Detection of virulence and β-lactamase encoding genes in Enterobacter aerogenes and Enterobacter cloacae clinical isolates from Brazil

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Short

communication

Detection

of

virulence

and

␤-lactamase

encoding

genes

in

Enterobacter

aerogenes

and

Enterobacter

cloacae

clinical

isolates

from

Brazil

Paola

Aparecida

Alves

Azevedo

_,

_João

_Pedro

_Rueda

_Furlan

_,

_Mariana

_{Oliveira-Silva}

_,

Rafael

Nakamura-Silva

_,

_Carolina

_Nogueira

_Gomes

_,

_Karen

_Regina

_Carim

_Costa

_,

Eliana

Guedes

Stehling

_,

_André

_{Pitondo-Silva}

a_{UniversidadedeSãoPaulo,FaculdadedeCiênciasFarmacêuticasdeRibeirãoPreto,DepartamentodeAnálisesClínicas,Toxicológicase} Bromatológicas,RibeirãoPreto,SP,Brazil

b_{UniversidadeFederaldoAmazonas,Manaus,AM,Brazil}

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Articlehistory:

Received2September2017

Accepted19April2018

Availableonline21May2018

AssociateEditor:ElizabethMarques

Keywords: Enterobactercloacae Enterobacteraerogenes Virulence Resistance

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EnterobactercloacaeandE.aerogeneshavebeenincreasinglyreportedasimportant

oppor-tunisticpathogens.Inthisstudy,ahighprevalenceofmulti-drugresistantisolatesfrom

Brazil,harboringseveral␤-lactamaseencodinggeneswasfound.Severalvirulencegenes

wereobservedinE.aerogenes,contrastingwiththeE.cloacaeisolateswhichpresentednone.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

BacteriabelongingtotheEnterobactergenusaregram-negative

facultativeanaerobesandwidelydistributedinnature.Inthe

lastdecades,speciesofthegenusEnterobacterhavearoused

greaterconcern,sincetheyareincreasinglyassociatedwith

nosocomial infections, especially in immunocompromised

patients.1_{EnterobacterspeciesaremembersofESKAPE}

(Ente-rococcusfaecium, Staphylococcus aureus, Klebsiellapneumoniae,

∗ _{Correspondingauthorat:DepartamentodeAnálisesClínicas,ToxicológicaseBromatológicas,FaculdadedeCiênciasFarmacêuticasde}

RibeirãoPreto,UniversidadedeSãoPaulo(USP),RibeirãoPreto.Av.doCafé,s/n,MonteAlegre,RibeirãoPreto,SPCEP:14040-903,Brazil.

E-mail:andre@pitondo.com.br(A.Pitondo-Silva).

Acinetobacterbaumannii,Pseudomonasaeruginosaand

Enterobac-ter species) which are described as the leading cause of

nosocomial infections throughoutthe world.2,3 _{Among the}

species,E.aerogenesandE.cloacaestandoutasopportunistic

pathogens,especiallyinpatientsonmechanicalventilation.4

Enterobactersp.,including E.aerogenesandE.cloacae

eas-ily acquire numerous genetic mobile elements containing

https://doi.org/10.1016/j.bjm.2018.04.009

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

resistanceandvirulencegenes,whichstronglycontributeto

theincreasedpathogenicityofthesebacteria.Thesespecies

are extended spectrum ␤-lactamase producers and posses

intrinsicresistancetoampicillin,amoxicillin,first-generation

cephalosporinsandcefoxitin,duetotheproductionof

consti-tutiveAmpC␤-lactamase.ThusEnterobactersp.maydevelop

antimicrobialresistanceduringtreatment,limiting

therapeu-ticoptions.3,4

Although studies have been pointing to the increasing

prevalence of E. cloacae and. E. aerogenes as opportunistic

pathogens,frequentlyassociatedwithmultidrug-resistance,

littleisknownabouttheirvirulencemechanisms.1_Therefore,

the aimof this study was to characterizeE. aerogenes and

E.cloacae clinical isolatesfrom Brazil regardingthe genetic

relationships, antimicrobial susceptibility profile, and the

presenceofvirulenceand␤-lactamaseencodinggenes.

Inthisstudyatotalof14Enterobacterisolateswereincluded,

beingeightE.cloacae(EC)andsixE.aerogenes(EA).Amongthe

isolates,sevenwere obtainedfrom malepatients, sixfrom

female patientsand onewas notinformed. These isolates

wereobtainedbetweenMarch2013toSeptember2016from

threeBraziliantertiaryhospitalslocatedindifferentregions:

thesoutheast(RibeirãoPretocity,SãoPaulostate),northeast

(Teresinacity,Piauístate)andthenorthregion(Manauscity,

Amazonasstate).Theseisolateswereobtainedfromdifferent

sourcessuchasurine,blood,analandnasalswabs,catheter

tip,oropharynxandgastrostomytube(Table1).

Bacteriaidentificationwereperformedbymatrix-assisted

laserdesorption–ionizationtimeofflightmassspectrometry

(MALDI-TOFMS) using the Vitek® MS system (bioMérieux,

Marcyl’Etoile,France),accordingtothemanufacturer’s

rec-ommendations.

Theantimicrobialsusceptibilitytestswereperformedby

disc diffusion in Mueller-Hinton Agar (Oxoid), as

recom-mendedbytheClinicalLaboratoryStandardsInstitute(CLSI).5

Thirty-two different antibiotic discs (Oxoid) were tested:

amikacin(30␮g),aztreonam(30␮g),cefaclor(30␮g),cefepime

(30␮g),cefixime(5␮g),cefotaxime(30␮g),ceftriaxone(30␮g),

ceftazidime (30␮g), cefuroxime (30␮g), ciprofloxacin (5␮g),

chloramphenicol (30␮g), doripenem (10␮g), doxycycline

(30␮g), ertapenem (10␮g), gentamicin (10␮g), imipenem

(10␮g),levofloxacin(5␮g),lomefloxacin(10␮g),meropenem

(10␮g),minocycline(30␮g),nalidixicacid(30␮g),

nitrofuran-toin(300␮g),norfloxacin(10␮g),ofloxacin(5␮g),

piperacillin-tazobactam(100/10␮g),streptomycin(10␮g),sulphonamide

(300␮g),tetracycline(30␮g),ticarcillin-clavulanate(75/10␮g),

trimethoprim (5␮g), tobramycin (10␮g) and

trimethoprim-sulfamethoxazole (1.25/23.75␮g). Thestrains E. coli ATCC®

25922 and ATCC® 35218,and Pseudomonasaeruginosa ATCC

27853 were used asquality controlfor these experiments.

Althoughcefuroximeisconsideredasintrinsicresistancefor

E.cloaceaeand E.aerogenesbythe CLSI,5 _{thisantibioticwas}

addedinthestudybecauseitisrecommendedbyMagiorakos

etal.6_{forthesespecies.Theisolateswereclassifiedas}

multi-drug resistant (MDR), extensively drug-resistant (XDR) and

pandrug-resistant(PDR),accordingtothecriteriaestablished

byMagiorakosetal.6_{Isolateswhichdidnotfittheprevious}

definitionsweredesignatedasnotclassified(NC).

GenomicDNAwasextractedusingthephenol/chloroform

methodasdescribedbyCovoneetal.7Theconcentrationand

purity were determined usinga DS-11+Spectrophotometer

(DeNovix,USA).

PCRreactionswereperformedforthedetectionofthe

fol-lowing␤-lactamaseencodinggenes:blaCTX-M-Gp1,blaCTX-M-Gp2,

Table1–GeneraldatarelatingtosixE.aerogenes(EA)andeightE.cloacae(EC)isolatesfromthisstudy.

Isolate Dateofisolation Sex Age Source Resistanceprofile(non-susceptible)

EA01 05/15/2013 F 39 Urine LMX,EST,DOR,CFC,CFM,CRX,DOX,NIT,SUL,SUT

EA02 05/06/2013 F 66 Urine CFC,CFM,DOX,NIT,SUL,TRI

EA03 04/30/2016 F NI Urine EST,CFC,CFM,DOX,MIN,NIT,SUL,SUT,NAL

EA04 05/05/2016 M NI Blood EST,CFC,DOX,MIN,NIT,SUL,SUT,NAL

EA05 09/15/2016 M 33 Cathetertip CIP,LEV,LMX,NOR,OFX,AMI,EST,GEN,TOB,DOR,ERT,MPM,CAZ,

CFC,CFM,CPM,CRO,CRX,CTX,ATM,PIT,TAC,DOX,MIN,TET,NIT,

SUL,SUT,TRI,NAL

EA06 04/29/2013 M 57 Nasalswab LMX,AMI,EST,GEN,TOB,DOR,ERT,IPM,CAZ,CPM,CRX,PIT,DOX,

NIT,SUL,SUT,TRI,NAL

EC01 04/20/2013 F NI Urine CFC,CFM,DOX,MIN,NIT,SUL,TRI,NAL

EC02 05/20/2013 F 57 Urine AMI,ERT,MPM,CFC,CFM,CRO,DOX,MIN,NIT,SUL,TRI,NAL

EC03 05/07/2016 NI NI Urine CIP,LEV,LMX,NOR,OFX,EST,TOB,CAZ,CFC,CFM,CPM,CRO,CRX,

CTX,ATM,PIT,TAC,DOX,MIN,TET,NIT,SUL,SUT,TRI,NAL

EC04 04/29/2013 F 43 Analswab CIP,LEV,LMX,NOR,OFX,EST,GEN,TOB,ERT,CAZ,CFC,CFM,CPM,

CRO,CRX,CTX,ATM,PIT,TAC,DOX,TET,NIT,SUL,SUT,TRI,CLO,NAL

EC05 05/06/2013 M 65 Analswab LMX,OFX,AMI,EST,GEN,ERT,CAZ,CFC,CFM,CPM,CRO,CRX,CTX,

ATM,PIT,TAC,SUL,SUT,TRI,NAL

EC06 02/23/2015 F 53 Oropharynx EST,CFC,CFM,CRX,DOX,NIT,SUL,SUT,TRI,NAL

EC07 03/13/2015 M 58 Cathetertip ERT,SUL,SUT,TRI

EC08 05/13/2015 M 51 Gastrostomytube CFC,CRX,NIT,SUL,SUT

Note:F,female;M,male,NI,not informed;AMI,amikacin;ATM,aztreonam;CFC,cefaclor; CPM,cefepime,CFM,cefixime;CRO,

ceftriax-one;CAZ,ceftazidime;CTX,cefotaxime;CRX,cefuroxime;CIP,ciprofloxacin;CLO,chloramphenicol;DOR,doripenem;DOX,doxycycline;ERT,

ertapenem;GEN,gentamicin;IPM,imipenem;LVX,levofloxacin;LMX,lomefloxacin;MPM,meropenem;MIN,minocycline;NAL,nalidixicacid;

NIT,nitrofurantoin;NOR,norfloxacin;OFX,ofloxacin;PIT,piperacillin-tazobactam;EST,streptomycin;SUL,sulphonamide;TET,tetracycline; TAC,ticarcillin-clavulanate;TRI,trimethoprim;TOB,tobramycin;SUT,trimethoprim-sulfamethoxazole.

blaCTX-M-Gp8,blaCTX-M-Gp9,blaSHV,blaPER,blaVEB,blaGES,blaKPC, blaVIM,blaOXA-48-like, blaIMP, blaSPM,blaSIM,blaGIM, andblaNDM

usingprimersandprotocolspreviouslydescribed.8–11_AllPCR

wereperformedusingpositiveandnegativecontrols.

DetectionofvirulencegeneswereperformedbyPCRforthe

followinggenes:allS(allantoinmetabolism),entB,ybtSandiutA

(siderophores), kfu (iron transport and phosphotransferase

function),mrkD(adhesintype3fimbriae),rmpA(regulatorof

mucoidphenotypeA)accordingtoCompainetal.,12_andycfM

(outermembranelipoprotein)andfimH(adhesivesubunitof

type1fimbriae)accordingtoElFertas-Aissanietal.13_AllPCR

wereperformedusingpositiveandnegativecontrols.

Confirmation of the amplified genes’ identity was

per-formedbysequencing.Oneofeachampliconwasrandomly

selectedandpurifiedusingtheIllustraTM _GFXPCRDNAand

GelBandPurificationKits(GEHealthcare,Buckinghamshire,

UK). The sequencing was performed in an automated

sequencer ABI 3500×LGeneticAnalyzer platform (Applied

Biosystems,USA).Thesequenceswereanalyzedusing

Chro-masPro version 1.7.6 software (Technelysium Pty.Ltd) and

comparedwiththe sequencesavailableinthe BLAST

algo-rithm(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

EnterobacterialRepetitiveIntergenicConsensusPCR

(ERIC-PCR)analysiswasperformedtoevaluatethegeneticsimilarity

among the bacterial isolates of this study. ERIC-PCR was

performed as previously described by Versalovic et al.14

using primers ERIC1R (5-ATGTAAGCTCCTGGGGATTCAC-3)

andERIC2(5-AAGTAAGTGACTGGGGTGAGCG-3).Bandprofile

analysiswas performed usingthe BioNumerics version 5.1

program(AppliedMaths,Keistraat,Belgium)forconstruction

ofthe similaritydendrogrambythe unweightedpairgroup

mean methodand Dice’s similarity coefficient. Onlybands

representingampliconsbetween500and3000bpwere

con-sideredforthisanalysis.TheERIC-PCRassayswereperformed

intriplicatetobesureofthereproducibilityoftheamplified

bands.

Among the 14 isolates, eight (57.1%) were classified

as MDR according to the criteria established by

Magio-rakos et al.,6 _{being five isolates of E. cloacae (EC02, EC03,}

EC04, EC05 and EC06) and three E. aerogenes (EA01, EA05

and EA06) (Fig. 1). All isolates showed non-susceptibility

(i.e. either intermediate or resistance) to sulphonamide;

12 (85.7%) isolates were non-susceptible to cefaclor and

nitrofurantoin; 11 (78.6%) to doxycycline; 11 (78.6%) to

cefixime,trimethoprim-sulfamethoxazole,trimethoprimand

nalidixic acid; nine (64.3%) to streptomycin; eight (57.1%)

to cefuroxime; six (42.9%)to lomefloxacin, ertapenem and

minocycline; five(35.7%)toceftazidime,cefepime,

ceftriax-one and piperacillin-tazobactam; four (28.6%)to amikacin,

gentamicin, tobramycin, ofloxacin, cefotaxime, aztreonam

and ticarcillin-clavulanate; three (21.43%) to ciprofloxacin,

levofloxacin, norfloxacin, doripenem and tetracycline; two

(14.3%)tomeropenemandjustoneisolate(7.14%)to

chloram-phenicol(Table1).

Theresistanceprofilefoundinthestudiedisolates

corrob-oratewithastudyconductedbyCabraletal.,4_{whichanalyzed}

aBrazilian collectionofE.aerogenesand E.cloacae complex

isolates,andfoundthelowestresistanceratesinamikacin,

gentamicin and tobramycin. However, theyfound low

lev-elsofresistancetotrimethoprim–sulfamethoxazolewhilewe

found71.4% resistanceforthis antibiotic.Fortunately,with

the exception ofisolateEA06,all isolateswere sensitiveto

imipenem,sincepapers havedescribed thatthis antibiotic

remainsoneofthemosteffectiveincombatingE.cloacae

infec-tions.However,thereareseveralworksreportingtheadaptive

responseofclinicalisolatesofEnterobactersp.toimipenemby

regulationofporins.4,15

In this study 16 different ␤-lactamase encoding genes

belongingtogroup2and3,accordingtoBushandJacoby16

wereinvestigated,andatotalof18ampliconsfromsix

differ-entgenes(blaCTX-M-Gp1,blaCTX-M-Gp2,blaSHV,blaVEB,blaGES,and

blaKPC)weredetected(Fig.1).Themostprevalentgenewas

Isolate EA03 EA04 EA05 EA02 EA01 EA06 EC01 EC03 EC04 EC05 EC02 EC06 EC08 EC07 RP NC NC NC NC NC NC MDR MDR MDR MDR MDR MDR MDR MDR

Blactx- Blactx- BlaSHVBlaVEBBlaGESBlaKPC MrkD Kfu EntB YbtS FimH YcfM AllS Virulence Genes found β−lactamases Gp1 Gp2 60 70 80 90 100 74.1 63.8 58.8 75.0 71.4 77.4 72.7 82.8 80.7 71.4 68.6 _80.0 53.0

Fig.1–DendrogramobtainedbyenterobacterialrepetitiveintergenicconsensusPCR(ERIC-PCR)analysisofsixE.aerogenes andeightE.cloacaestudiedisolatesfromBrazil.ClustersweredeterminedusingtheUnweightedPairGroupMean(UPGMA) methodandtheDicesimilaritycoefficient.Similarity(%)amongpatternsisrepresentedbythenumbersbesidethenodes. Foreachisolatetheirrespectiveresistanceprofileclassification(RP),␤-lactamase(whitebars)andvirulence(blackbars) encodinggenesarerepresented.MDR,multi-drugresistant;NC,notclassified.

blaSHV,detectedin10isolates(EA01,EA03,EA04,EA05,EA06,

EC04,EC05,EC06,EC07andEC08).Thefollowinggeneswere

foundintwoisolateseachblaCTXM-Gp1(EC04andEC08),blaKPC

(EC01andEC05)andblaGES(EC06andEC08).TheblaCTX-M-Gp2

andblaVEB geneswerefoundinjustoneisolateeach (EA04

andEA01,respectively)(Fig.1).TheblaCTX-M-Gp8,blaCTX-M-Gp9,

blaPER, blaVIM, blaOXA-48-like,blaIMP, blaSPM, blaSIM,blaGIM, and

blaNDM were not detected.These results are in contrast to

thosedescribedbyCabraletal.,4_{whichdidnotfindbla}

SHVin Entorobactersp.clinicalisolatesfromBrazil,buttheyfounda

highprevalenceofblaKPC.Inaddition,Pereiraetal.17studied

21 carbapenem-resistant E. aerogenes strains isolated from

a Braziliantertiary hospital inJuiz de Fora city located in

southeastBrazil.Theyobserved52.4%ofblaSHVand28.6%of

blaCTX-Mgroupsintheircollection.

Differentmechanismsofresistancemaybeassociatedwith

non-susceptibilityto ␤-lactam antibioticsin Gram-negative

bacteria;howevertheenzymatic,forintrinsicandacquired

␤-lactamases,isthemainmechanism.16,18_{Amongtheisolates,}

EC01andEA06presentedtheblaKPCgenebutdidnotpresent

non-susceptibility to extended-spectrum cephalosporins or

carbapenems, and the isolates EA05 and EA06 were

non-susceptibleto carbapenems and only the blaSHV gene was

detected. Therefore, the diversity of mechanisms, such as

porin loss, efflux pumpsor lack ofexpression ofacquired

genes and the presence ofother unsearched ␤-lactamases

maybeassociatedwiththeseinconsistenciesbetweengenes

foundandexpressedphenotype.3

Ninevirulencegeneswereinvestigatedand,surprisingly,

allE.aerogenesisolatespresentedoneormoregenes,whereas

noE.cloacaeisolatepresentedvirulencegenes.Thegenetic

similarity dendrogramconstructed withthe ERIC-PCR data

showed the formation of two large clusters, denominated

AandB,comprisingthe E.aerogenesandE.cloacaeisolates,

respectively(Fig.1).TheisolateEA05presentedseven

viru-lencegenes,followedbyEA03andEA04withsix,EA06with

two,andEA01andEA02withonegene(Fig.1).Theobtained

virulenceand␤-lactamaseencodinggenes’sequenceswere

depositedinGenBank(www.ncbi.nlm.nih.gov/Genbank)with

accessionnumbersMF622540toMF622551.

Theresults foundsuggest apredominance of virulence

genes in E. aerogenes when compared with E. cloacae. In

additiontothefewstudies investigatingvirulencegenesin

Enterobacter sp.,19,20 _{to our knowledgethere are no studies}

comparingthepredominanceofthesegenesinE.cloacaeandE.

aerogenes.However,asthenumberofisolatesfromthisstudy

doesnotallowestablishingastatisticalcorrelation onthis,

moredetailedstudieswithalargercollectionarerequired.

Theroleofthevirulencegenesfoundintheisolatesiswell

describedforKlebsiellapneumoniaestrains.ThegenesfimHand

mrkDencodingadhesinsoftype1and type3fimbriaeand

ycfMareoftenfoundinK.pneumoniaeandplayanimportant

roleintheprocessofbacterialadhesioninvarioushuman

tis-suesandalsointheformationofbiofilms.13 _Thegeneskfu,

entBandybtSareinvolvedintheproductionofsiderophores

whichareimportantmoleculesfortheacquisitionofironfor

bacterialmetabolism.Thesesiderophores,especiallyKfu,are

oftenfoundinhypervirulentstrainsofK.pneumoniae.12 _The

allSgeneassociatedwithassimilationofallantoinisclosely

associatedwithK.pneumoniaeisolatesfromliverabscesses.12

Hassanetal.19_{studiedthirty-twoisolatesofEnterobactersp.}

obtainedfromclinicalurinespecimensandidentifiedthefimH

gene in 40% ofthe isolates. Hussainet al.21 _studiedsome

virulencefactorencodinggenesof75 E.cloacaeandnine E.

sakazakiiandandobservedthatallisolateshavetheabilityto

producesiderophores.DespitetherelevanceofEnterobactersp.

asanosocomialpathogen,themechanismsofvirulenceof

thesespeciesarestillunclearduetothescarcityofstudiesin

thisarea.3

Inconclusion,ahighprevalenceofmulti-drugresistance

in the studied BrazilianEnterobacter species harboring

sev-eral␤-lactamaseencodinggeneswasfound.Moreover,several

virulence genes were observed in E. aerogenes, contrasting

withtheE.cloacaeisolates,whichdidnotpresent any

viru-lencegenes.Thecombinationofmulti-drugresistancewith

␤-lactamaseencodinggenesand associationwithvirulence

genes, especiallyin E. aerogenes, isworrying, since studies

haveshown anincreasingincidenceoftheseopportunistic

pathogenscausingnosocomialinfections.

Funding

ThisworkwassupportedbySãoPauloResearchFoundation–

FAPESP[grantnumber,2013/22581-5].

Ethical

Approval

EthicalapprovalwasreceivedfromtheSchoolof

Pharmaceu-ticalSciencesofRibeirãoPreto,UniversityofSãoPaulo,Brazil

[approvalno.:CEP/FCFRP362;CAEE36031914.9.0000.5403].

Acknowledgements

WearethankfultoDrJohannPitoutandDrAnaCristinaGales

forprovidingcontrolstrains.WealsowouldliketothankJohn

Carpenter(RibeirãoPreto,SP,Brazil)fortheEnglishrevision.

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