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
Original
article
Carbapenem-resistant
and
cephalosporin-susceptible:
a
worrisome
phenotype
among
Pseudomonas
aeruginosa
clinical
isolates
in
Brazil
Eloiza
Helena
Campana
a,∗,1,
Danilo
Elias
Xavier
a,1,
Fernanda
Villas-Boas
Petrolini
a,
Jhonatha
Rodrigo
Cordeiro-Moura
a,
Maria
Rita
Elmor
de
Araujo
b,
Ana
Cristina
Gales
aaUniversidadeFederaldeSãoPaulo,LaboratórioALERTA,DisciplinadeInfectologia,SãoPaulo,SP,Brazil bHospitalBeneméritaSociedadePortuguesadeBeneficênciaeHospitaldoCorac¸ão,SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received8July2016 Accepted7October2016 Availableonline1December2016
Keywords:
Gram-negativebacilli Carbapenemresistance OprDporin
a
b
s
t
r
a
c
t
Themechanismsinvolvedintheuncommonresistancephenotype,carbapenemresistance andbroad-spectrumcephalosporinsusceptibility,wereinvestigatedin25Pseudomonas aeru-ginosaclinicalisolatesthatexhibitedthisphenotype,whichwererecoveredfromthree differenthospitalslocatedinSãoPaulo,Brazil.Theantimicrobialsusceptibilityprofilewas determinedbyCLSIbrothmicrodilution.-lactamase-encodinggeneswereinvestigatedby PCRfollowedbyDNAsequencing.Carbapenemhydrolysisactivitywasinvestigatedby spec-trophotometerandMALDI-TOFassays.ThemRNAtranscriptionlevelofoprDwasassessed byqRT-PCRandtheoutermembraneproteinsprofilewasevaluatedbySDS-PAGE.Genetic relationshipamongP.aeruginosaisolateswasassessedbyPFGE.Carbapenemshydrolysis wasnotdetectedbycarbapenemaseassayinthecarbapenem-resistantand cephalosporin-susceptibleP.aueruginosaclinicalisolates.OprDdecreasedexpressionwasobservedinall P.aeruginosaisolatesbyqRT-PCR.TheoutermembraneproteinprofilebySDS-PAGE sug-gestedachangeintheexpressionofthe46kDaporinthatcouldcorrespondtoOprDporin. Theisolateswereclusteredinto17genotypeswithoutpredominanceofaspecificPFGE pattern.Theseresultsemphasizetheinvolvementofmultiplechromosomalmechanisms incarbapenem-resistanceamongclinicalisolatesofP.aeruginosa,alertforadaptationofP. aeruginosaclinicalisolatesunderantimicrobialselectivepressureandmakeawareofthe emergenceofanuncommonphenotypeamongP.aeruginosaclinicalisolates.
©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mailaddress:elocampana@gmail.com(E.H.Campana). 1 Theseauthorscontributedequallytothiswork. http://dx.doi.org/10.1016/j.bjid.2016.10.008
1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
58
braz j infectdis.2017;21(1):57–62Introduction
Pseudomonasaeruginosaisoneofthemostfrequentpathogens associated to nosocomial infections, especially among immunocompromisedpatients1 andexhibitsnotorious
ver-satility and capacity to acquire resistance mechanisms to antimicrobial therapy.2 Beta-lactam antimicrobial agents
are the main option to treat serious infection caused by this pathogen. However, the production of -lactamases, such ascephalosporinases and carbapenemases,has been intensely reported among clinical isolates of P. aeruginosa fromLatinAmericaandrepresentsthemosteffective mecha-nismof-lactamsresistancereportedamongGram-negative worldwide.3
Sincethe carbapenemsmolecules are moreresistant to hydrolysis activity by a great number of spread serine--lactamases, these drugs have a particular value in the treatment of infections caused by cephalosporinases pro-ducer strains,4 which remain susceptible to carbapenems.
In P. aeruginosa, the carbapenems resistance is modulated by acquired carbapenemases in association with intrin-sic mechanisms such as down-regulation or loss of OprD porin, efflux pumps hyperexpression, chromosomal AmpC -lactamaseproduction, and target alterations.2,3 However,
since carbapenemases have the ability to hydrolyze peni-cillins,cephalosporins,besidescarbapenems,Gram-negative bacteriacarryingacarbapenemase-encodinggenefrequently exhibitresistancetovirtuallyall-lactams.5
Giventhe importance ofcarbapenem forthe treatment ofinfectionscausedbyP.aeruginosa,itisessentialtoclarify the mechanismsinvolved inunusualand/orpoorly known phenotypes. Knowledge of these mechanisms alert for an adaptationtotheselectivepressureexertedbyantimicrobial and drug resistancedevelopment, thus affectingthe treat-mentofinfectionscausedbythesepathogensoftenrestricted toonlypolymyxins.
Theaimofthisstudywastoanalyzethepossible mecha-nismofantimicrobialresistanceinvolvedinclinicalisolates ofP. aeruginosathatexhibited anuncommon phenotypeof resistance: resistanceto carbapenems but susceptibility to broad-spectrumcephalosporins(Carb-R/Ceph-S).
Methods
Bacterialisolates,identificationandantimicrobial susceptibilitytesting
Between May, 2012 and March, 2013, a total of 25 P. aeru-ginosaclinicalisolatesexhibitingcarbapenemresistancebut broad-spectrum cephalosporin susceptibility (Carb-R/Ceph-S)were recovered from 18 different infectedpatients from threedistincthospitalslocatedinSãoPaulo,Brazil(Table1). The identification at the species level was confirmed by MALDI-TOF MS ina Bruker Daltonics Microflex LT MALDI-TOFusingtheBiotyperMALDI2.0(BrukerDaltonics,Bremen, Germany) as previously described.6 The susceptibility
pro-file was confirmed by broth microdilution to imipenem, meropenem, ceftazidime, cefepime, amikacin, gentamicin, and ciprofloxacin according the CLSI recommendations.7,8
AmericanTypeCultureCollection(ATCC)EscherichiacoliATCC 25922, P. aeruginosa ATCC 27853, and Staphylococcus aureus ATCC29213strainswereusedassusceptibilitytestingquality control.
ˇ-Lactamases-encodinggenesdetectionbyPCRandDNA sequencing
Acquired -lactamases encoding genes were investigated by PCR and DNA sequencing, as previously described, using primersfor:cephalosporinases(blaAmpC); (b) serino--lactamases (blaTEM, blaSHV, blaGES, blaCTX-M, blaBES, blaPER,
blaKPC, blaSME); (c) oxacillinases (blaOXA-1, blaOXA-2, blaOXA-3,
blaOXA-5, blaOXA-7, blaOXA-18, blaOXA-45, blaOXA-46, blaOXA-50,
blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, blaOXA-20, blaOXA-48,
blaOXA-62, blaOXA-143, blaOXA-198); (d) metallo--lactamases (blaIMP,blaVIM, blaSPM,blaGIM, blaSIM,blaNDM);and(e) class1 integron.9 Ampliconswere purifiedwith theQIAquick PCR
purification kit (Qiagen, Hilden, Germany) and sequenced in bothstrandsusing theAppliedBiosystems 3500genetic analyzerequipment(AppliedBiosystems,PerkinElmer,USA). Thenucleotidesequenceandtherespectivededucedamino acidsequenceswereanalyzedusingtheLasergenesoftware package (DNAStar, Madison,WI, USA) and compared with thesequencesavailableontheInternetusingtheBLASTtool (http://www.ncbi.nlm.nih.gov/blast/).
Carbapenemasehydrolysisassay
Carbapenemase activity was investigated in bacterial cell crude extracts by UV spectrophotometric assays against 100Mimipenemand100Mmeropenemin100mM phos-phatebuffer (pH7.0),aspreviouslydescribed.10 In parallel,
carbapenemhydrolysisinhibitionwasperformedby incubat-ing thewhole-protein extractwith25mMEDTAfor15min, previously to the assay with imipenem and meropenem. Imipenem hydrolysis was also investigated by MALDI-TOF MS according to the method described by Carvalhaes and colleagues.11
Relativegenetranscriptionallevel
Relative transcriptional levels of mexB, mexD, mexF, mexY, ampC, and oprD were determined and analyzed with Real Time 7500 (Applied Biosystems, Warrington, United King-dom) as previously described.12 Quantitative RT-PCR was
performed with Platinum SYBR Green Supermix (Invitro-gen,Carlsbad,USA).Accordingtopreviousstudies,reduced oprD and increasedampC transcription levels were consid-eredsignificantwhenitwas≤70%and≥10-fold,respectively, compared to their transcriptional levels in PAO1 strain.13
MexAB-OprM, MexCD-OprJ,MexEF-OprN, andMexXYefflux systems were considered overexpressed when mexB, mexC, mexE,andmexYtranscriptionallevelwereatleast2-,100-,100-, and4-foldhigherthanthoseinPAO1strain,respectively.14
AssessmentoftheOMPprofileandoprDanalysis
AmplificationofoprDgenewascarriedoutbyconventional PCRaspreviouslydescribed15andtheampliconsizeanalyzed
b r a z j i n f e c t d i s . 2 0 1 7; 2 1(1) :57–62
59
IMI MER CAZ CEP AMI GEN CIP oprD
P01 J.A.N. May7,12 Trachealaspirate H1 A 8 2 2 2 2 1 0.125 0.50
P02 J.A.O. Jun13,12 Trachealaspirate H1 B 8 8 4 8 4 2 0.5 0.43
P03 J.D.T. Set9,12 Urine H1 C 8 2 2 8 8 4 32 0.41
P04 A.E. Jun4,12 Trachealaspirate H2 D 16 4 8 8 8 4 0.25 0.10
P05 A.C.C. Jan28,13 Cathetertip H3 E 8 4 8 8 4 2 0.25 0.01
P06 A.C.C. Jan28,13 Cathetertip H3 F 8 8 8 8 8 4 0.5 0.18
P07 M.R.V. Jan28,13 Urine H3 G 8 4 8 8 4 2 0.5 0.02
P08 C.S.S. Jan31,13 Bloodstream H3 H 16 4 4 8 8 4 0.5 0.25
P09 A.C.C. Feb4,13 Cathetertip H3 E 16 8 2 4 4 2 0.25 0.07
P10 I.R. Feb7,13 Urine H3 L 8 2 2 4 4 2 0.125 0.37
P11 V.A.B. Feb8,13 Trachealaspirate H3 I 8 8 1 2 2 2 0.125 0.19
P12 V.A.B. Feb8,13 Trachealaspirate H3 I1 8 8 8 16 4 2 0.125 0.07
P13 A.C.L. Feb9,13 Respiratorytract H3 J 8 4 4 2 1 0.5 0.5 0.19
P14 V.A.B. Feb17,13 Trachealaspirate H3 I2 8 4 2 2 2 2 0.125 0.08
P15 D.G.O. Feb19,13 Trachealaspirate H3 M 8 1 1 8 8 4 16 0.01
P16 B.S. Feb19,13 Trachealaspirate H3 F1 16 8 4 8 8 4 0.5 0.14
P17 B.S. Feb21,13 Respiratorytract H3 F1 8 8 4 16 8 4 1 0.13
P18 SNa Feb24,13 Urine H3 N 8 4 1 2 >128 >64 16 0.26
P19 A.R.P.G Feb24,13 Trachealaspirate H3 O 16 4 4 8 2 16 16 0.50
P20 C.S.F. Feb25,13 Trachealaspirate H3 P 8 2 2 8 8 4 0.5 0.13
P21 A.C.L. Feb1,13 Trachealaspirate H3 K 16 8 2 4 1 1 0.25 0.25
P22 D.S.L. Mar1,13 Cathetertip H3 K 16 16 8 8 4 2 0.5 0.10
P23 A.J. Mar2,13 Urine H3 F1 8 4 4 4 16 0.5 8 0.01
P24 M.J.S. Mar11,13 Trachealaspirate H3 F1 16 16 4 8 4 1 0.5 0.14
P25 SND.R.C.a Mar13,13 Bloodstream H3 Q 16 16 2 8 4 2 0.25 0.47
a Newborn;unnamed(SN).
b BrothmicrodilutionaccordingtoCLSI(2012,2015).IMI,imipenem;MER,meropenem;CAZ,ceftazidime;CEP,cefepime;AMI,amikacin;GEN,gentamicin;CIP,ciprofloxacin.
60
braz j infectdis.2017;21(1):57–62 250 Kilo daltons MW P01 P02 P03 P04 MW P05 P06 P09 P07 P08 P11 P12 MW P14 P13 P21 P10 P15 P16 P17MW P18 P19 P20 P22 P23 P24 P25MW OprD (46kDa) OprF (32kDa) a * * * * 75 50 37 25 20 15Fig.1–OutermembranepatternofCarb-R/Ceph-SP.aeruginosaclinicalisolates.Outermembraneprofileswereanalyzedfor thepresenceoftheporin,OprD.PAO1*andATCC27853awereincludedascontrols.TheOprDandOprFproteinbandsare
indicatedwitharrow.MW,molecularweightproteinmaker(kDa).
by 1.5% agarose gel electrophoresis. The outer membrane proteins(OMP)wereanalyzedbySDS-PAGEina15% acryla-mideand0.3%N,N-methylenebisacrylamiderunninggel.The proteinswerestained withcoomassiebluer250and photo-graphedusingGelDocSystemImaging.P.aeruginosaPA01and ATCC27853wereusedasreferencestrains.16
Moleculartypingbypulsed-fieldgelelectrophoresis
GenomicDNAofisolateswasdirectlypreparedfroma bac-terialcellsuspensioninto agaroseblocksanddigestedwith SpeIrestrictionenzyme(NewEngland,Beverly,MA,USA). Elec-trophoresiswasperformedonCHEF-DRIII(BioRad,Richmond, CA).ThebandpatternswereanalyzedbyBioNumerics2.0 soft-ware(AppliedMaths,Belgium)17,18 andaccordingtoTenover
interpretivecriteria.19
Results
Carbapenem-resistancenotrelatedtocarbapenemase
The decreased susceptibility to at least one tested car-bapenemwasconfirmedaswellasthesusceptibilitytotested cephalosporins as shown inTable 1. All evaluated isolates exhibitedresistancetoimipenemwithMICrangevaryingfrom 8to16g/mL.Althoughfiveisolates(20%)showedmeropenem susceptibility(MIC≤2),mostisolateshadreduced suscepti-bilitytothisantimicrobial(MICs≥4g/mL).AllP. aeruginosa isolatesweresusceptibletoceftazidime(MIC≤8).Onlytwo isolatesexhibitedacefepimeMICequalto16g/mL,andwere classifiedasintermediateresistanttocefepimeaccordingCLSI breakpoints.8
All25P.aeruginosaisolatescarriedtheconstitutiveblaOXA-50 geneshowingseveralDNAsilentmutationsbutallofthem presented the predicted STYK motif commonly found in classD-lactamases(DBLs),whichhavenarrow-lactamase hydrolysis activity.20 A single isolate (P18) also had
posi-tivePCRforblaOXA-56gene.DNAsequencinganalysisofthe
blaOXA-56geneticcontextshowedthatitwascarriedbyaclass 1integron(namedIn163)aspreviouslydescribedbyCarvalho etal.amongSPM-1producingP.aeruginosaclinicalisolates.21
Nonetestedcarbapenemaseencodinggenesweredetectedby PCR.
To determine whether the carbapenem-resistance was mediatedbyanunknowncarbapenemase,invitrohydrolysis
of imipenem and meropenem by whole-protein crude cell extractwasinvestigatedbyspectrophotometricand MALDI-TOF MS analysis. None ofthe tested clinical sample were abletohydrolyzeimipenemandmeropenemmoleculesina spectrophotometricassay(datanotshown).Furthermore,no carbapenemhydrolysiswasdetectedbyMALDI-TOFMS.
Mechanismassociatedwithcarbapenem-resistance
TheexpressionofOprDwasestimatedbymRNA transcriptio-nallevelbyqRT-PCRandoutermembraneproteinpatternby SDS-PAGE.Ampliconswithsizehigherthanexpectedforwild typeP.aeruginosawereobservedforP05,P09,P13,P15,andP21 isolates.TheamplificationofoprDgenefailedintwoisolates (P07andP22).Relativetranscriptionallevelwasperformedto evaluateoprDdown-regulation.Relativetranscriptionallevel analysisusingP.aeruginosaPA01asreferencestain(baseline) showed that the transcriptional level of oprD gene among those isolateswassignificantlyreducedwhencomparedto wild-typePA01strain.
TheevaluationoftheOMPbySDS-PAGEstronglysuggested thatOprDwasvisuallyabsentorproducedatverylowlevels byallisolates(Fig.1).
Overall,themRNAtranscriptionlevelsofmexB,mexC,mexE, mexYandalsoofthe-lactamaseAmpCofmost Carb-R/Ceph-SP.aeruginosaisolateswereevenlowerthanthoseobserved forPAO1strain(datanotshown).
Geneticrelationship
Highclonaldiversitywasobservedbetweenthe25tested clin-icalisolatesofP.aeruginosa,with17differentPFGEpatterns withoutpredominanceofasinglepattern(Fig.2).
Discussion
Carbapenemshavebeenextremelyprescribedfortreatment ofP. aeruginosa infections.2 Carbapenemase productionhas
emergedasthemainmechanismofcarbapenemresistance amongclinicalisolatesofP.aeruginosa.However,lossofOprD has also contributed for carbapenemresistance, especially conferringresistancetoimipenem.Acquisitionofmutations, insertions, and/or deletions inthe oprD geneare the most commonmechanismsofOprDinactivationaswellas down-regulationofoprDtranscription.22
50 60 70 80 90 100 54.6 53.2 56.7 51.8 63.1 60.6 45.2 66.7 60.9 78.5 93.8 70.3 97.1 92.8 84.6 66.8 62.9 58.3 75.9 63.0 71.4 P02 P03 P04 P18 P12 P14 P11 P20 P25 P21 P22 P08 P07 P15 P16 P17 P06 P23 P24 P13 P01 P05 P09 P10 P19
Fig.2–Dendrogramshowingthegenotypicprofileof Carb-R/Ceph-SP.aeruginosaclinicalisolatesdeterminedby thePFGEtechnique.
This study evaluated clinical isolates of P. aeruginosa exhibitingreducedsusceptibilitytocarbapenemsnotrelated tocarbapenemaseproduction,whichhasbeenmostlyrelated tocarbapenemaseinBrazilianclinicalsettings.12,23The
emer-gence ofP. aeruginosa resistant to carbapenems associated withlossofporinhasbeendocumentedinclinicalsettings andalsocouldbeselectedinvitroafterlaboratory carbapen-emsexposure.InastudycarriedoutbyFowlerandcolleagues, whichevaluatedthemechanismofcarbapenemsresistance inselectedmutantsafterexposureofa cysticfibrosis clin-icalisolate to sub-inhibitoryconcentration of meropenem, demonstratedthatlossofOprDbyanISelement(ISPa8) dis-ruptionincreasedtheMICtoimipenemandmeropenemfrom 0.5and2g/mL,respectively,toupto16g/mL.24Inanother
workconductedbyOcampo-Sosaandcolleagues,OprD mod-ifications were also found among carbapenem susceptible isolates,withimipenemMICsvaryingfrom0.06to4g/mLin aclinicalsettingofmetallo--lactamase-negativeP.aeruginosa isolatedfromSpanishhospitals.16
In the present study we analyzed P. aeruginosa isolates resistant toatleast onecarbapenem withimipenem MICs rangingfrom8to16g/mLandmeropenemMICvaryingfrom
1to16g/mLthatremainedsusceptibletobroad-spectrum cephalosporins.Allisolatesanalyzedhadasignificantreduced expressionorlackofOprDporininP.aeruginosaouter mem-branebyexpressionofanulloralteredoprDgene.
We found a high diversity of genotypes among the OprD-deficientP.aeruginosa,whichwereisolatedfromthree different hospitalslocated in SãoPaulo city, Brazil.It may reveal an emergence of OprD-deficient P. aeruginosa in the Brazilian clinical scenario by a manner independently of a clone spread outwards, in accordance with previ-ous observation.25 TheabilityofOprD-mutant selectionby
carbapenemsexposuremayplayanimportantroleto OprD-mutant emergence and it should be considered regarding empiricalclinicalusageofcarbapenems.
Loss ofOprD has also been implicated in anincreased invivofitnessand/orvirulenceinamurineinfectionmodel. OprD-deficient P. aeruginosa strains were more resistant to lowpHenvironment,tonormalhumanserum,andpresented anincreasedcytotoxicityagainstmurinemacrophages.Ithas suggestedthattheassociationofantimicrobialresistanceand increasedsurvivalofthesestrainscanfavorthepermanence of this strains inthe hospitalsetting and maketreatment moredifficult.26TheincreaseinOprD-deficientsurvivalcan
alsoraisethepersistenceofthesestrainsinthehospital envi-ronment and favor the acquisition of new determinantof antimicrobial resistancebyhorizontal genetransfer.27 Itis
worthmentioningthatallisolatesevaluatedinthisstudywere susceptible to broad-spectrum cephalosporins (ceftazidime and cefepime). Aspreviouslydemonstrated, OprDmutants are resistant only to zwitterionic carbapenems, especially imipenemandthe cross-resistancetoothers-lactamshas notbeenobservedinthesestrains.Carb-R/Ceph-Sphenotype mightbeusedasaphenotypicbiomarkerforscreening OprD-deficientP.aeruginosaclinicalisolates.SincetheseP.aeruginosa donotproducehighlevelsofAmpC,theoreticallyceftazidime orcefepimecouldbeprescribedfortreatmentofsuch infec-tions.However,morestudieswould benecessarytoensure theefficacyofbroad-spectrumcephalosporinsfortreatment ofCarb-R/Ceph-SP.aeruginosainfections.
Conclusion
The carbapenem resistance of P. aeruginosa associated to OprD-deficientmaybecomemorefrequentinthehospitals scenarioasconsequenceofselectivepressureexertedby clin-icaluseofcarbapenems.Consideringthetherapeuticvalueof carbapenemsasoneofthelastoptionsforthetreatmentofP. aeruginosainfectiousandtheemergenceofP.aeruginosaasa publichealthproblem,rationalcarbapenemsusageis essen-tialtoreducetheselectivepressureoverP.aeruginosaclinical isolates.
Funding
ThisstudywassupportedbyFundac¸ãodeAmparoàPesquisa doEstadodeSãoPaulo(FAPESPgrantnumber:2010/12891-9) andConselhoNacionaldeDesenvolvimentoCientíficoe Tec-nológico.
62
braz j infectdis.2017;21(1):57–62Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
WewouldliketothanktheConselhoNacionalde Desenvolvi-mentoCientíficoeTecnológico(CNPq),MinistryofScienceand Technology(Brazil),forprovidingaresearchgranttoA.C.G. (305535/2014-5).WealsowouldliketothanktheCoordenac¸ão deAperfeic¸oamentodePessoaldeNívelSuperior(CAPES)and toCNPqforgrantingtheDoctoralscholarfellowshiptoE.H.C. andtoD.E.X.(CNPq141572/2008-6).
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