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Genomic Diversity within the Enterobacter cloacaeComplexArmand Paauwl*, Martien P. M. Caspers2, Frank H. J. Schuren2, Maurine A. Leverstein-van Hallr, AlexisDeléto¡|e3, Roy C. Montijn2, Jan Verhoefl, Ad C. Flu¡tt1 Department of Medlcal Mlcroblology, Unlverslty Medlcal centre Utrecht, utrechç The Netherlands, 2TNo Depârtment of Mlcroblology, zelst, The Nerherlands, 3 UniréBlodlvers¡té des Bactérles Pathogènes Emergentes, lnsfltut pasteur, pâr¡s, Frånce

Abstract

: lsolated as nosocomlal pathogens, but

, ible, ldentificatlon of specles based onbùt the taxonomy of isolates belonglng

B-asgd on genomic dlfferences lt is e previously defined (clonal andof the E cloacae complex have to b of'dlsagreements wlth known orowever,.further improved identifìcation les will 6e porriUtu based on novel

clt¡tlon3 Paauw A, cðspers MPMr Schuren FHJ, Levetsteln-van Hall MA, Delétolle A, et ¡1. (2008) Gênomlc Dlverslty wlthln the Entercbactet cloac¿e complex, pLosoNE 3(8): e3018, dol:'t0.137'l40urnal,pone,00030l8

Editorr Rosemary Jeanne Redfleld, Univeßlry of Brldsh Columbla, Canada

Received May 8, 2008; Accepled July 28, ZOOS¡ pubtishcd August Zl, ZO0B

coPy¡lght3 @ 20oB Pàauw et å1, Thls ls an open-access artlcle dlstrlbuted under the terms of the cl,eatlve commons AtÍlbutlon Llcense, which permltsunrestrlcted use, dlstrlbutlon, and leproductlon ln any medlum, provlded the orlglnal author and sou¡ce ar€ credlted,Fundlngi No gÍônts or contrlbutlons from lndustry were recelved by the authors, The study was funded by the Mlcroblology Departmenr of thc uMcu.Competlng lnt6rostr¡ The authors have declared lhat no competlng lntefests exlst.r E-mall: A,Pâauw@umcutrecht.nl

The filst mcthocl cnlailcd genoly¡ric iclcntificatir:n viat scquerrc-ing'of a llagment of thc hcat shock prorcin 60 gcnc (hsþ6Q, arr)was includccl becausc /rsTdll genoryping appcarccì to bc apromising rrovel mcthod, Using this appr.oach, thc E, doacaecomplcx was clividcd into l2 gcncLic clusrcrs (I-XÌI) ancl anunstablc sequcnce clustcr (XIII),flnblc l) [9],

l]he secorrd mcthod cousistccl olscqucnciug a lrngnrcnt olzy'ro.B,and possibly I'cplcsent¡ a good altcrnativc lor strâirr iclcntificririonbccausc of thc high rcsolution avirilal:lc to cliflbrcntiatc bctwccnclo¡ely lclated species [10],

Jhc third a¡rproach enrploycd M[,SA, a mor.c sophistìcatccltcchnique, With this methocl, scvcr.al widcly scparated gcnornicloci are analyzcd, rvhich minimizcs the cflcct of rccombinarioncvents on clustering analysis,

tr'inally, microarray-based CGI-I was also uscd; this approach isa ¡2owcrlul methocì lor pcr.florming genome-wide stuclics ondiflercnt bactelia I l-191.

lntroduction

Eil¿robacter cloqeat is a fhcultative nnacrobic Granr-ncgativcbaciJlus belonging to the family of Entnobactniaccae, Thc nomcn-cla loacae complcx is mainly basccl onwh bridizations nrrcl phcnotypic char-act spccies have bccn assigncd to tlrcDn.l¡robac l¿r c lo acae complex, includirrg Entqo baclcr øsburiae, Dt iltro-baelør cloacae, Enle¡oltacler hotmatclui, Dnterobacl¿r knbtí, Enluobaclnhulwìgìí, and Ertnoltacler nimìltre¡¡walis, QnIy lhtø.obacter isolares thatbclong to thc E, cloaca¿ com¡tlcx arc olclinical signitìcancc and al'cincrcasingly isolatccl as nosocomial pathogcns [4.,51, In survcillarrcestudics, En.letobaciar specics are oltcn not lur.thcl classilìcd bcyonclthe gcnus level probably because icìentification is di{licult,Enlerobaclcr spp. causes 70lo of nosocomial infections in intensivecare units in the USA [6,7].

¡\ccuratc spccics idcntifìcation is dcsirable lor detcrminingwhetbcr spccific species within thc D. cloacae complex alc mor.cp¡'one to causc infections, More precise identification of E, ckscaecomplex isolates may permit dillcrcntiation betlvecn nosocomialspecies and commensal or transitional species, Ilowcver, undl nowphcnotypic idcntification of species and subspccics within thc Z.

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I

il

ilt

IV

Table 1. The different clusters ofthe Enterobacter cloacoecomplex and their nomenclature, accoding hsp60genotyping by Hoffmann and Roggenkamp [9],

5pcclcs Rsference

Divers¡ty w¡lhln the ECC

mcthod for idcntification [9], isolatcs werc also classificd bascdon the rþoB scqucncc, Thirty-two dill'crent sequcnccs wcledctcctcd ìn thc sct ol l5B isolates aller anall'sis of' a 501 bpfragment of ry'03. Scqucnccs wcle comparccl rvith a databasc ofr/of scqucnces that rcprcscnts the major pbylogenctic c)adcs ìn thcfamily Enltrobacløi¿cn¿ [20]; this databasc is rnanagccl by thcPasteul l¡rsLit-utc iu Palis, Francc, The isolatcs wcrc idcntilìcd ns Ehormaeilnì (n = I I 7), D¡ùe¡obacbr øsburine (n = I 0), ¿, cloaca (n = 5),Ent¡robactn clnacae [V (n = 20), and Ent¡¡rol)actil ludwigií (n = 6) flableSl), ,4,s with lup60 genoq,ping, the rcsulLs ol r¡l,o3 ryping showedthat phcnotypic ìcìentification by thc Phoenix AutomatedMicrobiology System cannot discriminatc bctwecn clillcrcntspecies ancl subspecics of thc E, cloacat complcx. Furthcrmorc,thc rcsults also show cliscrcpancies betrvccn thc Þvo singlc gcncgenotyping methocls (Tablc Sl). Thcsc discrcpaucic,s may at lcasrin Jrart bc explained by recombination cverlt.s.

Reliability of DNA fragments for MLSA analysis of theEnterobocter cloacae complex

To minìmizc thc cflects of potcntial rccombinatiorr cvcntsbctwccn isolates, MLSA was uscd. Gcnc liagments of fìvcadditional gcncs ([wtl, gtrB, kuS, !t1trG, ancl rþlB) oÍ'bQ D. cloacatcorir¡rlex isolntcs wcre scqucncccl, Thcse isolatcs whclc sclcctcdbased on thcir dillbl'clrccs in hs1t60 scqucnce and a preliminaryanalysis of'the CGH data, Iror vniquc lsþ60 sequcrìcc, o:rc isolatcwas sclected, In addition, isolates with similat lqt60 sequenccs burdiffcrent pre.liminary CGFI clustering results were includccl withthe limitation that only one isolate was sclccted fì"om each cluster(data not shown). The acccssion numbcrs of the sequenced gcncfragmcnts are listed in Table 52. Initial analysis of rhe scqucncedatâ revcalccl that thc E, cloacae complex consisrs of 2 cladcs, To

thcr thc scqucncc cìata was appropriatc forclies, we asscssccl whcthcr positivc sc.lcction mightr data using tlvo population gcuctic tcsts: Tajima,snd lu's F' statisric [22], [30] flable 2), Tajima,s D

statistic only produccd a significant- (p<0.05) rcsuh lor rltc rþßgenc {ì'om clade l. This indicatcs that thc rþlB gcne rnigh[ buunder positive selcction, This finding is supportcd by a signilìcanrresult in Fut F test (p<0,01), In acldiüior.r, rlrc tlansirion/trarrsvcrsion r^tio olrl)ßwas cstimarcd by plotting thc h'nnsvcrsionand transitiou rates as a lunction ofthc genctic clistanccs (clata notshown). The data show no satrtrâtiotì for cithcr. cu,vc. Iìowcvcr,the tlansition/t¡ansversion t'ãtio was cstimatcd to bc l, inclicatinga non-random clistribution of mutatiorr frcqucncy. This supportsthc notion that thc rþß gene is under posit.ive sclcction, Thc onlyothcr signifìcant l¡u's F tcst was f'or the ry'oß fragmcnt ftom claclc i,indicatirrg that thesc isolatcs ar.c possibly under posirivc sclcctionor thât thi$ population expancls, Howcvcr, this rcsult wâs llotsuppoltcd by Taj.ima's o¡ thc K,,/K, rario (clatanot shown), Thcrcl"orc, ol rþoÙ is not likcly, Thcscrcsultr indicatc that îll xccpt lor rþlB arc sr.¡itablclor phylogcnctic nnalys ø comp.lcx,

'fJrc qualiry of thc phylogcnctic inl'ormation obtaincd lor rhcothcr' ¡'cma.ining gcnc$ was also analyzccl by plotting thctransvcrsio¡r and transition I'ates as a lunctio¡l ol tlrc gcncricd.isiancc (Figurc l). Transvcrsion ancl n.ansition ratcs incr.caseco¡cornitarrdy with geuctic distancc, indicating that thcr.c is litrlcor no saturation, Thc t¡ansitiorr/transvcrsion ratio was cstimatc(lto be 2. These data further support rhat ùe scqucnccs of thcremâining gencs can be used fo¡ phylogcnetic studies.

Phylogenetic analysesThe gcnetíc heterogenciry of ths .&', cloacae complcx was

an4lyzed by gcnerating phylogenetic o'ees basecl on thc MLSA

E, arbuñeÈ . :

E, kobel

E, cliooia"'yll. : ,,1 .tE, cloacoe lV

E. ludwlgll.

É, hormaechel subsp, ohoroe

E, homoechel subsp, hømøechel

E, hornaechel subsp, stelgewaltll

E, ctoaèqe tX

E. nlmlptessurolls

E, cloocqe subsp, cloocoe

E. cloacqe subsp, dlssolvens

.t?l

r4l1

t9J'

tel

:L4zl

t43l

t43l

f43l

Iel

l2l(4I

14l

fel

VI

vil

vil

IX

X

XI

XII

xlll E. cloqcce sequence crowd

dol:10.1 37 140u rn¿l.ponê.00030 1 B,r00l

Thc .E', cltacae complax could thus l¡e dividcd irrto twogcnctically distiuct clndcs that werc not prcviously lccognizcd,Thc youngcr t¡st cladc contains thc E, homu¿ehø spccics, Thisspccics is possibly rnore clinically relevanl bccause it rcplcsents thcmost fì'cquently cultured Ent¿roltøcl¿r specics in hospitals, Thesccorrd clacle ol'tlc,E', cllacaacomplcx consists olscvcral clonal anclhctcrogencous (sub)spccies, lcading us to pr.oposc a rcclefinition olspccies assignment within this genomical.ly divcrsc complex.

Results

ldentlflcatlon of isolates based on hspó0 sequencesThc popnlation súucturc of thc tr, cloane complcx in hospitds was

studicd using l58 isolrtes. Of thcsc, 120 (inclucling 27 outbr.ea.k Iisolates ancl 13 commcnsal fecal isolates) wcre obtaincd in thcUnivcrsity Mcdicai Ce¡rt¡e Utrecht, Uucchq Thc Nethcrlands(UMCUi ? I isolatq (irrclucling l0 outbrcak I isolatcs) lrom I I othcrDutch hospitals; l2 isolatcs lrom l2 diffcrcnr Europcan hospituls; 3isolahs lrom a¡r iuclustrial sitc, and 2 isolatcs wcr-c E, cloocat

ATCCI3047, (Sce Matcrials a¡rd Mcthods fo¡ morc inlormarionabout thc isolatc$, AII isolates wcre idcntilìcd its D, cloatae using rhcPhocnìx Automatcd Microbiology Sysrcm (Bccton Dickinso¡rBioscicnccs, Spalla, MD, U.S.), Thesc lcsulrs wcre comparecl roiclcntilicatio¡r bascd on sequcucing ofa prcviously dcscribcd 273 bplìagrmcnt oî luþ60 tlìât ilppeârcd to givc lcliable iclcnrilìcnf:ìon olEcloacae complcx isoìatcs [9]. Twcrrçninc diflcrcnt sequenccs wcÍcobtaincd and comparecl with scqucnccs desc¡il¡cd by l.Iollman andRoggcnkamp [9], Thc isolatcs wcrc idcnrilìcd as Z'. cloatafJl(n=26),D, cloacae I\,/ (n = I 3), E, cloacae D( (n = l), E, cloacae clnacae þ.t = 4), E,cloacae ¡lì¡¡olaerc (n=l), B. hormaccl¡ei oharae (n=13), E, lwnnauluiilcígautallü þ = 7B), Entcrobact¿r þøbd (n = 7), E ludwigií (n = 6), ancl D,ubuiac (n=9) flablc Sl), Thc lult60 gcnotyping data inclicatc thatphcnotypic idcntifìcatÌon using thc Phocr:ix Automated McrobiologySystem carurot cliscriminatc bctwecn dillerent s¡rccics and sr-rbspcciesof thc E claacøc complex.

ldentification of isolates based on rpoB seguencesBecausc idcntilìcation ol Ent¿robachri.acrac by sequencing of a

fiagrncnt of rþoB has bccn lcportcd as a robust and promising

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Dtverslty wlth¡n the ECC

Table 2. Polymorphlsms observed in seven protein-encodlng genes among 50 E,cloacae complex isolates.

Lén9thc¡ade 1 (n=30) (bp)

ryoq

hsp60 273

Máan " " Ðizexcluded from further analyses

tpl| 333

Clade 2 (n=20)

ln(luded foi analysls

lusA 633'syß

417 .

leuS 642

pytc 306

tpoï 501

.:,hsp60 : 273.

Meân 2772

excluded fiorn further analyses

No of vrrl¡ble no. ofiltos hrplotypes r T¡Jlma's D Fu'5 Fs

501

11

43

s4

I21'

26

,163

21

90

ì57

32

32

'Á4

376

9

9

l8'19

I'14

10

25

11

19

20

,15

18

17

20

7

0.01 53

0,091 0

0,0817

0,0227

0j0351

0,0842

0.0538

0,0006

0,0075

0,0008

0,0000

0.0000

0,0022

0.0017

0 (Tot!l) Totàl¡ynonymous non-¡ynonymouschanges chrngcr

-0,3246 -1.339

-0,1594 -1,437

-0,5045 -1,761

-0.4510 -1.8r7

-0i5739 -3.083

-0,0351 1,387

-0,406 1 - 1.391

-1,87843b -4,834c

-0,4267 - 1,3ó4

-0,3705 -3,52ó

0.1s23 -2,776

0,7711 -1,783

-0,0384 -8,191s

-05243 -3.707

-0.0700 - r,r'r 6

0,0319

0,2241

0,3399

0,1476

0.0686

0,t 7.l e

0,r 663

0,0018

0,0141

0,0175

0,0074

0,0000

0,0079

0,0082

333tpl9

"Olfference lndlcates that <lade 2 ls oldel than clade l,bP<0,05.

'P<o,ol,dolrl 0,1 371 {ournal.pone.000301 8.t002

0.0¡ó

0 tr29

0 c2t

00t5

ûM7

çcn¡tt, -s*-

ol#ff'"X

0.!120 0,02J r 0.0¡5t !,o.!El 0,0603 0,0722

K80 distance

Figure 1. Transltlons/transve¡slon vs genetic distance, From the six concatenated sequences used for phylogentic studles the transitlons (bluecrosses) and transverslons (green trlangles) afe plotted ag¿inst genetic dlstance accordlng to the KsO modó1, No saturation is detected and thetransitions/transverslon ratlo ls estlmated at 2.doi:t 0,1 371/Journå1,pone,000301 Lg00l

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data, Noncongrucncc bchvccn t¡ccs based on thc sequcnccs ofsinglc DNA lì'agmcnts iudicatcd thnt rccombination evcnts had

likclihood-t¡ec. This indicates thar rhe gcnctic relationshipbetwcen thc clustc¡s is perhaps uncc¡tain but that isolatcs incach cluster ale genetically similar to each other bccausc thcsamc isolatcs clustcr cogether in both lrees, The recombination/mutation rntio was 1,04 (95% conlidcncc intcrval 0.72-1,45),indicat.ing that lccombination evcnt¡ are involvcd in thc cvolutioirof thc Z, cloacae complcx. This may cxplain thc cliflcrencesbetwecrr thc phylogcnctic trecs, Dasccl on thc ClonalF¡.amc lreethc isolatcs, cxccpt onc wcrc dividcd into 7 scpar.ate clustcrs(Figure 2A+B),

Comparative genome hybrldlzation analysis

^ Thc six-DNA fr.agmcntr usccl for IvILSA present only a small

lraction of thc genome, To evaluatc whethcr MLSA results arc

Dlversiry withtn the FCC

Allcr anal¡zing all l5B slidcs, a total of 2,6 14 spots (85%) mcr thcquqlicy critcria a¡d wcre includcd in thc study p-ablc S3), Thcaverage sizc of thc DNA lì"agrrcnts spoftcd on thc süclcs rvas 1202 bp.E.ightccn laurdomly sclectcd DNA fingmcnts ol thc corc gcnomc (corcgcnome was dcfincd as gcncs plcsent in all isoìatcs with a likclihoocl ol')950/o) were sequcnced (Tablc S4), Thc avcragc sizc of lmgmcntsreprescnting the core genomc was llTB bp, No r.edunclancy wasdctccted in these sequcrrced DNA flagments, Thc ovcraìl re dunclancyof the MGA was calculated to bc less than 5.3%, thus a mzurimum of138 of thc 2,614 DNA fragmenls have a scqucnce ovcrlap wjrhanothcr insert, 1'he total gcnetic covcragc of cach of ùc scvcndifìþrent shai¡rs usecl for construction ol tIc ar.ray was calculated

[6,2a,] fltablc 3), 1'he gcne coverâgc variccl berwccn 550Á ancl 600/o

for the 7 isolatcs and thc pan-genomic covet.age ol' ü¡c E, cloacac

complex was cstimatccl to bc 55% to 60%r, Thc ca.lculatcd minjrralnuileotidc covcragc of thc corc gcnomc rvas 58%, Rcsr.rlts show thatm_o¡c than half of thc gcncs, but also norl-cncoding or othcr coclingD\A scquences, a¡'c rcprcscntccl in thc co¡rst¡uctcd MGA, For insc¡.liBnCl,0t4.D12 and EnOl,020I-I03, 980/0 and g7t\t ol'rhc pCR rcsultsco¡telatcd witì drc resulb of thc CGI-I anay, r.espectivcly. Thc highsirnlarity bctwee¡r CGI{ a¡¡d PCR rcsult¡ inclicatc rhat MG1hyþridization rcsul$ wclc consistcnt, Al.l rcplicatcs werc louncl in thcsarne cluster as the originals, Clcar.ly, CGI-I allay clata alc lcliablc l'orcoqtparaCivc genomic stuclics,

Olthc 2,6 l4 spots analyzcd, 1,358 wcrc consiclerccl to contain corcgenomc DNA fì.agments bccausc thcy wcrc prcsent jn all ìsoJatcstestpd. These lrag'ments wcr.c omitted for lurtJ¡er analysis bccauscthcy do not conribure to thc speciation of'thc E. cloacnc com¡:lcx.Prirrcipal component anal¡æis (PCA) showecl üat rhc isolatcs wcrc

Flgure 2, Phylogenetic veesol E,_cloacaecomplex,A) Phylogenetlctree based on results of MLSA clustered with Clonalframe, Numbers lndlcateconfidence values of the branches. { Solltalre lsolatà, Kl 2: t. ca¡ riz used,as the öutg roup. B) phylogenetlc tr"e uaseá ãn concat"nated seq uences ofslxgenefragmentscluste¡edwlththeMaxlmumLlkelihood,Eachcolordeplctspclústei accoroingtothecGH*rtitr.*iolitaireisolate, K1z:E.coliK12 used as the outgroup,doll 0,1 37 1 lJoumal,pone,O003Ol B,9OO2

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Dlvers¡ty with¡n the fCC

Table 3. Characterlstics of lsolates used for constructlon of DNA library and the calculated gene coverôge of each isolate,

l¡olatsno, Country Hospltsl year

I

1

l1

1ó

20

1

25

cluster' ryoBgenotyplng htp60genoryping

2

I

2

I

E, hotmalechel

E, homaechel

E¡ homaecl¡el

E, hotmaechel

Ê. Iicimqèc.hel

E. homaechel

E, êloqcoe lY

E, hotmaechel

PFGE- g€ne coverage plasmldrypa {%+/-sDb} pec

3l

48

19

142

146

1M't53

2002

2003

2002

2003

1997 .

NL

NL

NL,

NL

AUS

ITA

NL

POL

1998

2003

t 998

wound/sst'

rectal sw¡b

urlriÉi tract

pneumonla

:lvound/rii

blood

rectal swab

pneumonla

E, ho t naech el s t¿lg e Md I tll

E, homaechel stelgewallll'E,

ho.inløèchet oho¡oè

E, cloacoe lll

E, cloaèae lll

E,lþmoechel ohoroe

E. doacàe lV

E, humoechel stelgerwal¡ll

I 58.5+/-2.2

| 58.5+/-2,2

14

l5

17

27

53

56

56

60+/-4

58

60

56+/-3

62

posltlve

posltlve

negatlve

nega tive

negâtive

negatlve

posltlve

negôtlve

ðCluste/ accordlng MLSA results,blf repllcates âre tested the ständãrd devlatlon ls cålculated.'Wound or skln or soft tlssue lnfectlon, Abbrevlatlons, Ausi AusÌtla, ITA; ltäly, Nl-iThe Netherlands, and pO! poland,dol:t 0,'l 371ljournal.pone,ooo3Ol 8,tOO3

fi'agmcnts wcre likcly dclivccl lïom thc pQC plasmicl, These 106plasmid-dcrived DNA frnpnents wcrc rernoved fi'om lurther analysis.PCA of thc hybriclization lcsulrs of thc r.cmaining 1,150 DNAl'ragments showcd tlrat ûte E, eho¡a complcx is dividccl into [wogenetically clistinct clâdcs (Figure 3A), Subsequcnrl¡ thesc rcsulßwherc confìrrnecl with a phylogenctic tree crcatcd with hicrarchicalclustcring (not shown), Using the Pan program of'thc PIfYLIPsoftwarc package, a parsimony tree was computed (Figure 38) thatconfirmcd the genetic distinction bctween thc two clîdcs, Isolates thacwerc not previously typcd by MLSA wcre clustercd bascd on thelcsule of thc constructed l)arsimony trcc (Fig'r.rrc 4), Clustcls I-2rcprcscnt isolatcs from thc first cladc, whcr-cas isolatcs lrom thesccond cladc belong to chxtcrs 3-7 and tlìc solitairc isolatc, Ctustcnl-7 containcd 25, 91,6, 6, 9, 7, and l3 isolatcs, rcspcctivcly.

Comparison of the results of the differenr typingtechniques

Clustcr I isolatcs wcrc identifìcd as D, hormacchcí usìng rpoBscqucncing, bnt according to luþ60 genotypiug, all isolatcs wcrc .8,

cloacaefrI flfablc Sl), This clustcr was a scparate branch in thc first

cladc of both trces olr CGI-I ancl IvILSA rcsults. Clustcr 2 isolatcswcre all L, hornaechei isolatcs ilccordirry to rlnÙ, l:ut E, lnmnuh¿isleþmlallä and E. honnonhri oharae rccordÍ g ro hslt60 genotyping,Thcsc isolatcs wcrc highly similal accorcling to lvfLSA, butgcnetically hetcrogcncous accorcling to CGI-I. MLSA-bascdfurthcr subdivision of clustcr 2 was nol reliable. llowcver, thch'eó l¡ascd on CGI-I ¡csults showed that thc outbrcîk I isolatesco4sisted of a se¡rarate branch in clustcr 2, This clonality ofoutbrcak I isolatcs is also rupportcd by thc similarity ol thcir rþoBmcl luþ60 scqucnccs. Thc rcsults liom thc second clndc wcrc lessconglucnt in thc CGI{ [rcc. C]ustcr 5 jsolatcs lormcd a clistjnctcluster in CGIJ and MLSA and wcrc all idcnrificc) as E, asburhehyrpop and /uy'd't1, Clustcr 4, isolatcs wcrc all genotyp ed as E. lurlwigìiând clustercd toge thcr l¡orh in MLSA ancl CGI-I, Clusrcr 6 as wcllas blusto 7 wcr.c scpararc clusrcrs in IvILSA ancl CGIÌ but allbelorrged to E, cloocat IV accorcling to r/ioB gcnoryping, whcrcasaccbrding ro hslt60 genotyping all clLrster. 7 isolatcs tvuc ll. clnacae

W and all clustcr 6 isolates wcrc 4 &orsi, Isolates lì.om clustcr 3

wcre mixcd on CGFI analysis, indicating a hcterogcneous gr.oupconsisting of mulúplc species. Thc solitairc isolatc bclongcd to .0,

Flgure 3. Analyze of the comparative genome hybrldlzatlon results. Comparatlve genome hybrldization data clustered wlth pCA andmaximum Palslmony, Comparison of CGH data of 180 slides tested for 1150 DNA fragrñents A) w¡th PcA; fi) w¡ttt th. parsimony method, Both flgureishow that isolates of the E' cloocae complex a¡e dlvlded ln two separate clades, Red: Rrst clade wlth 116 dlfferent lsolates; Grèen: second cladjw¡th42 dlfferent lsolates,doirl 0,1371 {ournal,pone,00030l 8,9003

,j:,j!l ¡,*::

,

i

t.

I

j

¡.I,I

II

BA

'.t$ir: tlot oNE I www.ptosone,org August2008 | Volume 3 | lssue I I e3ot8

Figure 4, Trec constructed on basl¡ of the CGH result¡ of theParslmony method, The slanted cladogram deplcts genetlc relatlon-shlps between lsolates each color represents a different MLSA cluster.rSolltalre isolate ln MLSA.doi:10,1 371 {ourna1,pone,0003018,9004

asburíu accor)ing to rþoB but E, tloaa¿ III according to luþ60,

Furthermolc, this isolate also clustered separatcly.in the CGH trecand the maximum likclihood trec, indicating tlrat this specificisolatc may leprcscnt yet anothcr spccics,

Except fol a lack of congrucncc with clustcr 3 the rcsuls ofCGÍI and MLSA were ide ntical, This indicatcs that MISA rcsuluarc Âlso highly rcprcscntativc ol'genomic rclationships bcwccn E,cloacaa complex isolates, I'Iowevcr, thc fìndings with CGI-I orlvll-SÀ arc inconsistcnt with thosc obtained using fragmcnts olrþoß or hsþ60 fiable Sl),

Evolution of the Enferobacter cloacae complexTIre nrc¿rn pairwisc nuclcotide diversity (n) of thc 5Q E. ckacae

complex isolatcs was highcr within clade 2 tha¡¡ claclc I (0,047+/

-0,003 vs. 0,014+/-0,002), suggcsting that isolates lrom clade 2

arc oldcr fhan isolates lrom clacle l, Furthcrmorc, thc nunrbel olalleles was rclatively lower in cladc I comparcd to cladc 2 (25/30vs 20/20), which suppols this fìndirrg (tablc 2),

Selective markersIvIl,SA and CGII are costly aucl tirnc-consumirrg Echniqucs,

whulcas cfì"cctivc paticnt thcrapy rcquircs inexpcnsivc and rap.idrcsults, Thcrcfore! ne\,v approachcs that rapidly idcnti$ isolatcs at'c inclcmancl, Gcnctic tnarkcrs specific lor cach spcc¡cs may facilitalcquick and incxpensivc tcss, Spccific ma¡kcrs wclc idcnrifìcd withinthc sct of [,150 gencs thnt wcrc variably plcscnt among thc E. clo¿cae

cornplex isolatcs. Thi¡'tccn DNA lragmcn[s werc idcntiliccl that coulcl

¡rotcntial.ly serve as sclectivc markels for tllc lìnt cladc, and 2 potendalsclectivc markcrs were cletcctcd lol the sccond cladc (scnsitivity andspccilicity ol'100%) (Tablc {, Four DNA tagrnents wcrc idcntificdlor cluste¡ I (scnsitivity of 100% nnd specificity of =96%) and lourspccilic DNÀ ltagments were iclcntí{icd lor outbrcak I isolatcs

þcnsitivity of >95% and s¡rccificity of ag5t/o) $ablc a),

Dlscusslon

Thc rcsults of this study show that the CGFI approach with arandom library ol 7 dilfercnt stlains (B isolatcs) is a powclül

Diverslty wlthin the FCC

mcthod lol studying ncw or poolly charactcrizcd species such as

thoÀc that belong to the E, cloacae complex, Combincd wìthMLSA, this tcchniquc can hclp to acculately assign isolatcs to

speçics ol subspecies, In this stucly, the CGI-I nrray had n highresoluüion bccausc it was bascd on thc pt'cscncc or absctlcc ol'

1,150 difhrcnt gcne[ic clemcnts dcrivccl lrom clillcrcnt isolatcs,

whìch wcrc all cvaluatcd on an indiviclual basis, Thcrcfole, CGI-Iwas abìc to determine gcnctic cliffcrences betwec¡r dìflerenr lsolatcs

a¡¡d allowed unsupcrviscd comparison of' large scts ol isolatcs.

Howevcr, CGH had some l.imitations, One potcntial problcm lvasthc large numbcr of mobile clcments rcprescn[ccì on thc MGA,Thpsc mobile clemcllts may bias genetic relatiotrships bcrwccnisolates. If thesc limitations arc taken into account, the CGI-Iapproach can bc used to determine thc gcnctic rclationshi¡rbebwecn (sub)spccies and, imporlantly, to idenúly specilìc markersfor thcse (subþpecics. Although, thc E. cloaca¿ complex is

ge4etically hcterogencous, lhis stucly clclrly shows that clinicalisolates arc cvolutionary divided into nvo gcnctically clìst.inct

cladcs, wherc cladc I appcars to bc youngcr than cladc 2. Cladc I

isolatcs wcrc mosdy obtainccl lrom clinical scttings with hospital-izcd patients, which js consiclcrcd to bc an cnvironmcnt with highspccilic (antibiotic) pressurc, whcrcas thc scconcl cladc contai¡rs a

lcliitivcly largc numbcr of lccal isola(cs lrom paticr¡ts withoutgastr.o-intestifl al complain ls,

Retrospcctivcly, sevcn oI thc cight isolatcs usccl to construct theMGA belongcd to the fìr'st claclc. Thelclole, the CGI{ resuÌrs fbrthe sccond claclc shoulcl be intcrpre ted with caution. Aflcr.rcmovalof the pQC sequcnccs, which representccl the most importílntmobile clcmcnt prcsent in Lhc isolates, a similar clustering ofisolates, with thc cxccption o1'cluster 3, was obtained using cithcr'CÇI{ or MLSA (Figurc 2 and 4). Thc divcrsiry of thc second cladcwas larger when using MLSA thnn, but as mentioned previously,thig may lravc bccn duc to the limitccl numbcl' ol isolatcs ol thisclaile on thc MGA, With CGI-I, spccific gcnctic markcrs wc:.cidentifìcd that cnablcd diffctcntiation bcftvccn thc hvo claclcs. lnaddition, tlrrcc diflclcnt gcnctic markers wcrc idcnrifìcd thatcnablcd spccilìc iclentification of isolaLcs bclonging to clustcr LFoirr difle¡'cnt gcnctic rnâl'kcls wcl'c iclcntificcl rhat allowccldiscrÌmination o[ or¡Lbrcak I isolatcs fì'om all rcsrcd iiiolatcs,MLSA was only pcll'ormcd on onc oulbre¿ìk isolatc; rhcrclorc it is

unknown whcther aìl outbreak isolarcs clus[er üogcthcr. altcrMï,SA anal)nis. I-Iowcvcr', it is likcly that such clustcling occursbccause of thc clonality of'thc isolatcs. No spccifìc genct"ic malkcrswclc dctcctcd l'or clrìsl-crs of'thc second cladc; this may havc becrrduc to thc low numbcr of isolatcs of cladc 2 rcprcscntccl on thcMGA. In thc I'uture, a small array coulcl bc constructecl tl-rar

contains specilic markcrs fbr cliffcrcnt (.*rb)specics in olclcr tocorrcctly spcciate isolates bclonging to thc E, cloacac complcx, Thisrrcri mcthod to delì¡:c nnd idcntify ncw spccics may be extcrrclccl toothtr' micro-organisms.

Whcn combi¡rccì, thc MLSA and CGI-I mcthocls havc a

syncrgctic eflcct. Whcre thc CGI-I-allay offcls thc acìvanragc olbci¡rg able to clctcct spcciûc cìiscliminating markcrs, IvILSA is a

standardizecl mcthod whosc rcsults can bc usccl fbr aclclitionalcornparativc studics. MLSA analysis clivicled thc isolarcs into scvcnclustcrs. Htp60 and ry'oB gcnot¡:ing borh did not suflìcicnrlydiscriminate bctwccn thc subspccies ol E. hormn¿chei. Rccause CGI-land MISA also did nlso not discriminatc beh,vecn E. homta¿chei

subspccies, it is concludcd that thc current nomenclaturc lor.srtbspecies o{ E, horma¿thei is inadequate, Furthcrmore, idenrifica-tion based on sequcncing ol lu!60 and rþoB lragmcnts did noLalways give idenlical outcomcs, IJowevcr, this was mostly bascd oncliffqrenccs in nomenclah¡re betlveen genoryping mcthods (e .g. E,claacæ\,l / E, løbei a.nd E. homa¿cluí / E, ckacae III). If diflcrcnccs

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Table 4. Comparatîve results ûom the specific DNA fragrnents.

@,!-ooz.rÉ€€ì)õ-o5!Dorã

ldentifiaû-on no.DilA-frâgment SpxificiÇ [%) DNA sequence omparative ræulÉ 5pæics

B!-A5T ldent¡tytYPe 196) E-nluc

cac

Noa@

oçJoo

cô@

o@

first cìàde sÞédñc

EnC[03 ] G9

ffi.o12G4

EnCL025A1 0

EriO.ol8E|: ' -:

EnCI.ol2Bl l

End.O06À1? -

EnCl.001 D 12

!nQoÐe.2. -

EnCLo3{,E5

EnCl.020F8

Encl.ollGl lEnCL0o5B7

EnCl-oOrC12

*ä.onã aaue $èËitcEncl.ozlG7

'¡ncLôroæ ' '

.

nb

nb

nb

nb

nb

nb

nb

bx

bx

Fbx

bx

bx

100

100

100

10(}

100

l(X)

100

100

100

100

100

100

1m

parträl op€mn for PQQ pyroloqu¡nol¡ne quínone b¡oqythesis

partial pynoþuimlíne qu¡none ., .- :

nitrâte reducbse alphaJubun¡t

parù-al bcÈlCehyde dehydrogeæ , .'' .

TonB¿ependent siJerophore eeptormaþrfao-Irtator $perÉmly MFS (effiux pump)

partial lacyl-sn glycml3+hoÐhate acyluansfense

putatiì/e hdk-lurrÞhdDq AriC type

hypothetíøl prote¡n KPN_O3581

prote¡n tyro6lne ldnæe

aminqteminal doroin similar to tEnsciption regulatoß

rcpom reguhtor r<eîvc qrotein

pulalive sulfaÞse

Ktebs¡ella pneumon¡ae MGH 7857a

Pseudomonas:þutida F1

Enwoboctü sp- 638

tnttobøau sp. èlà ,

Entübøcter sp. _638

&rføoö.tdã sp.638

E 1tüoboctq sp- 638

Klebskllo pnatrionloe ttLGH 78518

Klùsiella pneumuiøe MGH 7857A

Bcherichio coi¡ Oltf,7 EDL933

Lireriø ¡nrc@ Clíp11262

entuobaìw sp- àie

Auomonas salmüicida A449

8t

.7989

ìbs, .

85

'.79-

a2

56.66

þ2

26

32'

66

0

i. ?3" .

o

.o,t'o

'rd' .,

-169,' -47., ',

-69

, .Íá'' ,.

-'19:,-ii ' '.

-t5

10()

10(}

panial pwaùve glutath¡one S-tr¿ruf,eæ

parthl oxidoedrrcÞ* aþhã- 1moþMoptsi;) subrinir

puanive outer membrane ushtr. protein

partial hypothetiøl protein

panial hypotheticl pmæin

Vypsin{ke ssine proteass

puÞ¡ive pil¡ uembly chapercne

putative GTP-b¡ndÍng factor

hypothetial protein BL¡477

KllA N-temiÈl doma¡rL N1R/P28 Dl.lA binding

Klebsk:llo pnumonbe MGH 7A57A

&rteJoó¿dü sp: 638

Kebsiella pneurioniae ¡¡gH ZaSZS

Klebsiella pneumoniae MGH 78574

Klebsiello pwonia e tvl61 7a57A

Yùs¡n¡o bücov¡ùi ATCC 43970

S erralía protea m aøl o rs 58khsíchio ali 5Bìñ d otto ct ù'¡ u m long u m NCC27 O 5

Acon tlu moúa poly phqgø mimîvirus

nb

nb

73

75

-49'-i.A

*17

-129

-12A

-65

-go

-352

I sp<if¡c

EnCl.015H7

EnCl.005F6

EnClJ05F4

EnO.ol8G3

ôutbræk I spsiF<

EnCl.o0lG3

EnO-oor Da

EftO-Oz7C17

EnCLo¡4H7

t00

too

99

99

99

97

95

u1

nb

nb

bx

bx

nb

bx

bx

45

T1

77

63

74

90

83

6(For sequene see, Supplement Table 52)

'bst h¡t in GenBank nb: nudeotidmudeotide blasq bE nudæddeprotein blðt (no hit wiù nudeotidmucleotide blast).

Iã9.K€J--rì

dcri:'¡ o.1 37l4ou mal.pone.OO03O'l 8-úO4

in nomcnclaturc arc cliscarclccl, only l.gwcrc misidcnti{ìecì bascd on scqucncìng oDiscrcpnncies arosc lor E, athuìae, E, clo

cliagnostic purposcs, scquenciug the S0l bp llagrnent of ry'oBrclnajns an acccptablc alternative,

Colrcct spccintion ol isolates is clinically I'elcvant. Thc currcutstudy showccl tlrat evcn whcn outbr.cak isolatcs rvcrc not incluclecl,isolates of'thc firrt clacle were isolatcd ncirrly Lwo timcs morc oltenthan isolates of'the scconcl cladc, Furthcrmore, it is norablc thatmorc tlran hall ol'thc 13 lccal ^0. cloatae isolücs lrom paticntswithout gastro-intestinal complaints werc lound in thc smallcr

In conclusion, MLSA combincd rvith CGII is a powerlulmctlrod fbr studlng spccics assignmcnc within thc E, cloata

Materials and Methods

Bacterlal strains

Europcan hospitals and 3 isolatcs flrom an inclustrial sitc, Filtally,E. cloacae ATCCI3047 and an ATCC 13047 conjugalt witl, rt.pQC plasmid were included,

Æ isolatcs wcre prcviously identificd ts E, clnacac using anautomated system and softwarc (Phocnix Automated Microbiology

Dlverslty withln the ÊCC

Systcm, Becton Dickinson Bioscicnccs, Sparla, MD, U.S,), ThcEnleroltacler specics lo¡m a hclcrogctric gloup ancì its norncuclaturcis still undelgoing altcrations. Isolatcs idcntifiecl with tltc phocnixapparatus as E. cloam¿ wcrc uscd ns a starting point, Whcn ls/60scquences jndicatcd thírt an isolâtc clid not belong to thc gcnusEnløobact¿r it was excludecl fi'om thc study.

In addition, E. cloaca¿ ATCC i307 wirh and wirhout peC wasused lor dcterminirrg the contribution ol DNA fragmcnts on themicroarlay that lvcrc derjved from this plasmid, Conjugation waspcrformcd as prcviously dcscr.ibcd [30], All isolatcs wer.c cul[urccl

gcncrated lì.om all postcrior samples. Congruence of thc obtainccltree was testccl by phylogcnic arralyses of thc conc¡tcnatccl

',,6,: pur oNE I www,ptosone,ors August2008 | Volume 3 | lssue 8 I e30t8

sequences of thc six genc h.a5'mcnts, Thc trcc was constructcclnsiug maximum likclihood statistics with thc ,,dnaml', program inPHYLIP. Pa¡'amctcl scttings wcre: t¡arrsition/transr.r¡ion ro-

bootstraps of the dara wcre perdormecl using scqboot from thePHYLIP sofrwarc packagc, Subsequcntly, thc output was uscd roconstruct trces wirh Splitsl'ree4. (htÞ://wvw.splitstrce.org) [86].

Microarray development

6 mm microtip, output intcnsity l, Gcneva, Switzcrland) nndfragmcnts ol approximatcly I,2 kb wcrc cxtrac[cd lrom agaroscgels (Qjaquick columns, Qjagen) ancl errd-repaircd (DNATerminator End Rcpair Kit, Lucigcn Corporation, Midcllcton,WI, U.S,). Bnd-repaircd lragments wcrc ligatccl into thcpSMART-FIC-I(an vector (Clonc-SMART, Lucigcn), Subsc"qucntly, tJre recombinnnt plasmicls wcrc hansformed inþ n, coli

Iinking to thc alclchydc-coatcd glass slidcs, PCR producrs wcrecthanol-pulifìcd and rcsuspenclcd in gxSSC (l xSSC: 0. lb MNaCl, 0,015 M sodiunr citr.atc, pFI 7), All PCR producrs wcrcplinLed on CSS silylatcd sliclcs (Dur.opcau Biotcclr, Nctwork,Dolcmbrcux, Belgium) by an ESI rhrec-axi¡ D!-B r.obot (Chip-Writcr Pro, Biorad, Flcrculcs, CA, U,S.) at a co¡ltrollecl humidityol55%. Sliclcs wcrc printcd in four.batches, aflcr which thcy wer.eblockcd and dcnarulcd (15 sec dip in boiling watcr) fbllowing themnnufacturer's instructions,

Labeling, hybridization and data acqulsitionLabeling, hybridization ancl data acquisitiou lvcrc pcr{olmccl as

prcviously clcsclibccl by Lcavis et al, [16], A Bioplirnc systcm(Invitrogcn) was nsccl to labcl 0,5 pg DNA, For normalization,cqual amounts ol'thc B liblary isolatcs (7 slr.ains) wcrr: nrixccl as thcrcfcrcncc pool and labelccl with Cy3 cìUTP, Tcstcr isolatcs wcrclabclcd with CyS dUTP, To rcst rcproducibiliry, lìftcc¡r isolatcswcrc testcd in duplicate, two in triplicatc âncl onc quadluplicatc,For all hybriclizations, Cy5 and Cy3 probcs wcrc combinccì with100 pg ycast IRNA, ch.iccl with a Spccdvac (30 min, at hightcmpcralulc), rcsuspcrrclcd in 40 ¡rl Easyhyb bufl'cr (Rochc,Alnrcrc, Thc Nctherlancls), and dcnnturccl for 2 min at 100'C.Plintcd slidcs wcre prc-hybriclizcd in 0,4,5 ¡rm-lìltercd prc-hybridization bullbr [ % ßSA, 5 xSSC, md 0. 1% socìium doclccylsulfate (SDS[ at42'C for 45 milr with rotat¡on, thcn wnshccl twiccwith pulilicd water (MilliQ Millipore, Billerica, MA, U,S,), driedwith N2, and pre-warmed at 42"C, Thc hybridization mix wasthcn pipcttcd on the printed sidc of thc slide, covered with ahybrislib, placccl ìn hybridization chambers (Corning Lifc ScicnccsB,V. Schiphol-Rijk, Thc Nethcllands), and incubated overnight at42'C in a watcr baù. Slidcs wcre thcn thoroughly washcdsequcntially in (a) lxSSC, 0,2%SDS for. l0 scc at 370C, þ)0,5 xSSC lor l0 sec at 37oC, and (c) tr,vice in 0,2 xSSC for l0 min

'@: ttot oNE I www,plosone,org 9

Dlverslty wlthln the FCC

at room tempcraturc, Slidcs wcrc dricd with N2 and scannccl usinga Scanan'ay Exprcss 680013 Microarray Analysis Systcm (pcrkinEln:crs Liflc Ànalytical Scicnces Inc., Wcllcslcy, MA, U,S,), Imagcswclc obtaincd aud quantificd with ImaGcnc 4,2 soflwarc(Biodiscovcry, Marina clel Rcy, CA, U,S.).

Processing and analysis of MGA dataIhlcrior spots (empty spotr or thosc whcn thc Cy3 signal was lcss

than 2 timcs thc background), wcre cxclucled li'om normalizationand data analysis p6], Each slidc was independcntly normalizcdto correct for individual differcnccs. CyS minus background/Cy3minus background r,atios wcrc calculatcd. Ratios were no¡malizcdby corrccting for thc ovcrall signal intensitics in both chanucls,Spots were selcctecl for further.analyscs whcn 95% of thc sliclcshad a hybridization rcsult l'or rhat spot. Next, thc dara wcrc log2transfolmed. The esti¡:ratcd probability of'pr.cscncc (npp) of caihinscrt was clctcrrninccl using(http: //falkow,stanlorcl.cdu /whWith GACl(-transfbrmarion, irr:ut:offs lor g¡ouping into prcsclragmcnts basccl on thc shapc ol'the clistribution. For hicra¡chicalclugts¡i¡g ancl plincipal compo îssclcctcd, Irr our casc thc gradccl csflom -0,5 to 0,5 in incr.cments rgto 5% or lcss EPP and 0,5 cor p.

corrcspondod to (500/o EPP, I to >50% Epp, aud blanls wcr.crcprcsentcd with a qucstion m¿rrk. http://cvolution.gcnctics.wa$hington.cdu/phylip.html f l 6,401,

Complcte linkagc hicr.archical clustering with pcarson cot.rcla-tion nncl plincipal componcnt analysis (PCA) wcr.c pcrformccl arrclvisualizcd with TIGR lvleV versjon 3,1 soliwarc (http://www,tm4,orglmcv.html), Data wcrc also analyzecl using Par.s l¡orn thc

tiou, gcnctics.wash in g-simony progr.am thatwith multiplc statcs,bccausc this isolatc

appcarccl to bc thc most divcr.sc allcr hicrarchical clustcr.ing. r\corisen¡us t¡'cc was corutructcd flom 50,000 trccs, All analyscsruccl all DNA flagments minus colc and minus pQC-rclatcd DNÂfrngrncnts, The out¡:ut fr.onr thc P¿trs aualys.is was usccl to constructtrces using SplitsTlcc4 ftup://www,spli*rrcc,olg) [361.

Validation of the arrayFor cnc otgun library, thc numbcr of

positivc h ) was dctcrrnincd and gcnccovcragc calculttcd nsing a lbrrnulapreviously t nl, [24]:

Qc = l-(t -(T+t-2(Ro/G))N

wherc Cç=gcnc covel.agc, T=genc sizc, I=inscrt size, l{O=lequired ovcrlap arrd G = gcnornc sizc, Thc zrverage gene size w¿rs

estimatccl to be 922 bp, which is thc avcragc gcne lcngth inEnlr,robaclnrsp,6S8. The avcrage lengú oIDNA lrngmcnts spotteclon the array was 1202 bp, bascd on scquenccs ol 425 DNAli'a5rments (data not sholvn), In contrast to oligonucleotìcle-basedhybridizations, only ovcrlaps bctwccn spottcd DNA ancl tcst DNA

August2008 | Volume 3 | lssue 8 | e30l8

arc ncccssary lor a random shotgun library based on DNA-DNAhybriclization, Thc assumption was madc that 100 nuclcotideswould be neccssaly lor a positivc hybliclization result [24,], Finaily,thc sizc of thc genomes ol thc Enlerobacl¡t isolatcs usecl to constructthc CGII-array wer.c cstir¡r¿¡ted to be 4,5 million bp based on thcwholc gcnome sequences ol Entnobad¿r sp, 638 and Enløobact¿r

sa/,azakií ATCC BAÀ-894 (+,518,712 and 4.,368,373 bp, respcc-tively), Next, the rninimal pcrccntage of coveragc of thc genomcwas defined as:

cM = (N- R)I/G x 100

whcle Cç1 rcpr-escnls tltc minimal coverâgc of thc core gcnomo, Nthc rrumber of'DNA li'agmcnts on the MGA that passecl qualirycontrol, R thc recìundnncy (l/numbcr of uniquc DNA frag'mcntssequcnced-l), I the DNA flagrment sizc, nnd G the genome size

[16]. To cvaluate thc robustness ofhybridization, thc pr.csence orabscnce in thc tested isola,tes of two DNA fragmcnts locatcd ontwo diflèr'cnt bidircctionnlly sequenccd DNA li"ngments wasconhrmcçl by PCR, The DNA fingrncnts chosen wcrc part oliry'l cncoclìng I{NIWPl and part ol o¡þ13 and lrln6a.¡-¡¡_e,

Determination and exclusíon of R-plasmid peC-relatedDNA fragments

Sincc this study focused on shrdying the genctic rclationshipsbetrvccn specics ol'thc E, cloacae complex, inter(brence by plasmidDNA should bc excluded. The large size of pQC (+/-300 kb)plohibits its scparation fi'om chromosomal DNA, Horvcvcr, DNAfra6'ments dcrived from pQC influcncc clustcring arralyscs,Thcleforc, DNA fi'agmcnts from pQC wcre rcmoved lor analyscs,Thc pailcd sluclent-T tcst þ<0.01) was used lbr th¡cc pairs olisolatcs to dctcrrninc which DN¿\ frag'mcnts wcrc der.ivccl fi.ompQC, Pairs ol isolatcs wcrc dcfiuccl as isolatcs obtaincd li.orn oncpaticnt with highly similal PIiGB pattcrns (in our casc all or¡tbrcakstlains) wherc one isolatc harbolecl pQC but thc othcr did nou, Tovcrify pQC specificily, all isolatcs werc hicrarchically clustcr.edusing tlrc ll8 DNA flagmenfs porcnriâlly derivcd fi.om pQC,

Referencesf , lìrcrìrrcl QJ, Mc\{hortcr AC, KnutronJK, Srcigcrwrlr t\G (1982) Ettlmicttio

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Bï!:f,'Jii å,Î:'ff ii t'#llÏ'{.liiÏ¿ii7, Sn'eit J\{, Joncr RN, Saclu H8, Friurchc 'I'lt (200,|) A¡rcssmcnt of prthogcn

occurrcrrccs and rqistancc prof¡lc¡ Âmong infecrcd plticnu in the inlcnsivc clrcunit: rcport. from thc SllN'l'RY Ânrinlicrobill Survcillmce Pl,ogr.am (NonhÄmcrica, 2001). lntJ Antlnicrob Âgcnrs 24: I I l-l lB,I rvm,':\:"1,#'i,ÌTi

t,ii,ll,iî?J,ld rhc Colilcrt-18 r¡atcm. lnr J Hyg

Diverslty w¡thln the ECC

'fwclvc DNA flagrnenLs that wcrc considcred not to bc spccilic lorpQC werc discarcled. Isolatcs that tcstcd posìtivc on l¡¡z1J-specilìcPCR wcrc consicìcrccl to car¡y pQC, as this gcnc is locatccl on thcplasmid [28,30],

Supportlng lnformation

Taþle SI Isolates used in this stuclyFoúnd at: doi: 10. l37ll.joulnal.pone.00030lB.s00l (0,05 lvlBxr¡)Table 52 Acccssion numbcls o{'scqucnccd genc fia6'mcntsFound at: doi:10,1371/joun'ral.pone,00030lB.s002 (0,04 MllxLs)

Taþle S3 FIag filtelcd, normalizcd 2log rranslcr.rcd data;* r'çplicatc slideFound atr doi;10,1371/joulnaì,pone,0003018,s003 (6,S5 MBXLS)

Taþle 54 Scqucnces o[ thc scquenccd DNA fragmcnts uscd inthe studyFound at: doi:l0.l3illjournal.ponc.00030l8.s004 (0.06 lvIBxls)Tsblc 55 Primcrs uscd lbr amplilicatiotr ancl scqr-rcncing,

Foirrd atl doi; 10, l37lljoumal,ponc.00030lB,s005 (0.02 ñfBxr"s)

Acknowledgments

We tha¡k'ljornme van der Bruggen lor, his comments and reading of tJre

manuscript,

Author ContributionsConceivcd ancl designecl the experiments:

^P MPMC F{JS RCIvI ,JV

ACF. Perlormcd dre experimenß: ^P

lvIPMC, Aualyzecl Lhe clara: ApMPMC

^CF, Contill¡utcd rragenrs/materials/nnalysis rools¡ Al, MPMC

AD ACF, lVrotc lhc papcl ÀP MPMC FIùS tvtALvFI AD ¿\CIiContributed to lrypothesis gcner¡tion nnd over:rll study design: AP ÌvfplvtCFIIfS MÂLvl'l AD RcM.JV

^Cr,

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