontonylon membranes (Bio-Rad) and hybridized with probesspecific for the 119887119897119886KPC-2 gene using the DIG DNA Labelingand Detection Kit (Roche Diagnostics GmbH MannheimGermany)

25 SNP Tree and cgMLST Analysis The single nucleotidepolymorphism (SNP) tree web server of the Center forGenomic Epidemiology (httpcgecbsdtudk) was used togenerate SNPs treeTheminimum coverage for SNP calls was10 the minimum distance between SNPs (prune) was 10 bpand the minimum distance to the end of sequence from areference sequence end was 0 bp Genome sequence of Kpneumonia 342 (CP000964) was used as reference for SNPcalls

A core genome MLST (cgMLST) scheme was definedusing the Ridom SeqSphere+ software (Ridom GmbH Mun-ster Germany) with default settings [15] The genome ofthe K pneumoniae KCTC2242 served as reference genomeand the following seven query genomes were used K pneu-moniae (strains NC 0185221 NC 0112831 NC 0225661NC 0168451 NC 0220821 NC 0096481 and NC 0127311)Default settings included the removal of the shorter of twogenes overlapping by more than four bases and of genes withan internal stop codon inmore than 80of all query genomesfrom the scheme [15]

3 Results

The strain MP14 was resistant to multiple antibioticsincluding ampicillin (MIC ge32mgL) ampicillin-sulbactam(ge32mgL) piperacillin-tazobactam (ge128mgL) cefoxitin(ge64mgL) ceftazidime (ge64mgL) cefotaxime (32mgL)cefepime (16mgL) aztreonam (ge64mgL) meropenem(ge16mgL) cotrimoxazole (ge32mgL) amikacin (ge64mgL) and levofloxacin (ge8mgL) butwas susceptible to gentam-icin (le1mgL) tigecycline (2mgL) and colistin (05mgL)

A total of 186 contigs representing 5661622 bases(5698 G+C ratio N50 = 156843) were obtained fromhybrid assembled sequences of strain MP14 (18376 times cov-erage) 5708 CDS 4 rRNA genes and 77 tRNA genes wereannotated for final contigs A genome tree composed ofstrain MP14 and 40 related strains obtained from the NCBIdatabase (date last accessed 1 October 2013) was constructedusing the UPGMA dendrogram based on average identity(ANI) value [16] computed by a BLAST algorithm (Figure 1)Strains in the NCBI database similar toMP14 were as followsKPNIH strains from outbreak patients in the USA [17]ST258-K28BO ST512-K30BO and ST258-K26BO isolatedfrompatients in Italy [18] andKPNV901664 a strain isolated

BioMed Research International 3

Antimicrobial class

Quinolone

TetracyclineTrim

etho-prim

Sulfon amide

Plasmids

Aminoglycoside

aac(3)-IIaaac(3)-Ivaaac(6rsquo)-Ib

aadA1aadA2

aadBaph(3rsquo) Iaaph(3rsquo)-

-Ic

aph(4)-IarmtBstrAstrB

DHA-1CARB-2

FOX-5CTX-M-14CTX-M-15

KPC-2KPC-3OXA-1OXA-9SHV-1

SHV-11SHV-28SHV-36SHV-45SHV-53SHV-60SHV-99

SHV-137TEM-1

TEM-154TEM-169

QnrA1QnrB4QnrS1

aac(6rsquo)Ib-croqxAoqxBfosA

mph(A)catA1catB3

cmlA1ARR-3tet(A)tet(B)tet(D)tet(G)dfrA1

dfrA12dfrA18dfrA21

sul1sul2sul3

HI1BAC2

Col(KPHS6)ColRNAIFIA(HI1)

FIB(pKPHS1)FIB(K)

FIB(pQil)FIB(Mar)

FII(K)FII

LM(pMU407)LM(pOXA-48

)R

X3N

MS (PRJN

A66159)

MG

H 78578

(PRJNA

57619)H

S11286(PRJN

A84387)

KPNIH

9(PRJN

A180469)

KPNIH

1(PRJN

A180462)

KPNIH

17(PRJN

A180475)

KPNIH

21(PRJN

A180479)

KPNIH

22(PRJN

A180480)

KPNIH

7(PRJN

A180467)

KPNIH

2(PRJN

A180463)

KPNIH

6(PRJN

A180466)

KPNIH

8(PRJN

A180468)

KPNIH

5(PRJN

A180465)

KPNIH

18(PRJN

A180476)

KPNIH

16(PRJN

A180474)

KPNIH

4(PRJN

A180464)

KPNIH

12(PRJN

A180472)

KPNIH

20(PRJN

A180478)

KPNIH

11(PRJN

A180471)

KPNIH

10(PRJN

A180470)

KPNIH

14(PRJN

A180473)

KPNIH

19(PRJN

A180477)

KPNIH

23(PRJN

A180481)

KPNMP14

(PRJNA

220252)ST

258-K28BO

(PRJNA

180984)ST

512-K30BO

(PRJNA

180981)K

PN V

901664(PRJN

A220269)

ST258-K

26BO(PRJN

A180983)

1191100241(PRJN

A200802)

KpQ3

(PRJNA

182046)N

TUH

-K2044

(PRJNA

59073)1084

(PRJNA

174151)KCTC 2242

(PRJNA

162147)JH

1(PRJN

A200779)

WG

LW5

(PRJNA

181876)W

GLW

3(PRJN

A181875)

WG

LW1

(PRJNA

181873)LCT

-KP214(PRJN

A89611)

1162281(PRJN

A67493)

WG

LW2

(PRJNA

181874)D

SM 30104

T(PRJN

A89609)

0002

Strains

Origin

MLST

USA

45U

SA38

China11

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

Korea258

Italy258

Italy512

USA

258Italy

258N

ether395

Spain37

Taiwan

23Taiw

an23

Korea275

USA

134U

SA134

USA

48U

SA48

China48

USA

133U

SA14

Korea3

120573-Lactam

FosMLB

Phenicol

RIf

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

Figure 1 A genome tree and resistome analysis of strain MP14 and similar strains obtained from the NCBI database The genome tree (left)was constructed using the UPGMA dendrogram based on average nucleotide identity values The scale bar indicates the difference of ANIvalues among strains In resistome analysis (right) boxes show the presence (gray and red) or absence (white) of the relevant genes for eachof the isolates Fos fosfomycin MLB macrolide-lincosamide-streptogramin B Rif rifampicin


Lhr

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

tnp1ISKpn7 ISKpn6

tnpARecD ProQ ProQ IstAIstB tnpAtnpR tnpA KPC

Tn4401

Figure 2 Comparison of genes identified in contigs containing the 119887119897119886KPC gene in strain MP14 (d) and representative strains showing highsimilarity KPNIH14 (a) KPNIH19 (b) KPNIH23 (c) ST258-K28BO (e) ST512-K30BO (f) KPN V901664 (g) and ST258-K26BO (h)

KpQ31191100241HS11286

ST512-K30BOST258-K26BO

ST258-K28BOKPN V90166

KPNIH1KPNIH14KPNIH19KPN MP14

KCTC2242JH1WGLW5

1084NTUH-K2044WGLW21162281

DSM30104T

LCT-KP214WGLW1WGLW3

MGH 78578MSK pneumoniae

K pneumoniaeK pneumoniaeK pneumoniae

K pneumoniaeK pneumoniae




K pneumoniaeK pneumoniaeK pneumoniaeK pneumoniaeK pneumoniae



K pneumoniae

002

(a)

China HS11286614

Italy ST258-K28BO

612208

USA KPNH1Spain KpQ3

KPN MP14 USA KPNIH14

USA KPNIH19

USA KPN V901664

63 11

613

2554

USA WGLW1

2USA WGLW3

197China LCT-KP214

2783

2726

2735 2675

2708 Taiwan 1084

219

Taiwan NTUH-K2044

Korea DSM30104USA 1162281

USA WGLW5

101

USA JHI

USA WGLW2

(b)

Figure 3 SNP tree (a) and cgMLST analysis (b) using whole genome sequences of the strain MP14 and similar strains obtained from theNCBI database

in the early 2000s in the USA The identity value betweenMP14 and these strains was over 998 in ANI analysisAll of those isolates carried 119887119897119886KPC-2 or 119887119897119886KPC-3 genes Allof these isolates were MLST ST258 except for one (ST512-K30BO) The K pneumoniae HS11286 strain carrying the

119887119897119886KPC-2 gene from China was ST11 A length of the Tn4401region harboring 119887119897119886KPC alleles was observed in some of thecompared strains including KPNIH strains 14 19 and 23ST258-K28BO ST512-K30BO ST258-K26BO and V901664(Figure 2) Furthermore the genetic environment around


M 1 2 1 2

(kb)

200150

100

50

(a) (b)Figure 4 PFGE patterns of S1-digested DNA fragments (a) andSouthern blotting with the 119887119897119886KPC-2 probe (b) Lane M lambdaladder (Bio-Rad) as a DNA size marker lane 1 a clinical isolate ofK pneumoniae lane 2 the strain MP14

Tn4401 was highly similar among the compared strainssuggesting the existence of a larger mobile element thanTn4401 carrying the 119887119897119886KPC gene

K pneumoniaKPNMP14 was themost similar to KPNIHstrains than other strains in SNP tree (Figure 3) Strain MP14 was clustered with Italy strains (ST258-K28BO ST258-K26BO and ST512-K30BO) and USA strains The result wassimilar to genome tree

A cgMLST scheme of K pneumoniae was defined usingNCBI data in this study Using K pneumoniae KCTC 2422strain as reference genome (4923 genes) and the genomeof further seven K pneumoniae strains as query genomeswe defined the standard set of 3548 genes for the cgMLSTscheme

The resistomes of strain MP14 and other isolates are pre-sented in Figure 1 The strain MP14 possessed the followingresistance genes four 120573-lactamase genes 119887119897119886KPC-2 119887119897119886SHV-11119887119897119886TEM-169 and 119887119897119886OXA-9 aac(61015840)-Ib aadA2 and aph(31015840)-Iaas aminoglycoside resistance-encoding genes mph(A) formacrolides oqxA and oqxB for quinolone catA1 for phenicolsul1 for sulfonamide and dfrA12 for trimethoprim TheKPNIH series isolated in the USA and three Italian strainshad very similar resistomes to that of strain MP14

We analyzed the WGS of the related strains using a webtool PlasmidFinder for characterization of plasmids [19]Plasmid-scaffold genes such as FII(K) FIB(K) FIB(pQil)and ColRNAI were present in the strain MP14 in all iso-lates of KPNIH strains compared and in the three Italianstrains (Figure 1) FIB(K) is known to be associated withplasmid pKPN3 identified in the USA (GenBank accessionnumber CP000648) and plasmid pKPN-IT identified inItaly (JN233704)19 FII(K) and FIB(pQil) are plasmid-scaffoldregions of plasmids pKpQil and pKpQil-IT (NC 014016 andJN233705 resp) which are approximately 115 kb sized plas-mids carrying the 119887119897119886KPC-3 gene [20] Based on hybridizationof S1-digestedDNA fragments of the strainMP14 the 119887119897119886KPC-2

probe hybridized with an approximately 120 kb plasmid(Figure 4)

4 Discussion

WGS was carried out for a KPC-2-producing K pneumoniaestrain that was isolated in Korea in 2011 Genome sequencesof 24 KPC-2-producing K pneumoniae strains (isolated inUSA and Italy) similar to Korean strain MP14 were obtainedfrom the NCBI database Strain MP14 could be comparedamong strains isolated in different countries or at differenttimes by whole genome sequences and various informationof strain could be analyzed The strain MP14 carried severalantimicrobial resistance genes for different antibiotic classesand showed multidrug resistance as expected

The cgMLST scheme in K pneumoniae isolate was con-structed in this study for the first time The cgMLST resultswere comparable to those of the SNPs tree in K pneumoniaeisolates (Figure 3) The cgMLST may be a promising toolfor standardized portable and expandable approach in Kpneumoniae isolates although this study has a limitationstemming from its small sample size

Both SNP and cgMLST based whole genome sequencessupported the closer relatedness MP14 strain KPN NIHstrains and Italy ST strains carrying 119887119897119886KPC genes Thesesuggested that the transmission of KPC-producing K pneu-moniae did not happen by mobile genetic elements butby spread of clone Although we could not obtain the fullsequence of the 119887119897119886KPC-2 gene-associated plasmids in theMP14 strain this strain was found to possess several plasmid-scaffold genes that were previously reported in plasmidspKpQil from Israel and pKpQil-IT from Italy which carriedthe 119887119897119886KPC-2 genes pKPN3 from the USA and pKPN-IT fromItalyThese findings suggest transfer of plasmids carrying the119887119897119886KPC gene together with the host strains from the USA orItaly to Korea

5 Conclusion

Whole genome sequencing and its in-depth analysis of theKPC-2-producing MDR K pneumoniae ST258 strain fromKorea made it evident that this was strongly related toMDR K pneumoniae strains from USA and Italy Globalspread of KPC-producing K pneumoniae is a worryingphenomenon and close inspection to stop their spread isstrongly warranted

Consider nucleotide sequence accession numbers thenucleotide sequence data reported in this paper are availablein the DDBJEMBLGenBank nucleotide database underaccession number ATAK00000000

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Yangsoon Lee and Bong-Soo Kim contributed equally to thiswork as first authors


Acknowledgment

This study is supported byKoreaCenters forDiseasesControland Prevention (Code 2012E4400100)

References

[1] H Yigit A M Queenan G J Anderson et al ldquoNovel carba-penem-hydrolyzing 120573-lactamase KPC-1 from a carbapenem-resistant strain of Klebsiella pneumoniaerdquo Antimicrobial Agentsand Chemotherapy vol 45 no 4 pp 1151ndash1161 2001

[2] L M Deshpande R N Jones T R Fritsche and H SSader ldquoOccurrence and characterization of carbapenemase-producing enterobacteriaceae report from the SENTRYAntimicrobial Surveillance Program (2000ndash2004)rdquo MicrobialDrug Resistance vol 12 no 4 pp 223ndash230 2006

[3] P Nordmann G Cuzon and T Naas ldquoThe real threat ofKlebsiella pneumoniae carbapenemase-producing bacteriardquoTheLancet Infectious Diseases vol 9 no 4 pp 228ndash236 2009

[4] G Patel S Huprikar S H Factor S G Jenkins and D PCalfee ldquoOutcomes of carbapenem-resistant Klebsiella pneumo-niae infection and the impact of antimicrobial and adjunctivetherapiesrdquo Infection Control and Hospital Epidemiology vol 29no 12 pp 1099ndash1106 2008

[5] J C Cai H W Zhou R Zhang and G X Chen ldquoEmergenceof Serratia marcescens Klebsiella pneumoniae and Escherichiacoli Isolates possessing the plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in intensive care units of aChinese hospitalrdquo Antimicrobial Agents and Chemotherapy vol52 no 6 pp 2014ndash2018 2008

[6] A Toth I Damjanova E Puskas et al ldquoEmergence of a colistin-resistant KPC-2-producing Klebsiella pneumoniae ST258 clonein Hungaryrdquo European Journal of Clinical Microbiology andInfectious Diseases vol 29 no 7 pp 765ndash769 2010

[7] L N Andrade T Curiao J C Ferreira et al ldquoDissemination ofbla119870119875119862minus2

by the spread of Klebsiella pneumoniae clonal complex258 clones (ST258 ST11 ST437) and plasmids (IncFII IncNIncLM) among Enterobacteriaceae species in Brazilrdquo Antimi-crobial Agents and Chemotherapy vol 55 no 7 pp 3579ndash35832011

[8] T Bogdanovich J M Adams-Haduch G Tian et al ldquoColistin-resistant Klebsiella pneumoniae Carbapenemase (KPC)-producingKlebsiella pneumoniae belonging to the internationalepidemic clone ST258rdquo Clinical Infectious Diseases vol 53 no4 pp 373ndash376 2011

[9] K H Roh C K Lee J W Sohn W Song D Yong and K LeeldquoIsolation of a Klebsiella pneumoniae isolate of sequence type258 producing KPC-2 carbapenemase in Koreardquo The KoreanJournal of Laboratory Medicine vol 31 no 4 pp 298ndash301 2011

[10] S K Hong D Yong K Kim et al ldquoFirst outbreak of KPC-2-producing Klebsiella pneumoniae sequence type 258 in ahospital in South Koreardquo Journal of Clinical Microbiology vol51 no 11 pp 3877ndash3879 2013

[11] A L Delcher K A Bratke E C Powers and S L SalzbergldquoIdentifying bacterial genes and endosymbiont DNA withGlimmerrdquo Bioinformatics vol 23 no 6 pp 673ndash679 2007

[12] R L Tatusov E V Koonin and D J Lipman ldquoA genomicperspective on protein familiesrdquo Science vol 278 no 5338 pp631ndash637 1997

[13] T Disz S Akhter D Cuevas et al ldquoAccessing the SEED genomedatabases via Web services API tools for programmersrdquo BMCBioinformatics vol 11 article 319 2010

[14] Y Lim Y Lee Y Seo et al ldquoLoss of blaVIM-2 and blaIMP-1 during the storage of Gram-negative bacilli antimicrobialsusceptibility of the gene- lost strain and location of the genein the cellrdquo Annals of Clinical Microbiology vol 16 pp 120ndash1252013

[15] T A Kohl R Diel D Harmsen J Rothganger K MeywaldWalter and M Merker ldquoWhole genome based Mycobacteriumtuberculosis surveillance a standardized portable and expand-able approachrdquo Journal of Clinical Microbiology 2014

[16] J Goris K T Konstantinidis J A Klappenbach T Coenye PVandamme and J M Tiedje ldquoDNA-DNA hybridization valuesand their relationship to whole-genome sequence similaritiesrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 57 no 1 pp 81ndash91 2007

[17] E S Snitkin A M Zelazny P J Thomas et al ldquoTrackinga hospital outbreak of carbapenem-resistant Klebsiella pneu-moniae with whole-genome sequencingrdquo Science TranslationalMedicine vol 4 no 148 Article ID 148ra116 2012

[18] F Comandatore D Sassera S Ambretti et al ldquoDraft genomesequences of two multidrug resistant Klebsiella pneumoniaeST258 isolates resistant to colistinrdquo Genome Announcements2013

[19] A Carattoli E Zankari and A Garcıa-Fernandez ldquoPlas-midFinder and pMLST in silico detection and typing ofplasmidsrdquo Antimicrobial Agents and Chemotherapy 2014

[20] A Garcıa-Fernandez L Villa C Carta et al ldquoKlebsiella pneu-moniae ST258 producing KPC-3 identified in Italy carries novelplasmids and OmpK36OmpK35 porin variantsrdquoAntimicrobialAgents and Chemotherapy vol 56 no 4 pp 2143ndash2145 2012

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Microbiology


Pulsed-field gel electrophoresis (PFGE) and MLST havebeen widely used to understand bacterial disseminationHowever to control the further spread of KPC-2-producingK pneumoniae a better understanding of their mode oftransmission is required With rapid technological advanceswhole genome sequencing (WGS) using a massive parallelsequencer is now becoming a standard protocol in bacterialtyping

Here we analyzed the whole genome sequence and resis-tome of the outbreak strainMP14 and compared it with thoseof KPC-2-producing isolates in the NCBI genome database(httpwwwncbinlmnihgovgenome) that showed highsimilarity to obtain insight into their mode of transfer

2 Materials and Methods

21 Bacterial Isolates and Antimicrobial Susceptibility TestingThe strain MP14 was isolated from the sputum sample ofa 72-year-old man with pneumonia in a Korean hospitalin 2011 He had no recent travel history The species wasidentified by conventional methods and the VITEK 32 GNsystem (bioMerieux Marcy lrsquoEtoile France) Antimicrobialsusceptibility testing was performed using the VITEK II N211system (bioMerieux)

22 Whole Genome Sequencing Genome sequence of MP14was obtained using a combination of Illumina Miseq (150 bppaired end) and Roche 454 (08 kb insert paired end)sequencing systems A total of 3445050 paired reads wereobtained from Miseq run (Q30 gt 78) and 199522 readswere obtained from 454 sequencing systems The sequencesobtained fromMiseq were assembled using the CLC genomicworkbench (CLC Bio Denmark) and the sequences fromthe 454 sequencing systems were assembled using GS DeNovo Assembler 23 (Roche Diagnostics Branford CT)The length of minimum contig was 500 bp and mismatchcost (2) insertion cost (3) deletion cost (3) length fraction(05) and similarity fraction (08) were used for assembly ofIllumina reads inCLCgenomicworkbench For assembly 454sequence reads the length of minimum overlap was 40 bpand the identity of minimum overlap was 90 Alignmentidentity score (2) and alignment difference score (minus3) wereused in GS De Novo Assembler Hybrid assembly of strainMP14 sequences from Miseq and 454 was conducted usingCodonCode Aligner (CodonCode Co MA) The genes wereidentified with Glimmer (maximum overlap length was 50minimum gene length was 110 and threshold score forcalling gene was 30) [11] and annotations were provided byhomology search against COGand SEEDdatabases (databaseobtained at 2012-1-28) [12 13] The whole genomes of thestrains sequenced in this study were compared with thereported genome sequences of K pneumoniae isolates in theNCBI genome database

23 Identification of Resistomes and Plasmids Antimicro-bial resistance genes and plasmid types were analyzedusing ResFinder and PlasmidFinder respectively and usingresources from the Center for Genomic Epidemiology

(httpwwwgenomicepidemiologyorg) ResFinder thresh-old of ID = 98 and PlasmidFinder threshold of ID = 95were selected

24 PFGE and Southern Blotting Whole genomic DNA ofisolates was digested with S1 nuclease (Invitrogen AbingdonUK) and PFGE was performed using a CHEF-DRII device(Bio-Rad Hercules CA) as described previously [14] Gelswith PFGE-separated fragments of DNA were blotted ontonylon membranes (Bio-Rad) and hybridized with probesspecific for the 119887119897119886KPC-2 gene using the DIG DNA Labelingand Detection Kit (Roche Diagnostics GmbH MannheimGermany)

25 SNP Tree and cgMLST Analysis The single nucleotidepolymorphism (SNP) tree web server of the Center forGenomic Epidemiology (httpcgecbsdtudk) was used togenerate SNPs treeTheminimum coverage for SNP calls was10 the minimum distance between SNPs (prune) was 10 bpand the minimum distance to the end of sequence from areference sequence end was 0 bp Genome sequence of Kpneumonia 342 (CP000964) was used as reference for SNPcalls

A core genome MLST (cgMLST) scheme was definedusing the Ridom SeqSphere+ software (Ridom GmbH Mun-ster Germany) with default settings [15] The genome ofthe K pneumoniae KCTC2242 served as reference genomeand the following seven query genomes were used K pneu-moniae (strains NC 0185221 NC 0112831 NC 0225661NC 0168451 NC 0220821 NC 0096481 and NC 0127311)Default settings included the removal of the shorter of twogenes overlapping by more than four bases and of genes withan internal stop codon inmore than 80of all query genomesfrom the scheme [15]

3 Results

The strain MP14 was resistant to multiple antibioticsincluding ampicillin (MIC ge32mgL) ampicillin-sulbactam(ge32mgL) piperacillin-tazobactam (ge128mgL) cefoxitin(ge64mgL) ceftazidime (ge64mgL) cefotaxime (32mgL)cefepime (16mgL) aztreonam (ge64mgL) meropenem(ge16mgL) cotrimoxazole (ge32mgL) amikacin (ge64mgL) and levofloxacin (ge8mgL) butwas susceptible to gentam-icin (le1mgL) tigecycline (2mgL) and colistin (05mgL)

A total of 186 contigs representing 5661622 bases(5698 G+C ratio N50 = 156843) were obtained fromhybrid assembled sequences of strain MP14 (18376 times cov-erage) 5708 CDS 4 rRNA genes and 77 tRNA genes wereannotated for final contigs A genome tree composed ofstrain MP14 and 40 related strains obtained from the NCBIdatabase (date last accessed 1 October 2013) was constructedusing the UPGMA dendrogram based on average identity(ANI) value [16] computed by a BLAST algorithm (Figure 1)Strains in the NCBI database similar toMP14 were as followsKPNIH strains from outbreak patients in the USA [17]ST258-K28BO ST512-K30BO and ST258-K26BO isolatedfrompatients in Italy [18] andKPNV901664 a strain isolated


Antimicrobial class

Quinolone

TetracyclineTrim

etho-prim

Sulfon amide

Plasmids

Aminoglycoside


aadA1aadA2


-Ic


DHA-1CARB-2




SHV-137TEM-1

TEM-154TEM-169

QnrA1QnrB4QnrS1


mph(A)catA1catB3


dfrA12dfrA18dfrA21

sul1sul2sul3

HI1BAC2


FIB(pKPHS1)FIB(K)

FIB(pQil)FIB(Mar)

FII(K)FII


)R

X3N

MS (PRJN

A66159)

MG

H 78578

(PRJNA

57619)H

S11286(PRJN

A84387)

KPNIH

9(PRJN

A180469)

KPNIH

1(PRJN

A180462)

KPNIH

17(PRJN

A180475)

KPNIH

21(PRJN

A180479)

KPNIH

22(PRJN

A180480)

KPNIH

7(PRJN

A180467)

KPNIH

2(PRJN

A180463)

KPNIH

6(PRJN

A180466)

KPNIH

8(PRJN

A180468)

KPNIH

5(PRJN

A180465)

KPNIH

18(PRJN

A180476)

KPNIH

16(PRJN

A180474)

KPNIH

4(PRJN

A180464)

KPNIH

12(PRJN

A180472)

KPNIH

20(PRJN

A180478)

KPNIH

11(PRJN

A180471)

KPNIH

10(PRJN

A180470)

KPNIH

14(PRJN

A180473)

KPNIH

19(PRJN

A180477)

KPNIH

23(PRJN

A180481)

KPNMP14

(PRJNA

220252)ST

258-K28BO

(PRJNA

180984)ST

512-K30BO

(PRJNA

180981)K

PN V

901664(PRJN

A220269)

ST258-K

26BO(PRJN

A180983)

1191100241(PRJN

A200802)

KpQ3

(PRJNA

182046)N

TUH

-K2044

(PRJNA

59073)1084

(PRJNA

174151)KCTC 2242

(PRJNA

162147)JH

1(PRJN

A200779)

WG

LW5

(PRJNA

181876)W

GLW

3(PRJN

A181875)

WG

LW1

(PRJNA

181873)LCT

-KP214(PRJN

A89611)

1162281(PRJN

A67493)

WG

LW2

(PRJNA

181874)D

SM 30104

T(PRJN

A89609)

0002

Strains

Origin

MLST

USA

45U

SA38

China11

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

Korea258

Italy258

Italy512

USA

258Italy

258N

ether395

Spain37

Taiwan

23Taiw

an23

Korea275

USA

134U

SA134

USA

48U

SA48

China48

USA

133U

SA14

Korea3

120573-Lactam

FosMLB

Phenicol

RIf

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae



Lhr

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

tnp1ISKpn7 ISKpn6


Tn4401


KpQ31191100241HS11286




KCTC2242JH1WGLW5

1084NTUH-K2044WGLW21162281

DSM30104T

LCT-KP214WGLW1WGLW3










K pneumoniae

002

(a)

China HS11286614

Italy ST258-K28BO

612208

USA KPNH1Spain KpQ3


USA KPNIH19

USA KPN V901664

63 11

613

2554

USA WGLW1

2USA WGLW3

197China LCT-KP214

2783

2726

2735 2675

2708 Taiwan 1084

219

Taiwan NTUH-K2044


USA WGLW5

101

USA JHI

USA WGLW2

(b)





M 1 2 1 2

(kb)

200150

100

50








4 Discussion




5 Conclusion








Acknowledgment


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Antimicrobial class

Quinolone

TetracyclineTrim

etho-prim

Sulfon amide

Plasmids

Aminoglycoside


aadA1aadA2


-Ic


DHA-1CARB-2




SHV-137TEM-1

TEM-154TEM-169

QnrA1QnrB4QnrS1


mph(A)catA1catB3


dfrA12dfrA18dfrA21

sul1sul2sul3

HI1BAC2


FIB(pKPHS1)FIB(K)

FIB(pQil)FIB(Mar)

FII(K)FII


)R

X3N

MS (PRJN

A66159)

MG

H 78578

(PRJNA

57619)H

S11286(PRJN

A84387)

KPNIH

9(PRJN

A180469)

KPNIH

1(PRJN

A180462)

KPNIH

17(PRJN

A180475)

KPNIH

21(PRJN

A180479)

KPNIH

22(PRJN

A180480)

KPNIH

7(PRJN

A180467)

KPNIH

2(PRJN

A180463)

KPNIH

6(PRJN

A180466)

KPNIH

8(PRJN

A180468)

KPNIH

5(PRJN

A180465)

KPNIH

18(PRJN

A180476)

KPNIH

16(PRJN

A180474)

KPNIH

4(PRJN

A180464)

KPNIH

12(PRJN

A180472)

KPNIH

20(PRJN

A180478)

KPNIH

11(PRJN

A180471)

KPNIH

10(PRJN

A180470)

KPNIH

14(PRJN

A180473)

KPNIH

19(PRJN

A180477)

KPNIH

23(PRJN

A180481)

KPNMP14

(PRJNA

220252)ST

258-K28BO

(PRJNA

180984)ST

512-K30BO

(PRJNA

180981)K

PN V

901664(PRJN

A220269)

ST258-K

26BO(PRJN

A180983)

1191100241(PRJN

A200802)

KpQ3

(PRJNA

182046)N

TUH

-K2044

(PRJNA

59073)1084

(PRJNA

174151)KCTC 2242

(PRJNA

162147)JH

1(PRJN

A200779)

WG

LW5

(PRJNA

181876)W

GLW

3(PRJN

A181875)

WG

LW1

(PRJNA

181873)LCT

-KP214(PRJN

A89611)

1162281(PRJN

A67493)

WG

LW2

(PRJNA

181874)D

SM 30104

T(PRJN

A89609)

0002

Strains

Origin

MLST

USA

45U

SA38

China11

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

USA

258U

SA258

Korea258

Italy258

Italy512

USA

258Italy

258N

ether395

Spain37

Taiwan

23Taiw

an23

Korea275

USA

134U

SA134

USA

48U

SA48

China48

USA

133U

SA14

Korea3

120573-Lactam

FosMLB

Phenicol

RIf

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae

K pneumoniae



Lhr

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

tnp1ISKpn7 ISKpn6


Tn4401


KpQ31191100241HS11286




KCTC2242JH1WGLW5

1084NTUH-K2044WGLW21162281

DSM30104T

LCT-KP214WGLW1WGLW3










K pneumoniae

002

(a)

China HS11286614

Italy ST258-K28BO

612208

USA KPNH1Spain KpQ3


USA KPNIH19

USA KPN V901664

63 11

613

2554

USA WGLW1

2USA WGLW3

197China LCT-KP214

2783

2726

2735 2675

2708 Taiwan 1084

219

Taiwan NTUH-K2044


USA WGLW5

101

USA JHI

USA WGLW2

(b)





M 1 2 1 2

(kb)

200150

100

50








4 Discussion




5 Conclusion








Acknowledgment


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Lhr

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

tnp1ISKpn7 ISKpn6


Tn4401


KpQ31191100241HS11286




KCTC2242JH1WGLW5

1084NTUH-K2044WGLW21162281

DSM30104T

LCT-KP214WGLW1WGLW3










K pneumoniae

002

(a)

China HS11286614

Italy ST258-K28BO

612208

USA KPNH1Spain KpQ3


USA KPNIH19

USA KPN V901664

63 11

613

2554

USA WGLW1

2USA WGLW3

197China LCT-KP214

2783

2726

2735 2675

2708 Taiwan 1084

219

Taiwan NTUH-K2044


USA WGLW5

101

USA JHI

USA WGLW2

(b)





M 1 2 1 2

(kb)

200150

100

50








4 Discussion




5 Conclusion








Acknowledgment


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2 1 2

(kb)

200150

100

50








4 Discussion




5 Conclusion








Acknowledgment


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgment


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Clonality and Resistome Analysis of KPC ...

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