Antifungal activities of tacrolimus in combination with antifungal agents against fluconazole-susceptible and fluconazole-resistant Trichosporon asahii isolates

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

Antifungal

activities

of

tacrolimus

in

combination

with

antifungal

agents

against

fluconazole-susceptible

and

fluconazole-resistant

Trichosporon

asahii

isolates

Thaís

Felli

Kubic¸a

_,

_Laura

_Bedin

_Denardi

_,

_Maria

_Isabel

_Azevedo

_,

_Vanessa

_Oliveira

_,

Luiz

Carlos

Severo

_,

_Janio

_Morais

_Santurio

_,

_Sydney

_Hartz

_Alves

a_{UniversidadeFederaldeSantaMaria(UFSM),CentrodeCiênciasdaSaúde,ProgramadePós-Graduac¸ãoemCiênciasFarmacêuticas,}

SantaMaria,RS,Brazil

b_{UniversidadeRegionalIntegradadoAltoUruguaiedasMissões(URI),Santiago,RS,Brazil}

c_{UniversidadeFederaldeSantaMaria(UFSM),CentrodeCiênciasdaSaúde,ProgramadePós-Graduac¸ãoemFarmacologia,Santa}

Maria,RS,Brazil

d_{UniversidadeFederaldeSantaMaria(UFSM),DepartamentodeMicrobiologiaeParasitologia,SantaMaria,RS,Brazil} e_{ComplexoHospitalarSantaCasa,PortoAlegre,RS,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14March2016 Accepted5August2016

Availableonline30September2016

Keywords: Susceptibility FK506 Resistance Synergism

a

b

s

t

r

a

c

t

Theantifungalactivityoftacrolimusincombinationwithantifungalagentsagainst dif-ferentfungalspecieshasbeenpreviouslyreported.Herewereporttheinvitrointeractions betweentacrolimusandamphotericinB,fluconazole,itraconazole,andcaspofunginagainst 30 clinicalisolatesofbothfluconazole-susceptibleandfluconazole-resistantTrichosporon asahii. Fortheseanalyses,weusedthe brothmicrodilutionmethodbasedonthe M27-A3 technique and checkerboard microdilution method. Tacrolimus showed no activity againstT.asahiistrains(minimalinhibitoryconcentrations,MICs>64.0␮gmL−1).However,a largersynergisticinteractionwasobservedbythecombinationstacrolimus+amphotericin B(96.67%)andtacrolimus+caspofungin(73.33%)againstfluconazole-susceptibleisolates. Combinationswithazoleantifungalagentsresultedinlowratesofsynergismforthisgroup (fluconazole+tacrolimus=40%anditraconazole+tacrolimus=10%).Antagonistic interac-tionswerenotobserved.Forthefluconazole-resistantT.asahiigroup,alltestedcombinations showedindifferentinteractions.Thesynergismshowedagainstfluconazole-susceptibleT. asahiiisolatessuggeststhatthepotentialantifungalactivityoftacrolimusdeservesinvivo

experimentalinvestigation,notably,thecombinationoftacrolimuswithamphotericinBor caspofungin.

©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/

by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:sydneyalves.ufsm@gmail.com(S.H.Alves).

http://dx.doi.org/10.1016/j.bjid.2016.08.008

1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Theincidenceofinvasivemycosescausedbyemergentfungal pathogenshasproportionallygrownwiththeincreased num-ber ofimmunocompromised hosts, suchas AIDS patients, transplantrecipientstreatedwithimmunosuppressivedrugs, andthoseoncancertherapy.1–3_{Trichosporonasahiiisthemost}

frequentlyinvolvedspeciesindisseminatedanddeep-seated trichosporonosisincludingsystemicinfectionsdueto thera-peuticfailureintransplantedpatients.2,3

Thefirst-linetreatmentfortrichosporonosisincludesthe useofazoleantifungalagents,sinceTrichosporonspp.is resis-tanttoamphotericinBand echinocandins.4–6 _However,the

frequentexposuretoazolescanleadtodevelopmentof sec-ondary resistance to azoles and sometimes to multidrug resistance,resulting intherapeutic failures7–9 _and

increas-ing mortality rates.1,10 _{Combination therapy is a rational}

alternativethathasbeen studiedtoimprovetheefficacyof antimicrobialtherapyfordifficult-to-treatinfections, includ-ingovercomingconcernsofantimicrobialresistance.11–13

Recentstudieshaveshownthatusingfungalcalcineurin pathwaysholdsgreatpromiseforthefuturedevelopmentof novel agents,including combination therapy with antifun-gals against fungal pathogens.14–17 The antifungal activity obtained by the combination of the calcineurin inhibitor tacrolimus(FK506)plusantifungalagentshasnotyetbeen evaluated against Trichosporon species. In this context, the aimofthisstudy wastoevaluatetheinvitroactivityofthe combinationofFK506withamphotericinB,fluconazole, itra-conazole, and caspofungin against fluconazole-susceptible andfluconazole-resistantT.asahiistrains.

Material

and

methods

Clinicalisolatesandmolecularidentification

Onegroupof30fluconazole-susceptible(FS)strainsof clin-ical isolates T. asahii maintained in the collection of the Department of Microbiology and Parasitology at the Fed-eralUniversityofSantaMaria,SantaMaria,RS,Brazilwere studied.Asecondgroupoffluconazole-resistantstrains(FR) (n=30) was obtained from the FS group after sequential exposuretogrowingconcentrationsoffluconazole,as previ-ouslydescribedbyFekete-Forgacsetal.,18_{withthefollowing}

modifications: the final concentration of fluconazole was 128␮gmL−1,andtheincubationtemperaturewas35◦Cwith shakingfor48h.CellsfromthisculturewereplatedonSDA plates,and asinglecolonywasdesignatedisolatedFR.The standardstrain T.asahii CBS2479was alsoincludedinthe susceptibilitytests.

Theidentityoftheseisolateswasconfirmedusingstandard microbiological and molecular methods. Total DNA was extractedaccordingtotheprotocoldescribedbyMolleretal.19

andKlassenetal.20_{withmodifications.Amplificationofthe}

IGS1 region(rDNAintergenic) wasperformedbyPCRusing the primers 26F (5ATCCTTTGCAGACGACTTGA-3) and 5SR (5AGCTTGACTTCGCAGATCGG-3).5 _{The PCR products were}

purifiedandsequenced.Thesesequenceshavebeendeposited inGenBank(http://www.ncbi.nlm.nih.gov/genbank/)withthe

followingaccessionnumbers:KR233249,KT438844,KR872655, KR872656, KR872657, KR872660, KR872661, KR233246, KR233247, KR233248, KT365854, KT365855, KR872662, KT438839, KT438840, KR872663, KR912056, KT438842, KT365856, KT438847, KR912058, KR912064, KT365858, KR912065, KT365859, KT438846, KT365860, KR912059, KT365861,KR912066. Chemicals

Amphotericin B(AMB)(SigmaChemicalCo.–St.Louis,MO, USA),fluconazole(FCZ)(SigmaChemicalCo.–St.Louis,MO, USA), itraconazole (ITZ)(FrangonofBrazil,Pharmaceutical, Ltd.,SãoPaulo,Brazil),caspofungin(CAS)(Merck,Darmstadt, Germany),andtacrolimus(FK506)(Janssen-Cilag Pharmaceu-tica,Beerse,Belgium)wereemployed.

Thestocksolutionsofthedrugswerepreparedindimethyl sulfoxide(DMSO,SigmaChemicalCo.),exceptforFCZ,which wasdilutedinsteriledistilledwater.FK506wasdissolvedin methanol.Workingsolutionswerepreparedaccordingtothe documentM27-A3oftheClinical andLaboratoryStandards Institute.21

Invitrosusceptibilityanddruginteractiontests

Susceptibilityassayswereperformedusingthebroth microdi-lutionmethod,asdescribedbythedocumentM27-A3ofthe ClinicalandLaboratoryStandardsInstitute.21_Thestrains

Can-didakruseiATCC6258andCandidaparapsilosisATCC22019were included in thetests as controls.The minimuminhibitory concentrations(MICs)weredefinedasthelowestdrug con-centrationabletoinhibit50%(forFCZ,ITZ,andCAS)or100% (for AMB and FK506) of fungal growth when compared to the growthofthecontrol.Thehighestconcentrationsused were16␮gmL−1forAMB,128␮gmL−1forFCZ,16␮gmL−1for ITZand64␮gmL−1 forCASandFK506.TheMICsemployed as indicators for resistance were: ≥2␮gmL−1 (AMB)5,22,23_;

≥64␮gmL−1 (FCZ)6,24_{; ≥1␮gmL}−1 _(ITZ),23 _{and ≥2␮gmL}−1 (CAS).5,23 _{After exposingthe strains togrowing}

concentra-tionsofFLZ,thestrainswereseparatedintwogroups:(a)FS group(fluconazole-susceptiblegroup),formedbythestrains notexposed toFLZ;and (b)FRgroup(fluconazole-resistant group)formedbythesamestrainsafterexposuretoFLZ,and nowshowingMICsrangingfrom64to128␮gmL−1.

In vitro combinations of antifungal agents with FK506 againsttheFSandFRgroupsofT.asahiiwereevaluatedbythe microdilutioncheckerboardmethod.25_{Forthecalculationof}

thefractionalinhibitoryconcentrationindex(FICI),MICvalues relatedto100%inhibitionofgrowthwereused.Synergismwas definedastheFICI≤0.5,indifferencewas0.5<FICI≤4.0,and antagonismFICI>4.0.TheFICIswerecalculatedforallwells alongtheturbidity/non-turbidityinterfacewherethelowest FICIasthefinalpointwasdetermined.

Statisticalanalysis

ThestatisticalanalysisusedtotesttheSusceptibilityofthe twogroups(susceptiblestrainsvs.resistantstrains)to treat-ment with individual antifungalagentswas analyzedwith thet-test. Statisticalsignificancewassetatp<0.05.Results

wereanalyzedusingthe softwareGraphPadPrism5(Graph PadSoftwareversion6.01,CA).

Results

Theinvitrosusceptibilitiesof30T.asahiiisolates(FS)against theantifungalagentsandFK506aredescribedinTable1.FS strainsshowedlowMICsrangeforFCZ(1.0–16.0␮gmL−1)and ITZ(0.13–1.0␮gmL−1)compared toFR strains: FCZ(64.0 to >128.0␮gmL−1)andITZ(0.5–16.0␮gmL−1).Interestingly,for AMB,thenumberofresistantisolatesdecreased(90–3.33%)in theFRgroup(MICrange=0.13–4.0␮gmL−1);andforCASthe MICsremainedsimilarinthetwogroups(MICrangeforFSor FR=4.0–16.0␮gmL−1).FK506didnotshowantifungalactivity againstFSandFRisolatesatthehighestconcentrationtested (MICs>64.0␮gmL−1).Thestatisticalanalysisshowed signifi-cantdifferencesbetweenthesusceptibilityofFSvs.FRgroups forazoleantifungalagents(FLZ:p<0.0001,ITZ:p<0.007),as wellasforAMB(p<0.0001)andCAS(p<0.0001).

TheMICs andcombinationsresultsofantifungalagents andtacrolimus againstT.asahii beforeand afterresistance induction are presented in Tables 2 and 3. FK506 com-binedwithAMBagainsttheFST.asahiiisolatesshowedthe highest percentage of synergism (96.67%), followed by the combination with CAS (73.33%). In the synergistic interac-tions betweenAMB+FK506 or CAS+FK506, the MIC values ofFK506 decreasedfrom 64␮gmL−1 to0.5␮gmL−1.In con-trast,intheFRgroupthemajorityofcombinationswithFK506 showedinteractions classifiedas indifferent:AMB (76.67%), CAS(73.33%),FCZ(63.33%)andITZ(50%).Antagonismswere notdetectedforcombinationswithFCZand CASbut were showedwithAMB(13.33%)andITZ(10%).

Discussion

T.asahiiisthemostfrequentlyinvolvedspeciesin dissemi-natedfungalinfectionsincludingcasesofsystemicinfection intransplantedpatientsrelatedtotherapeuticfailures.2,3_The

prognosisoftrichosporonosisisverypoor, showing mortal-ityratesashighas80%.1,10_{Thelackofresponsetotherapyis}

relatedtotherelativeresistanceofT.asahiitomanydifferent antifungals.3,4,7,9

Moreover,thecorrectidentificationofTrichosporonspecies isimportantbecausethesusceptibilityisspeciesdependent. Inagreementwithpreviousreports,10,26,27_{ourresultsconfirm}

thatT.asahiiclinicalisolatesseemtobemoreresistantinvitro

toamphotericinBthantotriazolecompounds(Table1). Interestingly,ourfindingsdemonstratedthatafter sequen-tial exposuretogrowingconcentrations offluconazole,the numberofresistantisolatestoAMB(MICvalues≥2␮gmL−1) decreasedfrom90%to3.33%(Table1).Althoughthisinverse relationship has been previously reported,5,7,27,28 _targeted

deletion of ERG3 or ERG11in Candida albicans and Candida glabrataseemstobeapotentialmechanismthatmayleadto increasedsusceptibilitytoAMBwithincreasedresistanceto azoles.29,30

Similarly, clinical failure and breakthrough infections with Trichosoporon have been reported with the use of echinocandins.3,31–34_{Asexpected,wefoundMICs≥4␮gmL}−1 for caspofungin in all our isolates (Table 1). The cases of intrinsic resistance to echinocandins already described for Cryptococcus spp., Trichosporon spp., Fusarium spp., and zygomycetesareassociatedwithinsufficientsensitivityofthe targetenzyme,beta1,3-d-glucansynthase,tothedrugora mutated form ofthe enzymethat precludes echinocandin binding.35

Regardingtheazoleantifungalagents,ourresults demon-strated that prolonged exposure of the clinical isolates to fluconazoleshowedanincreasedMICforitraconazole char-acterizingcross-resistanceamongazoles(Table1).Multidrug resistance to antifungal agents has been reported for T. asahii inpreviousstudies.4,7,9_{T. asahiiclinical isolatesfrom}

nongranulocytopenic patients showed reduced susceptibil-ityinvitrotoAMB,flucytosine,fluconazole,itraconazoleand ketoconazole.7_Kushimaetal.9_{describedthatlongtermuse}

offluconazoleinvivomayleadtoreplacementoftheamino acidERG11pandthus triggerT.asahiiresistancetomultiple drugs.ThisstudydemonstratedthattheMICsforotherazole antifungalagentsagainstT.asahiistrainsincreasedin paral-lelwiththeMICforfluconazole,whiletheAMBMICsdidnot significantlychange.9

Tacrolimus (previously known as FK506) is an effective immunosuppressant,obtainedfromStreptomycestsukubaensis,

and iswidelyusedforpreventionoftransplantrejection.36

Table1–InvitrosusceptibilityofTrichosporonasahiiisolatestoantifungalagentsandtacrolimus.

Agents Groupofisolates Geometricmean MICrange MIC50 MIC90 Number(%)ofresistantisolates

AMB FS 2.24 1.0–8.0 2.0 4.0 27(90) FR 0.41 0.13–4.0 0.25 1.0 1(3.33) FCZ FS 2.41 1.0–16.0 2.0 8.0 0(0) FR 64.00 64.0to>128.0 64.0 64.0 30(100) ITZ FS 0.27 0.13–1.0 0.25 0.50 1(3.33) FR 1.52 0.5–16.0 1.0 4.0 27(90) CAS FS 7.82 4.0–16.0 8.0 8.0 30(100) FR 7.29 4.0–16.0 8.0 8.0 30(100) FK506 FS ND >64.0 ND ND ND FR ND >64.0 ND ND ND

MICrange,minimalinhibitoryconcentrationrange(␮gmL−1);MIC50,MICatwhich50%ofisolatestestedwereinhibited;MIC90,MICatwhich 90%ofisolatestestedwereinhibited;AMB,amphotericinB;FCZ,fluconazole;ITZ,itraconazole;CAS,caspofungin;FK506,tacrolimus;FSeFR, fluconazole-susceptibleandfluconazole-resistantTrichosporonasahiiisolates;ND,notdetermined.

Table2–Minimalinhibitoryconcentrations(MICs)andcombinationsresultsofantifungalagentsandtacrolimusagainst

Trichosporonasahiibeforeresistanceinduction.

Isolates MICs(␮gmL−1)andcombinationsresultsbeforeresistanceinduction

AMB/FK506 FCZ/FK506 ITZ/FK506 CAS/FK506

Onitsown Inthe combination

Onitsown Inthe combination

Onitsown Inthe combination

Onitsown Inthe combination 06 2.00 0.50 S 8.00 1.00 I 1.00 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 11 2.00 0.25 S 2.00 1.00 I 1.00 2.00 I 16.00 8.00 I >64.00 0.50 >64.00 8.00 >64.00 0.50 >64.00 0.50 13 2.00 0.50 S 4.00 1.00 S 1.00 1.00 I 16.00 8.00 I >64.00 0.50 >64.00 4.00 >64.00 0.50 >64.00 0.50 19 2.00 0.25 S 4.00 1.00 S 1.00 1.00 I 32.00 8.00 S >64.00 0.50 >64.00 16.00 >64.00 0.50 >64.00 0.50 21 2.00 0.50 S 32.00 1.00 I 2.00 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 64.00 >64.00 0.50 >64.00 0.50 29 4.00 1.00 S 64.00 1.00 I 2.00 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 64.00 >64.00 0.50 >64.00 0.50 31 2.00 0.25 S 4.00 1.00 S 1.00 1.00 I 32.00 8.00 S >64.00 0.50 >64.00 16.00 >64.00 0.5 >64.00 0.50 32 4.00 1.00 S 16.00 1.00 I 2.00 1.00 I 32.00 16.00 I >64.00 0.50 >64.00 64.00 >64.00 0.50 >64.00 0.50 36 1.00 0.13 S 8.00 1.00 I 1.00 1.00 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 43 2.00 0.25 S 64.00 1.00 S 1.00 0.25 S 32.00 4.00 S >64.00 0.50 >64.00 4.00 >64.00 0.50 >64.00 0.50 44 4.00 0.25 S 8.00 1.00 I 1.00 2.00 I 32.00 16.00 I >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 46 2.00 0.50 S 4.00 1.00 I 1.00 1.00 I 32.00 16.00 I .>64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 47 8.00 0.50 S 32.00 1.00 I 2.00 1.00 I 64.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 48 2.00 0.50 S 8.00 1.00 I 1.00 1.00 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 49 2.00 0.25 S 16.00 1.00 S 1.00 1.00 I 32.00 8.00 S >64.00 0.50 >64.00 16.00 >64.00 0.50 >64.00 0.50 50 2.00 0.25 S 8.00 1.00 S 1.00 1.00 I 32.00 16.00 I >64.00 0.50 >64.00 16.00 >64.00 0.50 >64.00 0.50 51 4.00 0.25 S 2.00 1.00 I 1.00 1.00 I 32.00 16.00 I >64.00 0.50 >64.00 8.00 >64.00 0.50 >64.00 0.50 53 4.00 0.25 S 4.00 1.00 S 2.00 0.50 S 16.00 16.00 I >64.00 0.50 >64.00 16.00 >64.00 0.50 >64.00 0.50 54 2.00 0.50 S 8.00 1.00 I 1.00 1.00 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 56 2.00 0.50 S 8.00 1.00 I 1.00 0.50 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 58 2.00 0.50 S 16.00 1.00 I 1.00 1.00 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 59 1.00 0.50 I 16.00 1.00 S 1.00 0.25 S 64.00 8.00 S >64.00 0.50 >64.00 8.00 >64.00 0.50 >64.00 0.50 60 2.00 0.25 S 8.00 1.00 S 1.00 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 16.00 >64.00 0.50 >64.00 0.50 61 8.00 0.50 S 8.00 1.00 I 1.00 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 62 2.00 0.25 S 8.00 1.00 I 1.00 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 63 2.00 0.50 S 8.00 1.00 S 1.00 0.02 I 32.00 8.00 S >64.00 0.50 >64.00 16.00 >64.00 64.00 >64.00 0.50 64 2.00 0.25 S 4.00 1.00 S 0.50 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 8.00 >64.00 0.50 >64.00 0.50 65 4.00 0.25 S 8.00 1.00 I 0.50 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 32.00 >64.00 0.50 >64.00 0.50 66 2.00 0.50 S 8.00 1.00 I 1.00 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 64.00 >64.00 0.50 >64.00 0.50 67 1.00 0.25 S 4.00 1.00 S 1.00 2.00 I 32.00 8.00 S >64.00 0.50 >64.00 16.00 >64.00 0.50 >64.00 0.50

MICs,minimalinhibitoryconcentrationsasthelowestconcentrationthatshowed100%inhibitionoffungalgrowth(␮gmL−1);AMB, amphotericinB;FCZ,fluconazole;ITZ,itraconazole;CAS,caspofungin;FK506,tacrolimus;S,synergism;I,indifference.

Table3–Minimalinhibitoryconcentrations(MICs)andcombinationsresultsofantifungalagentsandtacrolimusagainst

Trichosporonasahiiafterresistanceinduction.

Isolates MICs(␮gmL−1)andcombinationsresultsafterresistanceinduction

AMB/FK506 FCZ/FK506 ITZ/FK506 CAS/FK506

Onitsown Inthe combination

Onitsown Inthe combination

Onitsown Inthe combination

Onitsown Inthe combination 06 1.00 0.50 I >128.00 32.00 S >16.00 2.00 S 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 11 0.25 0.25 I 128.00 32.00 S 4.00 1.00 S 8.00 4.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 13 0.50 0.50 I >128.00 64.00 I >16.00 4.00 S 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 19 0.50 0.25 I 128.00 64.00 I 4.00 2.00 I 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 21 0.25 0.25 I 128.00 32.00 S 2.00 1.00 I 32.00 8.00 S >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 29 1.00 0.25 S 128.00 128.00 I >16.00 8.00 I 16.00 32.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 31 0.50 0.13 S 128.00 128.00 I 4.00 0.50 S 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 32 0.25 0.50 I 128.00 128.00 I >16.00 4.00 S 16.00 32.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 36 0.50 0.25 I 128.00 64.00 I 2.00 2.00 I 16.00 4.00 S >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 43 0.25 0.25 I 128.00 32.00 S 2.00 1.00 I 16.00 4.00 S >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 44 0.25 0.25 I 128.00 32.00 S >16.00 2.00 S 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 46 1.00 1.00 I 128.00 128.00 I >16.00 2.00 S 16.00 16.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 47 4.00 0.25 S 64.00 64.00 I 2.00 2.00 I 16.00 16.00 I >64.00 5.00 >64.00 0.50 >64.00 0.50 >64.00 0.50 48 0.50 0.25 I >128.00 32.00 S 4.00 1.00 S 16.00 4.00 S >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 49 0.50 0.25 I 128.00 64.00 I 8.00 2.00 S 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 50 0.50 0.25 I 128.00 32.00 S >16.00 1.00 S 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 51 0.25 0.25 I 128.00 32.00 S 2.00 2.00 I 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 53 0.25 0.50 I 128.00 128.00 I 2.00 16.00 A 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 54 0.25 0.13 I 128.00 64.00 I >16.00 1.00 I 16.00 16.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 56 0.13 0.50 A >128.00 64.00 I >16.00 8.00 I 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 58 0.50 2.00 A 128.00 128.00 I 8.00 32.00 A 16.00 1.00 S >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 59 0.50 0.25 I >128.00 64.00 I 4.00 2.00 I 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 60 0.25 0.50 I 128.00 128.00 I >16.00 32.00 I 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 61 0.25 0.50 I >128.00 64.00 I >16.00 4.00 S 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 62 1.00 0.50 I >128.00 128.00 I 4.00 2.00 I 16.00 16.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 63 0.25 2.00 A >128.00 8.00 S 16.00 8.00 I 16.00 2.00 S >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 64 0.25 0.13 I 128.00 64.00 I 2.00 2.00 I 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 65 0.25 1.00 A >128.00 16.00 S 1.00 8.00 A 16.00 1.00 S >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 66 0.25 0.50 I 128.00 64.00 I 1.00 2.00 I 16.00 2.00 S >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50 67 0.50 0.50 I 128.00 32.00 S 8.00 2.00 S 16.00 8.00 I >64.00 0.50 >64.00 0.50 >64.00 0.50 >64.00 0.50

MICs,minimalinhibitoryconcentrationsasthelowestconcentrationthatshowed100%inhibitionoffungalgrowth(␮gmL−1);AMB, amphotericinB;FCZ,fluconazole;ITZ,itraconazole;CAS,caspofungin;FK506,tacrolimus;S,synergism;I,indifference;A,antagonism.

Thiscompoundexertsitseffectsbyblockingtheimmune sys-temthroughinhibitionofcalcineurin.37_{Moreover,thisprotein}

canalsoaffectessentialfunctionsofthefungalcellanditis intrinsicallyinvolvedinthegrowthandpathogenesisofthree majorfungalspecies:Cryptococcusneoformans,C.albicans,and

Aspergillusfumigatus.36,38_{Inthisstudy,FK506showedlow}

anti-fungalactivitywhentestedaloneagainstFSandFRT.asahii

isolates(MICs>64.0␮gmL−1).

However,against the FSgroup, the resultsofour study

(Table 2) demonstrate strong in vitro synergism of FK506

combinedwithdrugsthatwereineffectiveininhibitingthis groupofT.asahiiclinicalisolates(Table1)suchas caspofun-gin(73.33%)and AMB(96.67%).Highpercentagesynergistic interactionsforcaspofunginplusFK506havebeenpreviously reportedagainstC.neoformans,Fusariumspp.,andAspergillus

spp.11,14,39

The potential enhanced antifungal activity of caspo-fungin in combination with other antifungal agents and anti-calcineurindrugsagainstclinicalisolatesofFusariumspp. wasdemonstrated byShalitetal.11 _{Theassociationofthis}

echinocandinwithFK506appearedsynergisticagainstallthe isolatestested.11_{Theantifungaleffectexhibitedby}

immuno-suppressantscyclosporinAandFK506isprobablyrelatedto calmodulinactivatedproteinphosphataseinvolvedinfungal stressresponse,virulence,andantifungalresistance.36

Stein-bachet al.14 _{alsodemonstratedasynergistic interaction of}

cyclosporinand FK506 withcaspofungin againstA. fumiga-tus.Inthisstudy,thecalcineurininhibitorswere capableof causingadelayinfilamentationofA.fumigatus,which sug-gested that inhibition of this pathway may potentiate the actionofconventionalantifungalagentsincombination ther-apyagainstinvasiveaspergillosis.14

The pharmacokinetics of caspofungin is unaltered by coadministrationoftacrolimus,butcaspofunginmayreduce tacrolimusconcentrationsbyupto20%.40_Therefore,

monitor-ingstandardtacrolimusbloodconcentrationsandappropriate tacrolimusdoseadjustmentsarerecommendedforpatients receivingboththerapies.

On the other hand, the synergism observed with the associationofFK506andAMBcanbenefittheantifungal ther-apy regimen through the reductions in time to treatment response,dosewithassociatedtoxicity,costs,anddecreased potential of microorganism-acquired resistance. Although AMB has been reported to have an insignificant effect on tacrolimusmetabolism,41 _{this polyene iswell knownto be}

nephrotoxic. Therefore, monitoring of serum creatinine is probablywarrantedforpatientsreceivingbothdrugs.42

Studieshavealsoshownthatcalcineurininhibitorsmay exertasynergisticinteractionwhencombinedwith antifun-galazolesagainstCandidaspecies.15,16 _{However,ourresults}

demonstrated thatcombinations ofFCZor ITZwithFK506 producedinteractionsmainlyindifferentemphasizingalack ofeffectagainstFSandFRT.asahiiisolates(Tables2and3). ThepharmacokineticsinteractionsbetweenFK506andazoles arewellknownandshowthatazolesinhibitthemetabolism ofFK506,requiringmonitoringoftheplasmaticconcentration ofFK506.42

Inconclusion, ourfindings demonstrated that the com-bination of FK506 with AMB or CAS leads to high rates ofsynergism in vitro. These combinations against T. asahii

isolates that presented resistance to both AMB and CAS deserve attentionascandidatesforinvivostudiesfocusing

T.asahiiexperimentalinfections.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

AlvesSHthanksthefinancialsupportprovidedbythe Brazil-ianAgenciesFAPERGS(GrantProc.2261-12)andCNPq(Grant Proc.470229/2012-8).

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