βLactam antibiotics and vancomycin inhibit the growth of planktonic and biofilm Candida spp. : an additional benefit of antibioticlock therapy?

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InternationalJournalofAntimicrobialAgents45(2015)420–423

ContentslistsavailableatScienceDirect

International

Journal

of

Antimicrobial

Agents

j ou rn a l h om ep a ge :ht t p : / / w w w . e l s e v i e r . c o m / l o c a t e / i j a n t i m i c a g

Short

communication

␤

-Lactam

antibiotics

and

vancomycin

inhibit

the

growth

of

planktonic

and

biofilm

Candida

spp.:

An

additional

benefit

of

antibiotic-lock

therapy?

José

J.C.

Sidrim

a

_,

_Carlos

_E.C.

_Teixeira

a

_,

_Rossana

_A.

_Cordeiro

a

_,

_Raimunda

_S.N.

_Brilhante

a,∗

,

Débora

S.C.M.

Castelo-Branco

a

_,

_Silviane

_P.

_Bandeira

a

_,

_Lucas

_P.

_Alencar

a

_,

Jonathas

S. Oliveira

a

_,

_André

_J.

_Monteiro

b

_,

_José

_L.B.

_Moreira

c

_,

Tereza

J.P.G.

Bandeira

a

_,

_Marcos

_F.G.

_Rocha

a,d

a_Department_of_Pathology_and_Legal_Medicine,_College_of_Medicine,_Post_Graduate_Program_in_Medical_{Microbiology,}_Specialized_Medical_Mycology_Center, FederalUniversityofCeará,Fortaleza,CE,Brazil

b_Department_of_Statistics_and_Applied_Mathematics,_Federal_University_of_Ceará,_Fortaleza,_CE,_Brazil

c_Department_of_Pathology_and_Legal_Medicine,_College_of_Medicine,_Federal_University_of_Ceará,_Fortaleza,_CE,_Brazil d_College_of_Veterinary_Medicine,_Post_Graduate_Program_in_Veterinary_Sciences,_State_University_of_Ceará,_Fortaleza,_CE,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26May2014 Accepted1December2014

Keywords: Candidaspp. Biofilms Catheterinfection Antibiotic-locktherapy

a

b

s

t

r

a

c

t

Theaimofthisstudywastoevaluatetheeffectsofcefepime,meropenem,piperacillin/tazobactam(TZP) andvancomycinonstrainsofCandidaalbicansandCandidatropicalisinplanktonicandbiofilmforms. Twentyazole-derivative-resistantstrainsofC.albicans(n=10)andC.tropicalis(n=10)weretested.The susceptibilityofplanktonicCandidaspp.totheantibacterialagentswasinvestigatedbybroth microdi-lution.TheXTTreductionassaywasperformedtoevaluatetheviabilityofgrowingandmaturebiofilms followingexposuretothesedrugs.Minimuminhibitoryconcentrations(MICs)rangedfrom0.5mg/mL to2mg/mLforcefepime,TZPandvancomycinandfrom0.5mg/mLto1mg/mLformeropenemand thedrugsalsocausedstatisticallysignificantreductionsinbiofilmcellularactivitybothingrowingand maturebiofilm.Sinceallofthetesteddrugsarecommonlyusedinpatientswithhospital-acquired infec-tionsandinthosewithcatheter-relatedinfectionsunderantibiotic-locktherapy,itmaybepossibleto obtainanadditionalbenefitfromantibiotic-locktherapywiththesedrugs,namelythecontrolofCandida biofilmformation.

1. Introduction

Candidaspp.arethefourthandthirdleadingcausesof hospital-acquiredbloodstream and urinary tractinfections, respectively,

and most of these infections are associated with implanted

medical devices such as central venous and bladder catheters

owingtobiofilmformationwithinthesematerials[1].Arelevant characteristicofCandidabiofilmsisresistancetoantifungalagents, whichcanbeintrinsicoracquiredbytransferofgeneticmaterial betweenbiofilm cells [2]. Biofilm-associated Candida infections are usually difficult to diagnose, causing delayed therapy and highlethalityrates in hospitalised patients worldwide [3], and

∗Correspondingauthor.Tel.:+558533668319;fax:+558532951736.

E-mailaddress:brilhante@ufc.br(R.S.N.Brilhante).

theabilityof Candidaspp.toformdrug-resistantbiofilmsisan importantcontributingfactortohumandiseases[1].

In systemic infections, biofilms can also be polymicrobial,

formed by Candida spp. and bacteria [1]. Patients with

con-firmedorstronglysuspectedhospital-acquiredinfectionsprimarily

receive antibacterial therapy, including cefepime, meropenem,

piperacillin/tazobactam(TZP)andvancomycin[4],which,onthe otherhand,predisposesthemtotheoccurrenceofCandida infec-tionsbecauseitdecreasesmicrobialcompetitionwithinthehost’s

microbiome [1,3]. It has already been shown that

antibacte-rialdrugs can affectCandida biofilmformation. Tigecycline, for instance,ishighlyactiveagainstgrowingandmaturebiofilmsof Candidaalbicans[5],whilstrifampicincaninducebiofilm forma-tionbythisCandidaspecies[6].Thus,thisstudyaimedtoevaluate theeffectsof␤-lactams(cefepime,meropenemandTZP)and van-comycinonstrainsofC.albicansandCandidatropicalisinplanktonic andbiofilmforms.

http://dx.doi.org/10.1016/j.ijantimicag.2014.12.012

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J.J.C.Sidrimetal./InternationalJournalofAntimicrobialAgents45(2015)420–423 421

2. Materialsandmethods

2.1. Fungalstrains

Azole-derivative-resistantstrainsofCandidaspp.fromhuman casesof candidaemia (four C.albicans and sixC. tropicalis)and healthyanimals(sixC.albicansandfourC.tropicalis)wereincluded inthisstudy.Thestrainscamefromtheculturecollectionofthe SpecializedMedicalMycologyCenterofFederalUniversityofCeará (Fortaleza,Brazil)andwereselectedbasedontheirantifungal resis-tance[7].Theidentityofthestrainswasconfirmedaspreviously described[7].

2.2. AntimicrobialagentsandantifungalsusceptibilityofCandida planktoniccells

A stock solution of amphotericin B (AmB) (Sigma-Aldrich,

St Louis, MO) at 1mg/mL was prepared according to

Clini-caland LaboratoryStandards Institute(CLSI)guidelines[8] and wasusedas acontrol. Theantibacterial drugscefepime (Nova-farma,Anápolis,GO,Brazil),meropenem(AstraZeneca,Cotia,SP,

Brazil), TZP (Novafarma) and vancomycin (AstraZeneca) were

diluted with sterile distilled water as recommended by the

manufacturers. A stock solution of vancomycin was prepared

with distilled water at 50mg/mL as previously described [5]

and, based on this research [5], the other antibacterial drugs

were diluted to the same concentration. Serial two-fold

dilu-tionsof each drugwere prepared in RPMI 1640 medium with

l-glutamine and without sodium bicarbonate (Sigma-Aldrich,

St Louis, MO), buffered to pH 7.0 with 0.165M MOPS

(3-[N-morpholino]propane sulfonic acid) (Sigma-Aldrich, St Louis, MO).

Thesusceptibilityof Candidaspp.totheantibacterial agents wasinvestigatedbybrothmicrodilutionaccordingtoCLSI guide-lines[8].Thefinal inoculum wasdiluted withRPMI toreacha concentrationof0.5–2.5×103cells/mL.AmBwasalsotestedasa

controldrug.Todeterminethesusceptibilityofplanktoniccells,the

testedconcentrationrangeswere0.0039–4mg/mLforcefepime,

meropenem,TZPandvancomycinand0.03125–16␮g/mLforAmB.

Allisolatesweretestedinduplicate.Fortheantibacterialdrugs,

theminimuminhibitoryconcentration(MIC)wasdefinedasthe

lowest drug concentration capable of inhibiting 50% of fungal

growth[5]comparedwiththecontrolwell,whilstforAmBtheMIC wasdefinedasthelowestdrugconcentrationcapableof inhibi-ting100%ofplanktonicfungalgrowth[8].EscherichiacoliATCC 25922,StaphylococcusaureusATCC25923andCandida parapsilo-sisATCC22019wereincludedasqualitycontrolstrainsforeach test[8].

2.3. Biofilmformation

For biofilm testing, inocula were prepared as previously

described [9] with some modifications. Strains of C. albicans

(n=10)andC.tropicalis(n=10)weregrowninSabourauddextrose

broth (Himedia, Mumbai, India) at 30◦_C _for ₂₄_h _in _a _rotary

shaker at 150rpm. After this period, cells were collected by

centrifugation (3000rpm, 10min) and the pellet was washed

twice with phosphate-buffered saline (PBS). Suspensions were

adjusted to 1_×106_cells/mL _in _RPMI _medium _and _then ₁₀₀_␮_L

aliquots of inoculum were transferred to flat wells of 96-well polystyrene plates (TPP, Trasadingen, Switzerland). The plates wereincubatedat37◦_C_for₄₈_h_and_the_wells_were_washed_three

timeswith0.05%Tween20(Sigma-Aldrich,SãoPaulo,SP,Brazil) in Tris-buffered solution (Sigma-Aldrich, São Paulo, SP, Brazil)

to remove non-adherent cells. Biofilm viability was monitored

through the use of

2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)

(Sigma-Aldrich,StLouis,MO)asdescribedpreviously[9].

2.4. EffectofantibacterialdrugsongrowingandmatureCandida biofilms

TheabilityofthetesteddrugstoinhibitformationofC.albicans andC.tropicalisbiofilmswasevaluatedasdescribedpreviously[9]. Duringtheplateinoculationstep,100␮Laliquotsofthe antibac-terialsolutionswereaddedtoeachwell.Eachdrugwastestedat fivedifferentconcentrations(MIC/50,MIC/10,MIC,10×MICand

50× MIC). Biofilm formationwasthen performedaspreviously

described.Followingincubationat37◦_C_for₄₈_h,_the_effect_of_the

antimicrobialdrugsongrowingbiofilmswasevaluatedbasedon biofilmcellactivityusingtheXTTreductionassaywith75␮Lof XTTsalt solution(1mg/mLin PBS),6␮L ofmenadione solution (1mMinacetone)(Sigma-Aldrich,StLouis,MO)and50␮Lof ster-ilePBS,whichwereaddedtoeach well,followedbyincubation at36◦_C_for₅_h._The_metabolic_activity_of_biofilm_cells _was

mea-suredwithamicroplatereader(Epoch;Bio-Tek,Winooski,VT)at

492nm.

The inhibitory activity of the tested drugs against mature

biofilmsofC.albicansandC.tropicaliswasevaluatedaspreviously described[9].Theantibacterialdrugsweretestedatfivedifferent concentrations(MIC/50,MIC/10,MIC,10×MICand50×MIC).For

thispurpose,aliquotsof200␮Lofeachdrugwereaddedtoviable 48-h-oldbiofilmsgrowninflatwellsof96-wellpolystyreneplates, followedbyincubationat35◦_C_for₄₈_h._After_this_period,_inhibition

ofbiofilmmetabolicactivitywasmonitoredbyXTTreduction[9]

asdescribedabove.

Allbiofilmexperimentswereperformedinduplicateandwere

repeatedatthreeindependentmoments.Controlsweregrownin

mediumwithoutantimicrobials,andAmBwasusedasthecontrol drugforbiofilminhibition.

2.5. Statisticalanalysis

Inordertoverifydifferencesinabsorbancevaluestoevaluate theeffectsofantibacterialdrugsonbiofilmcellactivity,Student’s t-testforpairedsampleswasused.Foralloftheanalyses,a signifi-cancelevellowerthan5%indicatedstatisticallysignificantfindings (P<0.05).

3. Results

The antibacterialMICs against the20 tested Candida strains rangedfrom0.5mg/mLto2mg/mLforcefepimeandTZPandfrom

0.5mg/mLto1mg/mLformeropenem.TheMICsforvancomycin

rangedfrom0.5mg/mLto1mg/mLagainstC.albicansand from 0.5mg/mL to 2mg/mL against C. tropicalis. For AmB, the MICs rangedfrom0.5␮g/mLto2␮g/mLagainstC.albicansand from 0.5␮g/mLto4␮g/mLforC.tropicalis.

RegardingtheeffectsofantibacterialdrugsongrowingCandida

spp. biofilms, cefepime, TZP and vancomycin caused

statisti-callysignificantreductionsinbiofilmcellularactivityatMIC/10 (P<0.001), MIC(P<0.0001),10× MIC(P<0.0001) and 50×MIC

(P<0.0001),whilstmeropenemonlycausedsignificantreductions attheMICandhigherconcentrations(P<0.05).AmBsignificantly inhibitedgrowingbiofilmcellularactivityatalltested concentra-tions(P<0.01)(Fig.1).

Regarding mature Candida biofilms, cefepime, meropenem,

TZPandvancomycincausedstatisticallysignificantreductionsin biofilmcellular activityatMIC/10(P<0.05), MIC(P<0.01), 10×

MIC (P<0.0001) and 50× MIC (P<0.0001), but not at MIC/50

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422 J.J.C.Sidrimetal./InternationalJournalofAntimicrobialAgents45(2015)420–423

Fig.1.Effectofdifferentconcentrationsofcefepime,meropenem,piperacillin/tazobactam,vancomycinandamphotericinB(AMB)onthemetabolicactivityofgrowingand maturebiofilmsofCandidaspp.analysedbytheXTTreductionassay.Theminimuminhibitoryconcentration(MIC)isthatobtainedagainstplanktonicgrowthofCandidaspp. Lettersindicatestatisticallysignificantdifferencescomparedwiththecontrolandwithgrowthatdifferentantibacterialconcentrations(P<0.05).Thetestedantimicrobial concentrationsarepresentedinthefollowingordertop-down:50×MIC,10×MIC,MIC,MIC/10,MIC/50andcontrol.

inhibitedmaturebiofilmcellularactivityatalltested concentra-tions(P<0.05)(Fig.1).

4. Discussion

ThecapacityofCandidatoformbiofilmsonabioticandbiotic surfacesisanimportantvirulencefactorfortheestablishmentof recurringcandidiasis[10].Itiswellknownthattheuseofsystemic antibioticspredisposestheproliferationofCandidaspp.,hencethe occurrenceofcandidiasis[1,3].However,littleisknownaboutthe effectsofantibioticsongrowingandmaturebiofilmsofCandida spp.withinmedical devices,whicharecontinuouslyexposedto antibacterialdrugswhenpatientsareundertreatment.Thislack ofknowledgewasamotivationtoinvestigatetheinvitroeffects

ofcefepime,meropenem,TZPandvancomycin, whichare

com-monlyused againsthospital-acquiredinfections [4],onCandida biofilmsand,consideringthatthesedrugspredisposethe occur-renceofCandidainfections,itwasinitiallyhypothesisedthatthey

could induce biofilm formation or the maintenance of mature

biofilms.

Initially, the antimicrobials were tested against

azole-derivative-resistant C. albicans and C. tropicalis strains and all of them effectively inhibited the growthof planktonic Candida spp.,withMICsrangingfrom0.5mg/mLto2mg/mL.Althoughall testedantibacterialdrugswereabletoinhibittheinvitrogrowthof planktonicCandida,theMICssurpassedthedesirabletherapeutic bloodconcentrationsforalloftheseantibacterialdrugs[11–14]. Hence,theycannotbeusedtoeffectivelytreatCandidainfections.

The mechanisms through which these antibacterial drugs

inhibited fungal growth remain unknown. Only a few studies

havebeenperformedtacklingtheeffectsofantibacterialdrugson Candidabiofilmsandnoneofthemhaveaddressedpossible mech-anismsof action[5,6].However,it canbesuggested thatthese drugsactthroughunspecificmechanisms,withoutspecifictarget molecules,sinceextremelyhighconcentrationsofthefourtested drugswererequiredtocausethisgrowthinhibition.Thus,

consid-eringthis newpotentialuseofcefepime, meropenem,TZP and

vancomycin,itisnecessarytodesignspecificexperimental proto-colstoelucidatethemechanismsbehindtheobservedantifungal effect.

On the other hand, the results demonstrate that antibacte-rialconcentrationsaslowasMIC/10werecapableofsignificantly reducingthebiofilmcellularmetabolicactivitybothofgrowingand matureCandidabiofilms.Themostinterestingfindingofthisstudy wasthatantibacterialconcentrationslowerthantheMICsagainst

planktoniccellswereabletosignificantlydecreasetheinvitro via-bilityofgrowingandmatureCandidabiofilms.

Clinically,thesefindingsmaybeground-breakingsinceallof thetestedantibacterialdrugs(cefepime,meropenem,TZPand

van-comycin)arecommonlyusedinpatientswithhospital-acquired

infections[4]andarealsousedinantibiotic-locktherapy[15].This techniqueinvolvestheprolongedinstillationofasolution contain-ingextremelyhighconcentrationsofantimicrobialorantiseptic agents,100–1000-foldhigherthanthoseusedsystemically,within aninfectedintravascularcatheterasinanattempttosterilisethe interiorofthecatheterandcontrolbloodstreaminfections[5,15]. Thiskindoftreatmentappearstobeaviablealternativeinthose special cases in which the salvage of the catheter is desirable, althoughremovingthedeviceisthetreatmentofchoicefor per-sistentorcomplicatedbacteraemiaorfungaemiarelatedtoitsuse

[15].

Itis importanttoemphasisethattheuseof systemic broad-spectrum antibiotics is an important risk factor for developing

candidaemia and that biofilms play a major role in

main-taining bloodstream Candida infections [10]. Therefore,

consid-ering the low minimum antibacterial concentrations (MIC/10;

50–200␮g/mL) required to significantly decrease the viability of Candida biofilm cells, we believe that the use of cefepime,

meropenem,TZPandvancomycinintheantimicrobial-lock

solu-tionmightnotonlyaidthepatientinthemanagementofbacterial infectionassociatedwithindwellingcatheters,butalsocontrolthe formationofCandidabiofilmswithinthesemedicaldevicesduring antibacterialtherapy.However,morestudieswillbenecessaryto establishtheeffectsofthistherapyonCandidabiofilmsinsidethe patientduringtreatment.

5. Conclusion

Theantibacterial drugscefepime, meropenem,TZP and

van-comycinareabletodecreasetheinvitroviabilityofgrowingand

matureCandida biofilms. These resultsmay bring the

perspec-tiveofobtaininganadditionalbenefitwithantibiotic-locktherapy, namelythecontroloffungalbiofilmswhenusingthesedrugsin hospitalisedpatients.

Funding

ThisstudywassupportedbyCNPq, Brazil[PROTAX562296/

2010-7;504189/2012-3;307606/2013-9]andCAPES,Brazil[PNPD

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J.J.C.Sidrimetal./InternationalJournalofAntimicrobialAgents45(2015)420–423 423

Competinginterests

Nonedeclared.

Ethicalapproval

Notrequired.

References

[1]SardiJCO,ScorzoniL,BernardiT,Fusco-AlmeidaAM,MendesGianniniMJS. Candida species:current epidemiology, pathogenicity, biofilm formation, naturalantifungalproductsandnewtherapeuticoptions.JMedMicrobiol 2013;62:10–24.

[2]FanningS,MitchellAP.Fungalbiofilms.PLoSPathog2012;8:e1002585.

[3]WilleMP,GuimarãesT,FurtadoGHC,ColomboAL.Historicaltrendsinthe epidemiologyofcandidaemia:analysisofan11-yearperiodinatertiarycare hospitalinBrazil.MemInstOswaldoCruz2013;108:288–92.

[4]TorresA,FerrerM,BadiaJR.Treatmentguidelinesandoutcomesof hospital-acquiredandventilator-associatedpneumonia.ClinInfectDis2010;51(Suppl 1):S48–53.

[5]KuTSN,PalanisamySK,LeeSA.SusceptibilityofCandidaalbicansbiofilmsto azithromycin,tigecyclineandvancomycinandtheinteractionbetween tige-cyclineandantifungals.IntJAntimicrobAgents2010;36:441–6.

[6]VogelM,KöberlelM,ScäfflerH,TreiberM,AutenriethIB,SchumacherUK. RifampicininducedvirulencedeterminantsincreaseCandidaalbicansbiofilm formation.F1000Research,vol.2;2013.p.106,http://dx.doi.org/10.12688/ f1000research.2-106.v1.

[7]CordeiroRA,TeixeiraCE,BrilhanteRSN,Castelo-BrancoDS,PaivaMA,Giffoni LeiteJJ,etal.MinimuminhibitoryconcentrationsofamphotericinB,azoles andcaspofunginagainstCandidaspeciesarereducedbyfarnesol.MedMycol 2013;51:53–9.

[8]Clinical and LaboratoryStandards Institute. Reference method for broth dilutionantifungalsusceptibilitytestingofyeasts;approvedstandard.In: Doc-umentM27-A3.3rded.Wayne,PA:CLSI;2008.

[9]ChatzimoschouA,KatragkouS,SimitsopoulouM,AntachopoulosC,Georgiadou E,WalshTJ,etal.Activitiesoftriazole–echinocandincombinationsagainst Can-didaspeciesinbiofilmsandasplanktoniccells.AntimicrobAgentsChemother 2011;55:1968–74.

[10]Cuéllar-CruzM,López-RomeroE,Villagómez-Castro JC,Ruiz-BacaE. Can-didaspecies:newinsightsintobiofilmformation.FutureMicrobiol2012;7: 755–71.

[11]LamothF, BuclinT,PascualA,Vora S,Bolay S,DecosterdLA,etal. High cefepimeplasmaconcentrations andneurological toxicityinfebrile neu-tropenicpatientswithmildimpairmentofrenalfunction.AntimicrobAgents Chemother2010;54:4360–7.

[12]AmericanSocietyofHealth-SystemPharmacists.Therapeuticmonitoringof vancomycininadultpatients:aconsensusreviewoftheAmericanSociety ofHealth-SystemPharmacists,theInfectiousDiseasesSocietyofAmerica, andtheSocietyofInfectiousDiseasesPharmacists.AmJHealthSystPharm 2009;66:82–98.

[13]NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem. ClinInfectDis2008;47:32–40.

[14]Burgess DS, Waldrep T. Pharmacokinetics and pharmacodynamics of piperacillin/tazobactamwhenadministeredbycontinuousinfusionand inter-mittentdosing.ClinTher2002;24:1090–104.