brazjinfectdis2018;22(1):47–50
w w w . e l s e v i e r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Brief
communication
Isolation
of
bla
NDM
-producing
Enterobacteriaceae
in
a
public
hospital
in
Salvador,
Bahia,
Brazil
Maria
Goreth
Barberino
a,
Silvia
de
Araujo
Cruvinel
a,
Célio
Faria
b,
Marco
Aurélio
Salvino
a,
Marcio
de
Oliveira
Silva
c,∗aHospitalUniversitárioProf.EdgarSantos,Salvador,BA,Brazil bLaboratórioCentralDeSaúdePública,Brasília,DF,Brazil cHospitalSãoRafael,Salvador,BA,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received3July2017 Accepted20October2017
Availableonline13November2017
Keywords:
NewDelhiMetallo-b-lactamase Carbapenemase
Antimicrobialresistance
a
b
s
t
r
a
c
t
Carbapenemaseshavegreatimportanceintheglobalepidemiologicalscenariosince infec-tionswithcarbapenemase-producingbacteriaareassociatedwithhighmortality,especially inhospitalizedpatientsinintensivecareunits.Thisstudydescribestwomicroorganisms producersoftheNewDelhiMetallo-b-lactamase,KlebsiellapneumoniaeandCitrobacter fre-undii,fromtwopatientsadmittedtoapublichospitalinSalvador,Bahia.Thesearethefirst clinicalcasesofNewDelhiMetallo-b-lactamasedescribedinmicroorganismsinthenorth andnortheastBrazil.Theisolateswerecharacterizedbyantimicrobialsusceptibilitytest, withresistancetoall-lactamsincludingcarbapenems,negativeModifiedHodgeTestand thesynergytestwithEthylenediaminetetraaceticacid,PhenylboronicAcidandCloxacillin waspositiveonlywithEthylenediaminetetraaceticacid(differenceof>5mminthe inhibi-tionzonebetweenthediskwithoutandwiththeinhibitor).AnalysisbymultiplexPCRfor
blaIMP,blaVIM,blaNDM,blaKPCandblaOXA-48enzymesconfirmedthepresenceofblaNDMgene. ThisreportoftwodifferentNewDelhiMetallo-b-lactamase-producingmicroorganismsina differentregionofBrazilconfirmstheriskofspreadingresistancegenesbetweendifferent speciesandemphasizestheneedforpreventionandcontrolofinfectionscausedbythese pathogens,whichhavelimitedtreatmentoptionsandhavebeenlinkedtohighmortality rates.
©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Carbapenemases have great importance in the global epi-demiologicalscenariosinceinfectionswith carbapenemase-producing bacteria are associated with high mortality, especiallyinhospitalizedpatientsinintensivecareunits.In addition,therearefewtreatmentoptionsavailable,coupled
∗ Correspondingauthor.
E-mailaddress:oliveirasm@yahoo.com.br(M.O.Silva).
with the potential for transmission of resistance to other speciesthroughmobilegeneticelements.1
Todate,researchhasshownthatresistanceto-lactam antibioticsinEnterobacteriaceaecouldbemediatedby differ-ent-lactamases,includingthosethatdegradecarbapenems, called carbapenemases. Further, the Metallo--lactamase (MBLs)areclassifiedasClassBofAmbleranddifferfromother carbapenemasesbyusingzincattheactivesite,which facili-tatesthehydrolysisoftheantibiotic.2
https://doi.org/10.1016/j.bjid.2017.10.002
1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
48
braz j infectdis.2018;22(1):47–50In2009,agroupofresearchersfromIndiadescribedanew variantofMBL,calledNewDelhiMetallo--lactamase(NDM), encodedbythe gene blaNDM.3 TheNDMis endemicinthe Indiansubcontinentandsinceitsfirstdescriptionithasbeen reportedincases ofinfectionsworldwide, oftenassociated withinternationaltravel,medicaltourismandpotential expo-suresintheBalkansandtheIndiansubcontinent.4Bytheyear
2012,therewerenoreportsofmicroorganismsproducingthis enzymeinSouthAmericaandAntarctica.5,6Thefirst
descrip-tionofNDMinBrazilwasinProvidenciarettgeriandEnterobacter hormaecheiisolatedfromRioGrandedoSulStatein2013.After thefirstreportanactivesurveillancewasinitiated,andthere have been other reports involving NDM-producing Entero-bacteriaceae,suchasEnterobactercloacae,Morganellamorganii, Escherichiacoli,andKlebsiellapneumoniae.7
These are the first clinical cases of NDM described in microorganisms derived from samples from two patients admittedtotheFederalUniversityHospitalinSalvador,State ofBahia. ThesearethefirstcasesofNDMdescribed inthe northandnortheastBrazil.
Patient
A
Thefirstpatientwasa25-year-oldmanadmittedtothe onco-hematologicalwardatthehospitalonJuly17th2015.Hehad apreviousdiagnosisofxerodermapigmentosumsince child-hoodand was diagnosed with pancytopeniathree months beforeadmissionduetoepisodesofdiffusebleeding (epis-taxis,earandgingivalbleeding).Healsohadinfectedulcers attheleftleg.Previouslyhewasadmittedtoanother hospi-tal,wherehewastransfused,treatedforfebrileneutropenia anddiagnosedwithbonemarrowaplasiainabonemarrow aspirateinMay25th2015,andthentransferredtoour hospi-tal.Duringthehospitalizationhewasdiagnosedwithacute myeloidleukemiaonJuly23rd2015,andchemotherapywas initiated.Duringtreatment,heevolvedwithfebrile neutrope-niaandbloodculturesaswellascultureofthecatheterwere drawninAugust8th 2015.Thesamecarbapenemresistant strainofK. pneumoniaegrew from thesesamplesand were investigatedforthe presence ofcarbapenemase (described below).Antibiotictherapywasintroducedwithpolymyxinand tigecycline,butthepatientdiedonday40ofhospitalization.
Patient
B
The second patient was a 75-year-old man with previous diagnosisofsystemicarterialhypertension,admittedtoour hospitalon July 28th 2015due to a15-day history of pro-gressivemuscularweaknessinlowerlimbs,associatedwith urinaryretention.Priortoadmissioninourhospitalhewas diagnosedwith Guillain-Barrè Syndrome inanother hospi-talwherethepatientwastreatedwithimmunoglobulinand ceftriaxone.Thepatientwasthentransferredtoourhospital usingurinarycatheter,whichwaswithdrawnatadmission. Bloodandurinaryculturesfromadmissiondaywerenegative. Duringhospitalization, heevolvedwithrespiratorydistress beingmovedtotheintensivecareunit(ICU)ofthehospital, butwasdischarged4dayslaterwithnoneedofinvasive ven-tilatorysupport.SevendaysafterICUdischargehepresented
withfeverand theurinaryculturecollected fourdayslater (onAugust13th2015)grewCitrobacterfreundiiresistanttoall carbapenems,andwasalsoinvestigatedforthepresenceof Carbapenemase.Thisculturewasinterpretedascolonization, andthepatientdidnotpresentanyotherepisodeoffever.
Theidentificationandantimicrobialsusceptibilitytestsof thebacteriaisolatedfromthetwopatientswereperformed on Vitek 2 (bioMerieux– Marcy I’Etoile, France) and inter-pretedinaccordancewiththeparametersoftheClinicaland LaboratoryStandardsInstitute.8BothK.pneumoniaeisolates
showed resistancetoall-lactams including carbapenems, aminoglycosidesandfluoroquinolones.TheisolatedC.freundii
showed resistancetoall-lactams including carbapenems, butretainedinvitrosensitivitytoaminoglycosidesand fluoro-quinolones.Theminimuminhibitoryconcentrations(MICs)to carbapenemswereconfirmedusingEtest(bioMerieux–Marcy I’Etoile,France).ThethreeisolatesanalyzedshowedMICsto ertapenem,meropenemandimipenemhigherthan32g/mL. Afterconfirmingtheresistancetocarbapenems,Modified HodgeTest(MHT)andphenotypictestingdiscssynergywith meropenem, imipenem,ertapenem and with and without Ethylenediaminetetraaceticacid(EDTA)0.1M,cloxacillinand phenylboronicacidwereperformed.TheMHTwerenegative andsynergytestsshowedincreasedinhibitionzone(>5mm) incarbapenemsdiscsinthepresenceofEDTA,EDTA-freein relationtothediscs,andwerenegativewithphenylboronic acidandcloxacillinforthemicroorganismstested.
TheisolateswerethenanalyzedbymultiplexPCRforblaIMP,
blaVIM, blaNDM, blaKPC and blaOXA-48 enzymes, as described below.9
The bacterial lysates were prepared by suspending the colony in500mLofdeionized water (Milli-Qsystem, Milli-pore, Bedford,MA)and subjected toboilingfor15minand thenfrozen.TheK.pneumoniaeATCC700603strainwasused asanegativecontrolintheanalyses.EachPCRtubecontained 20Lofthereactionmixture.PCRwasperformedusing2X READYMIXKappaTaqcontainingDNApolymerase(0.05U/uL, 1.25Uper25L)reactionbufferwithMg2+and0.4mMofeach dNTP, with loading dye (KappaBiosystems, Japan), primers (Table1)and2Lofbacteriallysates.Allreactionswere sub-jectedtoreactioncontrolamplificationusing16Sgene.
Thesolutionsweresubjectedtothefollowingcycling con-ditions:initialdenaturationof95◦C(2min,1cycle);followed by40cycles:denaturation95◦C(30s),annealing55◦Cor57◦C (30s)andextension72◦C(2min,1cycle);andafinal exten-sion cycle of 72◦C (2min). We used the gradient thermal cycler Mastercycler® System,Eppendorf,Germany.ThePCR product (ampliconusing 15Lingel) were visualizedafter electrophoresis(2%agarose),andstainedwithethidium bro-mide(10mg/mL)inKodakdocumentationsystem(GelLogic 100ImagingSystem).
Fig.1showsbandingpatternsofmultiplexPCRreactions forthegenesstudied.TheblaNDMgenewasamplified, con-firmingtheenzymaticprofileofmicroorganisms.
Enterobacteriathatcarryresistancegenetocarbapenems, includingblaNDM,areconsideredanimportantpublichealth problembecauseofthepossibilityofexpansionofthegenes and the clinical impact on the management of infections associated withthese microorganisms, which are typically resistanttoalmostallantibiotics.12
brazj infect dis.2018;22(1):47–50
49
Table1–ThemultiplexPCR(carbapenemaseand16Sgenes)byusingspecificoligonucleotideprimers.
Gene Primer Product(pb) Reference Positivecontrols
16S U3 ∼900 JamesGetal.,201010 –
U4
blaKPC
KPC-F
1011 YigitHetal.,200111 K.pneumoniae
IOC4955 KPC-R blaNDM NDMF 512 GenBanka KP772192.1 E.cloacaeCCBH10892 NDMR blaOXA-48 OXAF 440 GenBanka KP849468.1 K.pneumoniae CCBH9976 OXAR1 blaVIM VIMF 332 GenBank a KR259332.1 P.aeruginosa CCBH11808 VIMR2
blaIMP IMPF2 440 GenBankaKF745070.2 K.pneumoniaBR01
IMPR
a Accessionnumberofthesequenceusedforprimerdesign.
blaKPC (1011bp) M Nº 2 3 4 5 1 Negative control Positive control 5843 5939 5958 + + + + + + + + + + - -- -- -+ + + Sample 16s
(~900bp) blaKPC blaNDM blaOXA-48 blaVIM blaIMP
M M M M M 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 blaNDM (512bp) blaOXA (440bp) blaVIM (332bp) blaIMP (440bp)
Fig.1–MultiplexPCRassayforsimultaneousdetectionof16SrRNA(∼900bp),asanamplificationcontrol,and
carbapenemasegenes.BlaKPC:Lane1,K.pneumoniaIOC4955(positivecontrol);Lane2,K.pneumoniaATCC700603(negative control);Lane3,5843;Lane4,5939;Lane55958.BlaNDM:Lane1,E.cloacaeCCBH10892(positivecontrol);Lane2,K.
pneumoniaATCC700603(negativecontrol);Lane3,5843;Lane4,5939;Lane55958.BlaOXA-48:Lane1,K.pneumoniae
CCBH9976(positivecontrol);Lane2,K.pneumoniaATCC700603(negativecontrol);Lane3,5843;Lane4,5939;Lane55958. BlaVIM:Lane1,P.aeruginosaCCBH11808(positivecontrol);Lane2,K.pneumoniaATCC700603(negativecontrol);Lane3, 5843;Lane4,5939;Lane55958.BlaIMP:Lane1,K.pneumoniaBR01(positivecontrol);Lane2,K.pneumoniaATCC700603 (negativecontrol);Lane3,5843;Lane4,5939;Lane55958.LaneM,100-bpDNAladder(Invitrogen).Theelectrophoresiswas runina2%agarosegel,whichwasstainedwithethidiumbromide.
AfterthefirstNDMisolatein2009inIndia,theNDMhas beendetectedineverycontinent,includingBrazil.InSouth America,blaNDMgenewasfirstreportedin2012inUruguay,a countrythatborderstheRioGrandedoSulstate,whereNDM wasfirstdescribedinBrazil.13Inaddition,inbothaccounts,
thegenewasisolatedfromP.rettgeri.Sincethen,other NDM-producingEnterobacteriaceae(M.morganii,E.cloacaecomplex) were isolated in this state from clinical and surveillance culturesin different hospitals.14 Morerecently, it was first
isolatedfromasurveillanceswab,theblaNDM-geneinK.
pneu-moniae,inRiodeJaneiro.Thisisolatecarriedaresistancegene alreadyfoundinaKPCstrainin2010inthe stateofMinas
Gerais,highlightingtheabilityofmicroorganismstoacquire anddisseminatesuchresistancegenes.15
This report of NDM isolates in two different microor-ganisms, K. pneumoniae and C. freundii, confirms the risk of spread of resistance genes between different species. The rapid spread of genes conferring resistance to car-bapenems,blaKPC,blaNDM,amongothers,isofgreatconcern because the emergence of multi-resistant microorganisms is a threat to public health due to the shortage of new antibiotics in development.16 This report emphasizes the
need for strict measures to prevent and control infec-tions, which can minimize the spread ofgenes conferring
50
braz j infectdis.2018;22(1):47–50resistance to carbapenems among different species of Enterobacteriaceae.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
r
e
f
e
r
e
n
c
e
s
1. HidronAI,EdwardsJR,PatelJ,etal.NHSNannualupdate: antimicrobialresistantpathogensassociatedwith healthcare-associatedinfections:annualsummaryofdata reportedtotheNationalHealthcareSafetyNetworkatthe CentersforDiseaseControlandPrevention,2006–2007.Infect ControlHospEpidemiol.2008;29:996–1011.
2. QueenanAM,BushK.Carbapenemases:theversatile beta-lactamases.ClinMicrobiolRev.2007;20:440–58. 3. YongD,TolemanMA,GiskeCG,etal.Characterizationofa
newmetallo-beta-lactamasegene,bla(NDM-1),andanovel erythromycinesterasegenecarriedonauniquegenetic structureinKlebsiellapneumoniaesequencetype14from India.AntimicrobAgentsChemother.2009;53:5046–54. 4. WalshTR,WeeksJ,LivermoreDM,TolemanMA.
DisseminationofNDM-1positivebacteriaintheNewDelhi environmentanditsimplicationsforhumanhealth:an environmentalpointprevalencestudy.LancetInfectDis. 2011;11:355–62.
5. JohnsonAP,WoodfordN.Globalspreadofantibiotic resistance:theexampleofNewDelhimetallo-b-lactamase (NDM)-mediatedcarbapenemresistance.JMedMicrobiol. 2013;62:499–513.
6. StruelensMJ.TheEuropeanNDM-1SurveyParticipantsNew Delhimetallo-beta-lactamase1-producing
Enterobacteriaceae:emergenceandresponseinEurope.Euro Surveill.2010;15:19716.
7.AiresCA,PereiraPS,deAraujoCF,etal.Multiclonalexpansion ofKlebsiellapneumoniaeisolatesproducingNDM-1inRiode Janeiro,Brazil.AntimicrobAgentsChemother.2017;61, http://dx.doi.org/10.1128/AAC.01048-16,pii:e01048-16. 8.ClinicalandLaboratoryStandardInstitute.Performance
standardfor188antimicrobialsusceptibilitytesting;23rd informationalsupplement.CLSI189documentM102-S53. Wayne,PA:CLSI;2015.p.2015.
9.Faria-JuniorC,RodriguesLDO,CarvalhoJOD,etal.
NDM-producingEnterobacteriaceaestrainsamongHospitals inBrasília,Brazil.JMicrobiolExp.2016;3:00083.
10.JamesGS.UniversalbacterialidentificationbyPCRandDNA sequencingof16SrRNAgene.In:SchullerM,SlootsTP, HollidayCL,JamesGS,CarterIWJ,editors.PCRforclinical microbiology.NewYork:Springer;2010.
11.YigitH,QueenanAM,AndersonGJ,etal.Novel carbapenem-hydrolyzingbeta-lactamase,KPC-1,froma carbapenem-resistantstrainofKlebsiellapneumoniae. AntimicrobAgentsChemother.2001;45:1151–61. 12.GaibaniP,AmbrettiS,BerlingeriA,etal.Outbreakof
NDM-1-producingEnterobacteriaceaeinnorthernItaly,Julyto August2011.EuroSurveill.2011;16:20027.
13.Carvalho-AssefAP,PereiraPS,AlbanoRM,etal.Isolationof NDM-producingProvidenciarettgeriinBrazil.JAntimicrob Chemother.2013;68:2956–7.
14.RozalesFP,RibeiroVB,MagagninCM,etal.Emergenceof NDM-1-producingEnterobacteriaceaeinPortoAlegre,Brazil.Int JInfectDis.2014;25:79–81.
15.FerreiraAM,MondelliAL,CamposE,etal.Firstreportofa clinicalisolateofNewDelhimetallo--lactamase-producing KlebsiellapneumoniaeinBrazil.JHospInfect.2016;94: 73–4.
16.WiseR.Theurgentneedfornewantibacterialagents.J AntimicrobChemother.2011;66:1939–40.
