Isolation of blaNDM-producing Enterobacteriaceae in a public hospital in Salvador, Bahia, Brazil

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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,∗

a_Hospital_{Universitário}_Prof.Edgar_Santos,_Salvador,_BA,_Brazil b_Laboratório_Central_De_Saúde_Pública,_Brasília,_DF,_Brazil c_Hospital_São_Rafael,_Salvador,_BA,_Brazil

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Articlehistory:

Received3July2017 Accepted20October2017

Availableonline13November2017

Keywords:

NewDelhiMetallo-b-lactamase Carbapenemase

Antimicrobialresistance

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b

s

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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.

Carbapenemases have great importance in the global epi-demiologicalscenariosinceinfectionswith carbapenemase-producing bacteria are associated with high mortality, especiallyinhospitalizedpatientsinintensivecareunits.In addition,therearefewtreatmentoptionsavailable,coupled

∗ _{Corresponding}_author.

E-mailaddress:[email protected](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

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In2009,agroupofresearchersfromIndiadescribedanew variantofMBL,calledNewDelhiMetallo-␤-lactamase(NDM), encodedbythe gene blaNDM.3 TheNDMis endemicinthe Indiansubcontinentandsinceitsﬁrstdescriptionithasbeen reportedincases ofinfectionsworldwide, oftenassociated withinternationaltravel,medicaltourismandpotential expo-suresintheBalkansandtheIndiansubcontinent.4_By_the_year

2012,therewerenoreportsofmicroorganismsproducingthis enzymeinSouthAmericaandAntarctica.5,6_The_ﬁrst

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.8_Both_K._pneumoniae_isolates

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,meropenemandimipenemhigherthan32␮g/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 20␮Lofthereactionmixture.PCRwasperformedusing2X READYMIXKappaTaqcontainingDNApolymerase(0.05U/uL, 1.25Uper25␮L)reactionbufferwithMg2+_and_0.4_mM_of_each dNTP, with loading dye (KappaBiosystems, Japan), primers (Table1)and2␮Lofbacteriallysates.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 15␮Lingel) 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

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brazj infect dis.2018;22(1):47–50

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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 GenBanka_KF745070.2 _K._pneumonia_BR01

IMPR

a _Accession_number_of_the_sequence_used_for_primer_design.

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.13_In_addition,_in_both_accounts,

thegenewasisolatedfromP.rettgeri.Sincethen,other NDM-producingEnterobacteriaceae(M.morganii,E.cloacaecomplex) were isolated in this state from clinical and surveillance culturesin different hospitals.14 _More_recently, _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

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resistance to carbapenems among different species of Enterobacteriaceae.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

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