␤ -lactamase producers by the EDTA/DDSTtest clinical isolates as ametallo- Misidentification of pan drug-resistant Klebsiellapneumoniae INFECTIOUS DISEASES

braz j infect dis.2015;19(1):102–104

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Letters

to

the

Editor

Misidentification

of

pan

drug-resistant

Klebsiella

pneumoniae

clinical

isolates

as

a

metallo-

␤-lactamase

producers

by

the

EDTA/DDST

test

Carbapenemase-producing Enterobacteriaceae may exhibit

susceptibilitytocarbapenems.Forthisreasonwiththerecent

spread ofNDM-1 amongEnterobacteriaceae, the phenotypic

detection of metallo-beta-lactamase (M␤L)-producing has

beenrecommendedbyBrazilianAgencyofSanitary

Surveil-lance(ANVISA).1

In2013,twopandrug-resistantKlebsiellapneumoniae(KPN1

andKPN2)isolateswererecoveredfromurine(cystostomy)of

a75-year-old malepatient hospitalizedina tertiary

teach-ing hospital in Florianópolis, Santa Catarina, Brazil. Both

isolateswerephenotypicallyidentifiedasM␤Lproducersby

ethylenediaminetetraaceticacid(EDTA)/double-disksynergy

test(DDST)andforwardedtoourlaboratoryforfurther

charac-terization.IdentificationofbothisolatesasK.pneumoniaewas

confirmed by MALDI-TOF MS (Bruker Daltonics, Germany),

accordingtothemanufacturer’srecommendations.Both

iso-latesshowedanidenticalpatternbyERIC-PCR.Theminimal

inhibitory concentrations (MICs) for selected antimicrobial

agentsweredeterminedbybrothmicrodilutionaccordingto

ClinicalandLaboratoryStandardsInstitute–CLSI.TheMICs

were interpreted according to the CLSI guidelines,2 _except

fortigecycline,whichinterpretationwasperformedaccording

totheEuropeanCommitteeonAntimicrobialSusceptibility

Testing – EUCAST. Both isolates were fully resistant to all

broad-spectrumcephalosporins,aztreonam,gentamicin,

flu-oroquinolones,meropenem,ertapenem,andpolymyxinBas

showninTable1.Theisolatesshowedintermediateresistance

toimipenemandamikacin.WhileKPN1wassusceptibleto

tigecycline,KPN2becameresistanttothisagent.

TheisolateswerealsoscreenedforES␤Lproductionbydisk

approximationandthesynergismwasobservedonlywhen

amoxicillin/clavulanicaciddiskwastested15mmapartof

ceftriaxone,ceftazidimeandcefepimedisks.Thephenotypic

detectionofM␤LwascarriedoutbytheEDTA/DDSTand

con-firmedbyertapenemhydrolysisassayusingMALDI-TOFMS

(BrukerDaltonics,Germany)aspreviouslyreported.3_Although

both isolatesshowed anincreasein the inhibitionzoneof

ceftazidime/EDTA(6mm)andmeropenem/EDTA(7mm)disks

comparedtotheceftazidimeandmeropenemdisks,

respec-tively,hydrolysisofertapenemwasnotobserved,suggesting

thatbothisolateswerenotM␤Lproducers.

Thesearch for ␤-lactamaseencoding geneswas carried

outbyPCRfollowedbyDNAsequencingofamplicons.Both

K. pneumoniae isolates carried blaCTX-M-15, and the narrow

spectrum-␤-lactamases encoding genes, blaSHV-11, blaTEM-1

andblaOXA-1.ThepresenceoftheplasmidmediatedqnrS1gene

andamutationSer83IleingyrAwerealsodetected,justifying

thequinoloneresistanceexhibitedbybothKPNisolates.The

analysisoftheoutermembraneproteinsprofilebySDS–PAGE

showedthatbothisolateslostthemajorporinsOmpK35and

OmpK36.Sequencinganalysisoftheompk35andompk36genes

revealedthepresenceoftheinsertionsequenceIS1between

thepromoterregionandthestartcodonoftheompK35gene,

and the presenceofthe IS908disruptingthe ompK36gene.

These results corroborated with the absence of the major

porins on the SDS–PAGE gels, sincethe presence ofthe IS

resultedinnon-functionalOmpK35andOmpK36porins.

Themisidentificationoftwopan-resistantK.pneumoniae

isolatesasM␤LproducersbytheEDTA/DDSTisinagreement

withpreviousstudythatreportedfalse-positiveresultswhen

EDTAwasemployedforidentificationoftheM␤Lproduction.4

ThisfactmayresultfromthebactericidaleffectofEDTAwhich

actsincreasingthemembranepermeability.5_{Basedonthat,a}

disk containedonly100mMofEDTAwasalsotestedinthe

presentstudy,confirmingtheirbactericidaleffect,since

con-siderableinhibitiondiameterzonesof18mmforKPN1 and

16mmforKPN2wereobserved.

ThisstudyreportedamisidentificationofM␤Lproducers

byEDTA/DDST inK. pneumoniaeclinical isolates,as

recom-mendedbyANVISA.1_{Thepan-resistantphenotypeobserved}

between the two K. pneumoniae strains isolated in our

b r a z j i n f e c t d i s . 2 0 1 5; 1 9(1) :102–104

103

Table1–AntimicrobialsusceptibilityprofileandresistantdeterminantsamongtheK.pneumoniaeisolatesmisidentifiedasM␤LproducersbyEDTA/DDST.

Isolate MALDI-TOF MSID

MIC(␮g/mL)/susceptibilitycategorya

CEP CAZ CTX FEP AZT IPM MEM ETP AK GEN LEV CIP TGC PO

KPN1 K.pneumoniae ≥256[R] ≥256[R] ≥256[R] ≥256[R] ≥32[R] 2[I] 8[R] 64[R] 32[I] ≥64[R] ≥128[R] ≥128[R] 1[S] 32[R] KPN2 K.pneumoniae ≥256[R] ≥256[R] ≥256[R] ≥256[R] ≥32[R] 2[I] 8[R] 64[R] 32[I] ≥64[R] ≥128[R] ≥128[R] 4[R] 32[R]

Isolate MALDI-TOFMSID DiametervariationwithEDTA(mm) Porinlesiontypeb _{Resistantdeterminants}

CAZ MEM OmpK35 OmpK36

KPN1 K.pneumoniae 7 6 IS1(−5) IS908(+726) CTX-M-15,TEM-1,SHV-11,QnrS1,OXA-1

KPN2 K.pneumoniae 7 6 IS1(−5) IS908(+726) CTX-M-15,TEM-1,SHV-11,QnrS1,OXA-1 a _{Abbreviations:CEP,cephalotin;CAZ,ceftazidime;CTX,cefotaxime;FEP,cefepime;IPM,imipenem;MEM,meropenem;ETP,ertapenem;AK,amikacin;GEN,gentamicin;LEV,levofloxacin;CIP,}

ciprofloxacin;TGC,tigecyclin;PO,polymyxinB.

b _{Porinlesiontype:+and−indicatethepositionupstreamanddownstreamofstartcodon;numbersbetweenparenthesescorrespondthenucleotideinsertionposition;IS}

nindicatethemutation

104

clinicallyavailablefortreatingsuchinfections.Probably,the

isolatesalsopossessotherresistancemechanismsthathad

contributedtotheirmulti-drugresistantprofile.Thechelating

agentssuchasEDTAcanincreasetheoutermembrane

perme-ability,facilitatingtheentryofantibiotics.4,5 _Basedonthat,

diskcontainedonly100mMofEDTAshouldbeused

addition-ally,toconfirmtheir bactericidaleffect; thereforeattention

shouldbetakenbytheroutinelaboratoriestoavoidthereport

offalsepositiveresults.

Conflicts

of

interest

A.C.G. recently received research funding and/or

consulta-tionfeesfromAstraZenecaandMerckSharp&Dohme.Other

authorshavenothingtodeclare.

Acknowledgments

WethankAnaCarolinaRamosdaSilvaforherassistancein

theSDS–PAGEgelsandtoLorenaCristinaCorrêaFehlbergfor

reviewingthismanuscript.

r

e

f

e

r

e

n

c

e

s

1.AgênciaNacionaldeVigilânciaSanitária(ANVISA).Medidasde Prevenc¸ãoeControledeInfecc¸õesporEnterobactérias Multiresistentes.Brasília,Brasil:ANVISA;2013.

2.ClinicalandLaboratoryStandardsInstitute(CLSI).Performance standardsforantimicrobialsusceptibilitytesting;approved standard–twentyfourtheditionM100-S24.Wayne:CLSI;2014.

3.CarvalhaesCG,CayôR,AssisDM,etal.Detectionof SPM-1-producingPseudomonasaeruginosaandclassD ␤-lactamase-producingAcinetobacterbaumanniiisolatesbyuse ofliquidchromatography–massspectrometryand

matrix-assistedlaserdesorptionionization-timeofflightmass spectrometry.JClinMicrobiol.2013;51:287–90.

4.ChuYW,CheungTK,NganJYW,KamKM.EDTAsusceptibility leadingtofalsedetectionofmetallo-␤-lactamasein

PseudomonasaeruginosabyE-testandimipenem-EDTAdisk method.IntJAntimicrobAgents.2005;26:340–1.

5.RatkaiC,QuinteiraS,GrossoF,MonteiroN,NagyE,PeixeL. Controllingforfalsepositives:interpretingMBLEtestandMBL combineddisctestforthedetectionof

metallo-beta-lactamases.JAntimicrobChemother. 2009;64:657–8.

DandaraCassu-Corsi∗,1_{, WillamesM.B.S.Martins}1

LaboratórioALERTA,Disciplinade Infectologia,Departamento de Medicina,UniversidadeFederaldeSãoPaulo-UNIFESP,

SãoPaulo,SP,Brazil

MaraCristinaScheffer

LaboratóriodeMicrobiologia,DivisãodeAnálisesClínicas, Hospi-talUniversitário,UniversidadeFederaldeSantaCatarina-UFSC, Florianópolis,SC,Brazil

RodrigoCayô, AnaCristinaGales

LaboratórioALERTA,Disciplinade Infectologia,Departamento de Medicina,UniversidadeFederaldeSãoPaulo-UNIFESP,

SãoPaulo,SP,Brazil ∗_{Correspondingauthor.}

E-mailaddress:dandara.corsi@gmail.com(D.Cassu-Corsi).

1_{Theseauthorscontributedequallytothiswork.}

Received7August2014

Accepted20August2014

Availableonline13October2014

http://dx.doi.org/10.1016/j.bjid.2014.08.008