fmicb-08-02101 November6,2017 Time:16:42 #1 ORIGINALRESEARCH published:07November2017 doi:10.3389/fmicb.2017.02101 Synergistic Antifungal Effect of Fluconazole Combined with Licofelone against Resistant Candida albicans XinningLiu1,2,TaoLi3,DecaiWang1,YileiYang4,WenwenSun1,JianqiaoLiu5and ShujuanSun4* 1DepartmentofClinicalPharmacy,TaishanMedicalUniversity,Taian,China,2DepartmentofMicrobialandBiochemical Pharmacy,SchoolofMedicineandPharmacy,OceanUniversityofChina,Qingdao,China,3IntensiveCareUnit,Qianfoshan HospitalAffiliatedtoShandongUniversity,Jinan,China,4DepartmentofPharmacy,QianfoshanHospitalAffiliatedto ShandongUniversity,Jinan,China,5GeneralPractice,ShandongProvincialHospital,Jinan,China Candida albicans (C. albicans) is one of the important opportunistic fungal pathogens that is closely associated with disseminated or chronic infections. The objective of this study is to evaluate the synergistic antifungal effect of licofelone, which is dual microsomal prostaglandin E2 synthase/lipoxygenase (mPGES-1/LOX) inhibitor in combination with fluconazole against C. albicans. Here our results showed that Editedby: JackWong, licofelone (16 µg/mL) can synergistically work with fluconazole (1 µg/mL) against TheChineseUniversityofHongKong, planktoniccellsoffluconazole-resistantC.albicans.Thetwo-drugcombinationinhibited HongKong theC.albicansbiofilmformationover12h,andreducedtheexpressionofextracellular Reviewedby: TaissaVila, phospholipase genes, biofilm-specific genes and RAS/cAMP/PKA pathway related UniversityofTexasatSanAntonio, genes.Inaddition,thetwo-drugcombinationinhibitedthetransitionfromyeasttohyphal UnitedStates growthform,anddecreasedthesecretedaspartylproteinaseactivity,whilenotaffecting ChamindaJayampathSeneviratne, NationalUniversityofSingapore, the drug efflux pumps activity. Galleria mellonella model was also used to confirm Singapore the antifungal activity of the drug combination in vivo. This study first indicates that *Correspondence: the combination of fluconazole and licofelone has synergistic effect against resistant ShujuanSun [email protected] C.albicansandcouldbeapromisingtherapeuticstrategyfortheantifungaltreatment. Specialtysection: Keywords:biofilms,cyclooxygenase(COX),drugresistancemechanisms,enzymeinhibitor,phospholipase Thisarticlewassubmittedto Antimicrobials,Resistance andChemotherapy, INTRODUCTION asectionofthejournal FrontiersinMicrobiology Candida albicans is one of the important fungal pathogens that forms biofilm on the surface of Received:04August2017 catheters and other medical devices. It is also believed to be mainly implicated in disseminated Accepted:13October2017 or chronic fungal infections in ill and immunocompromised individuals (Carrillo-Munoz et al., Published:07November2017 2006). Currently, the antifungal drugs used in a clinical practice include azoles, amphotericin Citation: B, and echinocandins. Amphotericin B is a potent antifungal agent against an array of yeast Liu X,Li T,Wang D,Yang Y,Sun W, andfilamentousfungalpathogens,however,theapplicationislimitedbythesignificanttoxicities Liu JandSun S(2017)Synergistic suchasrenaltoxicity,infusionreactions,andhepatotoxicity(ClementsandPeacock,1990).Both AntifungalEffectofFluconazole amphotericin B and echinocandin drugs also have minimal gastrointestinal absorption and are CombinedwithLicofeloneagainst onlyavailableasparenteralformulations,whilefluconazole,oneofthecommonlyusedfirst-line Resistant Candidaalbicans. drug of the azole family in clinical prevention and treatment of Candida infections, is readily Front.Microbiol.8:2101. doi:10.3389/fmicb.2017.02101 absorbedwithhighbioavailability(NettandAndes,2016).Thewide-spreaduseofantifungaldrugs FrontiersinMicrobiology|www.frontiersin.org 1 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #2 Liuetal. SynergisticEffectofDrugCombination has increased the incidence of candida-resistance, ultimately thatphosphatidylcholine-specificphospholipaseD1isimportant leadingtorefractoryfungalinfections(Chenetal.,2012;Pfaller for yeast to hyphal transitions under certain conditions in et al., 2015). In the United States, fluconazole-resistance has C. albicans (Hube et al., 2001). The gene expression levels caused significant additional hospitalization costs and deaths includingRAS/cAMP/PKApathwayrelatedgenes(RAS1,CYR1, (Sagatovaetal.,2015).Therefore,seekingnovelagentcombined TPK2), biofilm-specific related genes (EFG1, BCR1, ALS1, withfluconazoleagainstresistantC.albicansisaurgentneed. ALS3, HWP1), and secreted aspartyl proteinase (SAP) genes Licofelone, a dual mPGES-1/LOX inhibitor is the terminal (SAP1, SAP2, SAP3, SAP4) were assessed by RT-PCR. The enzyme in the biosynthesis of Prostaglandin E2 (PGE ). morphogenesisofC.albicanswasalsoobservedbyfluorescence 2 Previous studies have noted that PGE , as the final product microscope. We also detected the effect of the two-drug 2 of arachidonic acid metabolic pathway can mediate several combinationonextracellularphospholipaseactivitiesbyeggyolk biological phenomena (Harizi et al., 2008). It is also associated agar method and measured the drug efflux pumps activity by with the ability of C. albicans to switch between yeast rhodamine6Gassay. and hyphal growth forms. Recent studies indicated that PGE synthesized from host and fungal can promote cell 2 MATERIALS AND METHODS adhesion, germ tube formation and enhance fungal resistance in C. albicans (Kalo-Klein and Witkin, 1990; Noverr et al., Strains and Media 2001).RAS/cAMP/PKApathwayisimportantformediatingthe transition from yeast to hyphal growth form in C. albicans EightclinicalisolateswereobtainedfromShandongQianfoshan and is known to regulate the expression of many biofilm- HospitalinChina.Sixresistantstrains,Candidaalbicans(CA10, specific genes including EFG1, BCR1, ALS1, ALS3, and HWP1 CA16), Candida glabrata (CG2, CG3) and Candida parapsilosis (Hogan and Sundstrom, 2009). Latest study shows that PGE (CP2, CP3); and two sensitive strains, Candida albicans (CA4, 2 increased cAMP level, activated PKA in C. albicans-infected CA8) were used in this study. Their susceptibilities were macrophages, and therefore stimulated C. albicans germ tube determined according to Clinical and Laboratory Standards formation (Yun et al., 2016). Based on this, we hypothesize Institute document M27-A3 (Wayn, 2008) with C. albicans that the regulation of PGE influencing biofilm formation (ATCC 10231) as the quality control strain. The strains may be associated with the2 RAS/cAMP/PKA pathway. In were maintained at −80◦C and subcultured at least twice several studies, the synergistic effects of cyclooxygenase (COX) on the yeast–peptone–dextrose (YPD) solid medium at 35◦C. inhibitors combined with antifungal drugs against C. albicans Licofelone and fluconazole were purchased from Shanghai biofilmandplanktoniccellshavebeenobserved.Thesepotential Boylechem Co., Ltd., China and Dalian Meilun Biotech Co., antifungal activities are supposed to be associated with the Ltd., China, respectively. The stock solution was prepared regulation of PGE production (Alem and Douglas, 2004; de accordingtothemanufacturer’sinstructions.Briefly,fluconazole 2 Quadros et al., 2011; Rusu et al., 2014; Liu et al., 2016). was dissolved in sterile deionized water at room temperature, A dual mPGES-1/LOX inhibitor, licofelone can suppress PGE and licofelone was dissolved in dimethyl sulfoxide (DMSO) 2 formation (Koeberle et al., 2008) and has recently succeeded with 0.2% Tween 80. Each stock solution was prepared at in reaching the required criteria in phase III clinical trials for a final concentration of 2560 µg/mL and stored at −20◦C osteoarthritis (Payandemehr et al., 2015), but no researches until needed. RPMI1640 was used as a diluent medium in regards with its antifungal activity and mechanism against for drugs and strains. The minimal inhibitory concentration planktonicandbiofilmcellsofC.albicanshavebeencarriedout (MIC) was defined as the lowest concentration of the drug sofar. that inhibited fungal growth by 80% (MIC80) compared In this study, we first evaluated the in vitro efficacy of with that of the growth control and sessile MIC (sMIC) licofelone alone and in combination with fluconazole against was read as the lowest concentration that produced a 50% C. albicans by the checkerboard microdilution method, and reduction in growth compared with that of the drug-free observed their antimicrobial effects on biofilm formation. In control. Before conducting the large number of experiments, addition, the toxicity of C. albicans treatment was investigated the MIC and sMIC of which fluconazole and licofelone used by Galleria mellonella model in vivo, survival analysis, and alone in the experiments was first evaluated as a preliminary histology.Thefungalburdendeterminationwasusedtoconfirm experiment. the antifungal effect of combined treatment during C. albicans MIC Determination by Broth infections. Related antifungal mechanisms were also explored Microdilution Assays in this study. Various virulence factors, other than biofilm formation,havebeencontributedtoC.albicanspathogenicityin TheMICsoflicofeloneandfluconazoleagainstC.albicansstrains thehosts,suchassecretedaspartylproteinaseandphospholipase weredeterminedbythebrothmicrodilutionmethodaccordingto activity (Garcia-Vidal et al., 2013; Cui et al., 2015). Aspartyl theClinicalandLaboratoryStandardsInstitute(CLSI)standard proteinase secreted from C. albicans is directly related to M27-A3 (Lockhart et al., 2011). For the checkerboard assays virulence properties such as adhesion, tissue invasion and (Kuykendall and Lockhart, 2016), the final concentration of immune evasion (Braga-Silva and Santos, 2011). C. albicans fluconazole and licofelone used were 0.125–64 µg/mL and produces phospholipases, which can destroy cell membranes 2–128µg/mL, respectively, for resistant candida strains (CA10, duringhostcellinvasion(Ghannoum,2000).Ithasbeenshown CA16, CG2, CG3, CP2, CP3); and 0.03125–16 µg/mL and FrontiersinMicrobiology|www.frontiersin.org 2 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #3 Liuetal. SynergisticEffectofDrugCombination 2–128µg/mL, respectively, for sensitive candida strains (CA4, Efficacy of Fluconazole and Licofelone in CA8). All drugs were diluted in 50 ml sterile RPMI 1640 G. mellonella Infected with C. albicans by medium. Fluconazole and licofelone were added to the wells Survival Assay in the second to eleventh columns and A through G lines of the 96-well plate, respectively. 100 µl of C. albicans cell Galleriamellonellainthefinalinstarslarvalstageofdevelopment suspensions (0.5∼2.5 × 103 cells/mL) were then inoculated was used as previously described with some modifications (Mylonakis et al., 2005; Amorim-Vaz et al., 2015). Larvae were into each well of 96-well plates. Each wells were filled with RPMI-1640 to a final volume of 200 µl except the control divided into four groups: growth control group (PBS only), plate. Negative controls were performed in 200 µl RPMI1640 fluconazole (160 µg/mL) treated group, licofelone (80 µg/mL) treated group, and fluconazole (160 µg/mL) with licofelone in A12-H12 wells, and growth controls were performed in 100 µl RPMI1640 and 100 µl microorganisms in the H1 well. (80 µg/mL) treated group. Control group of larvae inoculated The plate was incubated at 35◦C for 24 h. Both visual reading wasstudiedinparallelineveryinfectioninvestigation.Thelarvae were stored in wood shavings in petri dishes at 35◦C in the and optical density (OD) were performed to determine the dark condition for 2 days before the experiments. Larvae with MIC value (Bertout et al., 2011). OD was measured on the dark spots or apparent melanization were excluded. Resistant absorbance at 492 nm on a microplate reader. MIC endpoints C. albicans isolates (CA10) were grown overnight on liquid were defined as the lowest concentration of drugs causing 80% Sabouraud medium, and the inocula were suspended in PBS decreaseinviabilitycomparedtothedrug-freecontrol(MIC80). buffer supplemented with 20 µg/mL of ampicillin to prevent Allexperimentswereperformedintriplicate.Druginteractions bacterialcontamination.10µlofeachlarvasuspension(5×106 wereinterpretedbythefractionalinhibitoryconcentrationindex (FICI) model and the percentage of growth difference ((cid:49)E) cells/mL)wereinjectedofthroughthelastleftprolegusinga10-l model.TheFICI≤0.5representssynergy,FICI0.5<FICI≤4 syringe (Gaoge, China). The infected larvae were incubated at representsnointeraction,andFICI>4.0representsantagonism 35◦Cfor2h,andthentreatedwithdifferentgroupofantifungal (Odds, 2003). The (cid:49)E model was defined by the following drugs.Thedeathoflarvaewasmonitoredbyvisualinspectionof equation: (cid:49)E = E − E . The E value was theircolor(brown-darkbrown)each24hfor4days.Thesurvival predicted measured experiments were terminated at 4 days after infection. Each calculated by the data obtained directly from experiments. Statistically significant interactions of <100% were considered experiment groups contained 20 larvae (0.2–0.25 g in weight) andtheexperimentwasrepeatedthreetimesusinglarvaefrom weak, those from 100 to200% were considered moderate, and thoseof>200%wereconsideredstrong,aspreviouslydescribed differentbatches. (Katragkouetal.,2015). Fungal Burden Determination Fungal burden was determined by colony-forming unit (CFU) sMIC Determination by Broth countsfor4daysafterinfection(Krezdornetal.,2014).Larvae Microdilution Assays weredividedintofourgroups:growthcontrolgroup(PBSonly), Candidaalbicansbiofilmwasdevelopedusingaslightlymodified fluconazole (160 µg/mL) treated group, licofelone (80 µg/mL) method described above (Gu et al., 2016; Sun et al., 2017). treated group, and fluconazole (160 µg/mL) with licofelone The sMICs of fluconazole and licofelone against C. albicans (80µg/mL)treatedgroup.Eachlarvawasinfectedwith5×106 (CA10, CA16, CA4, CA8) biofilms were tested. The biofilms cells/larvaofresistantC.albicans(CA10)andincubatedat35◦C were formed over three time intervals (8, 12, and 24 h) at for 2 h, then treated with different drug. At every 24 h, 3 35◦C by pipetting 100 ml of the standardized cell suspension larvae from each group were selected with no discrimination, (103 cells/mL) into selected wells of 96-wellplate. After each suspended in 1 ml of PBS-ampicillin, and gently homogenized adhesion phase (8, 12, and 24 h), the cell suspensions were forafewseconds.Themixwas10-folddilutedwithPBSbuffer gentlywashedthreetimeswithsterilephosphate-bufferedsaline and 10 µl of these dilutions were inoculated on the sabouraud (PBS) and the planktonic yeast were removed. Fluconazole agarplates.Theplateswereincubatedat35◦Cfor24handthe and licofelone were then added to the corresponding wells number of CFUs was counted. The results were expressed as of 96-well plate in serially double-diluted concentrations. The meanstandarddeviation(SD).Eachexperimentgroupcontained final concentration of fluconazole and licofelone in wells was 20 larvae (0.2–0.25g in weight) and experiment was repeated ranged from 1–512µg/mL and 2-128 µg/mL for each isolate, threetimesusinglarvaefromdifferentbatches. respectively. The control wells were filled with 200 µl RPMI Histological Study 1640 only. The plates were then incubated for another 24 h at 35◦C in an orbital shaker. A colorimetric reduction assay was ToevaluatethepresenceofresistantC.albicans(CA10)intissue carriedoutwith2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H- of G. mellonella, three larvae from different groups (infected tetrazolium-5-carboxanilide(XTT)accordingtotheprotocolof only; and infected and treated with licofelone, fluconazole and Meloetal.Theabsorbancewasmeasuredwithamicrotiterplate licofelone-fluconazole)werecollectedatday3afterinfection.The readerattheabsorbanceof492nm,andthedrugconcentration larvae were preserved in 70% ethanol and cut into 8-µm-thick thatbroughtaboutareductioninabsorptionby50%contrasted sections using a cryostat. The samples were dried naturally at to that in the control well was reported as the sMIC endpoint. room temperature for 2 days, stained with Periodic Acid Schiff Eachtestwasperformedintriplicate. reagent (PAS), and dehydrated with increasing concentrations FrontiersinMicrobiology|www.frontiersin.org 3 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #4 Liuetal. SynergisticEffectofDrugCombination of ethanol and xylol (70, 80, 90, 96, and 100%). Finally, the suitableforuseasanendogenouscontrolgene(Caoetal.,2012; samples were fixed in neutral balata and dried naturally at Buetal.,2016).PrimersusedinthisstudyareinSupplementary roomtemperaturefor2days.Thestainedtissueswereobserved Table S1. The experiment was repeated on 3 independent under an Olympus FSX100 fluorescence microscope with ×4.2 occasions. objectives.Theexperimentwasrepeatedthreetimesusinglarvae fromdifferentbatches. C. albicans Morphogenesis Analysis Themorphogenesisanalysiswascarriedoutasdescribedbefore Phospholipase Activity of C. albicans (Pierceetal.,2015).ThecellsofresistantC.albicans(CA10)were Treated with Licofelone grownovernightat30◦CinRPMI1640andtreatedwithdifferent drugs including growth control (drug-free) group, fluconazole Phospholipase activity of resistant C. albicans (CA10) treated (1µg/mL)group,licofelone(16µg/mL)group,andfluconazole withdifferentdrugswasdetectedbyeggyolkagarplatemethod (1 µg/mL) combined with licofelone (16 µg/mL) group in the withsomemodifications(NiewerthandKorting,2001).Theegg well plates. The treated suspensions were then examined by yolk medium consisted of 15 g/L peptone, 3 g/L beef extract fluorescence microscope to determine the percentage of hyphal ointment, 5 g/L NaCl, 15 g/liter agar, 10 g/L glucose, and 10% sterile egg yolk emulsion. Each culture with 107 CFU/mL formation. The experiment was repeated on 3 independent occasions. were treated with different drug group including fluconazole (1 µg/mL) group, licofelone (16 µg/mL or 32 µg/mL) group, Rhodamine 6G Efflux Assays fluconazole (1 µg/mL) combined with licofelone (16 µg/mL or 32 µg/mL) group, and growth control (drug-free) group, Thedeterminationofthefunctionalactivityofdrugeffluxpumps transferred into separate eppendorf tubes, and then incubated affected by licofelone was carried out (Sun et al., 2015). Briefly, at 35◦C for 24 h. Then, 10 µl suspension of each tubes were resistantC.albicans(CA10)cellswereshakenovernightat35◦C inoculatedontothemedium,theplateswereincubatedat35◦C in YPD broth and then washed three times with PBS. The for 72 h. The diameter of the precipitation zone (a) and the cells were harvested by centrifugation and adjusted to 1 × 107 diameteroftheprecipitationzoneplusthediameterofthecolony cells/ml in PBS. In order to assess the efflux of Rh6G, we set 2 (b) were measured. Phospholipase zone (Pz) was designated groupsinthisexperiment:growthcontrolgroup(drug-free)and as Pz = a/b, as described above. According to this definition, licofelone(16µg/mL)group.Rhodamine6G(finalconcentration phospholipase index was scored and categorized as follows: of 10 µM) was added to each cell suspension. After incubating negative(Pz=1);verylow(Pz=0.90to0.99);low(Pz=0.80 55 min at 35◦C, C. albicans cells were collected by centrifuging to 0.89); high (Pz = 0.70 to 0.79), and very high (Pz ≤ 0.69), at 3,000 rpm for 5 min, washed three times with PBS, and aspreviouslyreported(Priceetal.,1982).Eachexperimentwas resuspended with PBS containing 5% glucose. Five hundred tested in triplicate and the phospholipase activity value was microlliterofeachsamplewerewithdrawnatevery10min.The recordedastheaverageofthe3measurements. mean fluorescent intensity (MFI) was immediately determined usingtheflowcytometerwithexcitationat488nmandemission at 530 nm at each specific time intervals. The experiment was Real-Time Quantitative PCR repeatedon3independentoccasions. mRNA expression of secreted aspartyl proteinase-related genes (SAP1, SAP2, SAP3, and SAP4), RAS/cAMP/PKA pathway Statistical Analysis relatedgenes(RAS1,CYR1,TPK2),andbiofilmformationrelated Data are presented as mean ± SEM. All experiments were genes (EFG1, BCR1, HWP1, ALS1, ALS3) was measured by performedintriplicate.Graphsproduction,datadistributionand RT-PCR.ResistantC.albicans(CA10)cellsweregrowntomid- log phase in RPMI-1640 medium at 35◦C after treatment with statistical analyses were performed using Graph Pad Prism 5. fluconazole(1µg/mL)alonegroup,licofelone(16µg/mL)alone Afterensuringdataconformedtoanormaldistribution,before group and fluconazole (1 µg/mL) combined with licofelone andafterdatatransformation,analysisofvariance(ANOVA)and (16 µg/mL) group. Cultures without drugs were served as t-tests were used to investigate significant differences between independent groups. The G. mellonella survival curves were controls.TotalRNAwasextractedandisolatedfromcellsusing analyzedbytheKaplan–Meiermethod,andfungalburdenswere RNA pure yeast kit (DNase I) (CWBiotech, Beijing, China). analyzedusingat-testtomeasurestatisticaldifferencesbetween A diluted RNA was reverse transcribed to cDNA using first- strandcDNAsynthesisSuperMixkit(CWBiotech)at42◦Cfor twoindependentgroups.P<0.05wasconsideredasstatistically 30minfollowing85◦Cfor5minaccordingtothemanufacturer’s significant. instructions. RT-PCR reactions were performed using cDNA, ultra SYBR mixture (with ROX) (CWBiotech), and primers RESULTS with an ABI ViiA 7 (Applied Biosystems) sequence detection system (CWBiotech). An aliquot of 25 µl PCR mix was used foreachgeneandthecyclingconditionswere95◦Cfor10min, MICs Determined by Broth Microdilution followed by 40 cycles of 95◦C for 15 s and 60◦C for 1 min. Assays ACT1 was found to be performed well under all experimental The susceptibilities of 8 Candida isolates (CA4, CA8, CA10, conditions, therefore, in this study, ACT1 was chosen to be CA16, CG2, CG3, CP2, CP3) were assessed under planktonic FrontiersinMicrobiology|www.frontiersin.org 4 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #5 Liuetal. SynergisticEffectofDrugCombination FIGURE1|Druginteractionsoffluconazoleandlicofeloneareinterpretedbythe(cid:49)Emodel.Three-dimensionalplotsoffluconazolecombinedwithlicofeloneagainst CandidaalbicanswerecreatedbyusingMATLABprogram.Theconcentrationsoffluconazoleandlicofelonearedepictedonthexaxisandyaxis,respectively,and theE-valuesobtainedforeachcombinationisdepictedonthezaxistoconstructathree-dimensional(3D)graphic.Peaksabovethe0planerepresentsynergistic combinations.Thecolor-codingbarontherightindicatesthattheclosertothetopofthebar,themoreeffectivethedrugcombination.(A)The(cid:49)EmodelofCandida albicans10,(B)the(cid:49)EmodelofCandidaalbicans16. states. For all of the strains, the MIC of licofelone used alone C. albicans (CA10, CA16) and sensitive C. albicans (CA4, was 128 µg/mL, and the MICs of fluconazole alone were CA8), the sMIC of fluconazole and licofelone used alone was rangedfrom1to512µg/ml(Figures1A,B).Susceptibilityassay 512µg/mLand128µg/mL,respectively.Thesusceptibilitytest showed that the combination of licofelone and fluconazole has showed that licofelone (16 µg/mL) combined with fluconazole strongsynergisticantifungaleffectsagainstresistantC.albicans: (1 µg/mL) had synergistic antifungal effects against C. albicans the MIC of fluconazole alone against resistant C. albicans biofilms (FICI < 0.5) at both 8 and 12 h time point. However, 80 (CA10,CA16)were>512µg/mL,whereaswhencombinedwith after 12 h formation, the combined effect of fluconazole and licofelone (16 µg/mL), the MICs of fluconazole were decreased licofelone on biofilm has gradually reduced. The combined to 1 and 0.5, respectively. These effects were also illustrated by antifungal effect was barely observed on the biofilm formed the FICI in vitro: the FICI for the resistant C. albicans (CA10, over 24 h with FICI > 1, suggesting that the two-drug CA16)strainsis0.127and0.250,respectively(Table1),however, combination has synergistic antifungal effect at the early stage, the FICI of sensitive C. albicans (CA4, CA8) and non-albicans but not the mature biofilm. The following experiments were (CG2,CG3,CP2,CP3)strainswereall>0.5,indicatingthatthe performed in resistant C. albicans (CA10) isolate due to its two-drug combination exhibited no antifungal activity against strong susceptibility to the combination of licofelone and thesensitiveC.albicansandnon-albicans.Theseresultswerealso fluconazole.TheSMICsofcombinedantifungaldrugsareshown interpretedby(cid:49)Emethod. inTable2. sMIC Determined by Broth Microdilution Efficacy of in G. mellonella Infected with Assays C. albicans The susceptibilities of 4 C. albicans (CA4, CA8, CA10, CA16) Invivoeffectoffluconazole(160µg/mL),licofelone(80µg/mL), isolates were assessed under biofilm states. For the resistant and their combination on infected G. mellonella treated was TABLE1|ThecombinedantifungaleffectsoffluconazolecombinedwithlicofeloneagainstCandidaalbicans. Drug Strains MICs(µg/mL) LAtheory BItheory MICA CA MICB CB FICI IN (cid:54)SYN Fluconazole+Licofelone CA10(R) >512 1 128 16 0.127 SYN 2704% CA16(R) >512 0.5 128 32 0.250 SYN 1112% CA4(S) 1 0.5 128 64 1.000 NI <100% CA8(S) 2 1 128 64 1.000 NI <100% CG2(R) 128 32 128 64 0.750 NI <100% CG3(R) 64 16 128 64 0.750 NI <100% CP2(R) 128 2 128 64 0.516 NI <100% CP3(R) 128 4 128 64 0.531 NI <100% EightisolateswithdifferentsusceptibilitiesinvolvedthreeCandidaspp.CA,Candidaalbicans;CG,Candidaglabrata;CP,Candidaparapsilosis.MICsdenotetheMIC80 ofeachdrugaloneorincombinationagainsttheisolatesandareshownasthemedianofthreeindependentexperiments;S,susceptible;R,resistant.TheFICIvalueis themedianofthreeindependentexperiments;FICI≤0.5forsynergism,FICI>4.0forantagonismand0.5<FICI≤4.0fornointeraction. FrontiersinMicrobiology|www.frontiersin.org 5 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #6 Liuetal. SynergisticEffectofDrugCombination TABLE2|AntifungaleffectoffluconazolecombinedwithlicofeloneagainstbiofilmofresistantCandidaalbicans. Strains Times(h) SMIC(µg/mL) LAtheory MICA CA MICB CB FICI IN CA10(R) 8 >512 2 >128 32 0.252 SYN 12 >512 4 >128 32 0.254 SYN 24 >512 256 >128 128 >1 NI CA16(R) 8 >512 4 128 16 0.133 SYN 12 >512 4 128 32 0.258 SYN 24 >512 256 128 128 >1 NI CA4(S) 8 >512 8 128 16 0.141 SYN 12 >512 16 128 16 0.156 SYN 24 >512 256 128 128 >1 NI CA8(S) 8 >512 4 128 8 0.070 SYN 12 >512 8 128 16 0.141 SYN 24 >512 256 128 128 >1 NI Timeindicatestheincubationperiodofbiofilmformation.SMICswerereadasthelowestconcentrationsthatproduceda50%reductioningrowthcomparedwiththatof thedrug-freecontrol.MICA,TheMICsoffluconazoleusedalone;MICB,TheMICsoflicofeloneusedalone;CA,TheMICsoffluconazolecombinedwithlicofelone;CB, TheMICsoflicofelonecombinedwithfluconazole;IN,interaction;SYN,synergism;NI,nointeraction.TheFICIvalueisthemedianfromthreeindependentexperiments. FICI≤0.5forsynergism,FICI>4.0forantagonismand0.5<FICI≤4.0fornointeraction. FIGURE2|SurvivalcurveofdifferenttreatmentonG.mellonellainfectedwithresistantC.albicans(CA10).Theconcentrationofyeastcellswas5×106cells/larva. Thecurveswereconsistedgrowthcontrol(PBS)group,fluconazole(160µg/mL)group,licofelone(80µg/mL)groupandlicofelone(80µg/mL)combinedwith fluconazole(160µg/mL)group.Thesefourcurveswereputinthesamecoordinatesystemtocomparethesurvivalrates.∗P<0.05comparedtothecontrol, fluconazolealoneandlicofelonealonegroup.Theexperimentwasrepeatedon3independentoccasions(n=3).Valuesrepresentthemeansstandarddeviations fromthreereplicates. FrontiersinMicrobiology|www.frontiersin.org 6 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #7 Liuetal. SynergisticEffectofDrugCombination thegrowthcontrolgroupby1.3and4-fold,respectively.Thedrug combinationgroupwasalsohighest(P<0.05).Thesurvivalrate of larvae treated with licofelone alone group was a little higher thanthatoflarvaetreatedwithfluconazolegroupalone,thatmay beexplainedbythefactthattheantifungalactivityoflicofelone aloneisstrongerthanfluconazole. Fungal Burden Determination After 3 days of infection, the fungal burden was determined by recovering yeast cells from the larvae infected with resistant C.albicans(CA10).ThenumberofCFUsinlarvaewereincreased over the time of infection (Figure 3). Infected larvae that were treated with fluconazole (160 µg/mL) in combination with licofelone (80 µg/mL) exhibited lower fungal burden FIGURE3|Effectofdrugcombinationonlarvalburdensofresistant thanfluconazolegroup.Thetwo-drugcombinationsignificantly C.albicans(CA10).Alllarvaewereinfectedwith5×106cells/larva C.albicans,thetreatmentconsistedofcontrol(PBSalone)group,fluconazole decreasedCFUnumberbyalmost4-foldcomparedtothecontrol (160µg/mL)alonegroup,andfluconazole(160µg/mL)combinedwith group(P<0.05).TheCFUnumberofthecontrolgroupwasalso licofelone(80µg/mL)group.∗P<0.05comparedtothecontrol,and slightlydecreased,whichmaybecausedbyhemocytesinlarvae. fluconazolealonegroup.Theexperimentwasrepeatedon3independent occasions(n=3).Valuesrepresentthemeansstandarddeviationsfromthree replicates. Histopathology Study Histopathologic staining of larvae infected with resistant C. albicans (CA10) and treated with different drugs was determined,andthemelanizationofG.mellonellainfectedwith performed at 3 day post infection. The differences in shape resistant C. albicans (CA10) was observed. The selection of andcytoplasmicstainingweredetected.Bothyeastandfilament drug concentration is not shown in this research. The results forms of C. albicans formed clusters in infected larvae. In the showedthatafter2daysinfection,allofthedrug-treatedgroups treatment with antifungal drugs, the formation of a majority showedattenuatedmelanization(Figure2).Specifically,thetwo- of yeasts was decreased in the combination of fluconazole drug combination group significantly reduced the melanization (160µg/mL)andlicofelone(80µg/mL)group(Figures4G,H). comparedtothefluconazolegroupandwasthehighestsurvival In addition, licofelone alone group (Figures 4E,F) decreased rate(P<0.05).After4dayinfection,thesurvivalrateoflarvae more yeast cells than fluconazole alone group (Figures 4C,D). treatedwithfluconazoleandcombinationgroupwashigherthan The growth control (treatment with the PBS alone) group FIGURE4|HistopathologystudyofinfectedG.mellonellatreatedwithdifferentdrugs.After72hofinfection,larvaeinfectedwith5×106cells/larvaofresistant C.albicans(CA10)wereprocessedforhistopathologyasdescribed.(A,B)Untreatedcontrolsgroup;(C,D)larvaetreatedwithfluconazole(160µg/mL)group; (E,F)larvaetreatedwithlicofelone(80µg/mL)group;(G,H)larvaetreatedwiththecombinationoffluconazole(160µg/mL)andlicofelone(80µg/mL)group.The experimentwasrepeatedon3independentoccasions. FrontiersinMicrobiology|www.frontiersin.org 7 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #8 Liuetal. SynergisticEffectofDrugCombination TABLE3|ExtracellularphospholipaseactivityofC.albicanstreatedwith licofeloneandfluconazole. Group(µg/mL) Precipitation Phospholipase zone activity Control 0.61 High Fluconazole(1) 0.62 High Licofelone(16) 0.76 High Fluconazole+Licofelone(16) 0.91a Verylow Licofelone(32) NZb Fluconazole+Licofelone(32) NZb Theprecipitationzonerepresentstheratioofthediameterofthecolonytothe cloudyzonepluscolonydiameter.aP<0.05comparedtothecontrol.bNZ,no FIGURE5|RelativegeneexpressionlevelsofSAP1,SAP2,SAP3,andSAP4 inresistantC.albicans(CA10).Cellsweretreatedwithfluconazole(1µg/mL), zoneofprecipitation. licofelone(16µg/mL)andtheircombination.TotalRNAwasextractedand reversetranscribedtocDNAforfurtherreal-timequantitativePCRtodetect (Figures 4A,B) has higher levels of infection than any other geneexpressionlevels.∗P<0.05comparedtothecontrol,fluconazolealone group. The mean size of the infected areas was smaller andlicofelonealonegroup.Theexperimentwasrepeatedon3independent occasions(n=3).Valuesrepresentthemeansstandarddeviationsfromthree in the combination group compared to the fluconazole replicates. group. Effect of Licofelone on Extracellular Phospholipases Activity of Resistant C. albicans The extracellular phospholipase activity of resistant C. albicans (CA10) was measured (Table 3). There were no obvious differences in average Pz between control group, fluconazole (1 µg/mL) group, and licofelone (16 µg/mL) group. The precipitationzoneofthecombinationoffluconazole(1µg/mL) and licofelone (16 µg/mL) group was significantly increased FIGURE6|RelativegeneexpressionlevelsofRAS1,CYR1,TPK2,EFG1, to 0.91 compared to the control group (P < 0.05). No BCR1,HWP1,ALS1andALS3inresistantC.albicans(CA10).Cellswere precipitation zone was observed in licofelone (32 µg/mL) treatedwithfluconazole(1µg/mL),licofelone(16µg/mL)andtheir combinedwithfluconazolegroup(P<0.05)andlicofeloneused combination.TotalRNAwasextractedandreversetranscribedtocDNAfor alone(32µg/mL)group. furtherreal-timequantitativePCRtodetectgeneexpressionlevels.∗P<0.05 comparedtothecontrol,fluconazolealoneandlicofelonealonegroup.The experimentwasrepeatedon3independentoccasions(n=3).Values Effect of Fluconazole-Licofelone on SAP representthemeansstandarddeviationsfromthreereplicates. Gene Expression Levels To investigate the effects of licofelone and fluconazole on resistant C. albicans (CA10) secreted aspartyl proteinase The results showed that the combination of licofelone with activities, the expression of 4 SAP family genes was analyzed fluconazolegroupdown-regulatedtheRAS1,CYR1,TPK2,BCR1, by RT-PCR. The results showed that both licofelone combined HWP1, ALS1 and ALS3 expression in comparison with control withfluconazolegroupandfluconazolealonegroupcandown- group(Figure6).Thecombinationgroupsignificantlydecreased regulate the expression levels of SAP1, SAP2, SAP3, and SAP4 the expression level of RAS1 (3-fold), CYR1 (10-fold), TPK2 comparedtothecontrolgroup(Figure5).Thelevelofdecreased (3-fold), BCR1 (4-fold), HWP1 (3-fold), ALS1 (2-fold) and expressioninthecombinationgroupwasmoreremarkablethan ALS3 (8-fold) compared to the fluconazole group (P < 0.05). thefluconazolealonegroup(P <0.05).Theexpressionlevelof While the expression of RAS1, CYR1, TPK2, ALS1, ALS3, SAP1inthefluconazolealonegroupwassignificantlydecreased and HWP1 expression on licofelone alone were higher than comparedtothecontrolgroup(P<0.05).Whiletheexpression fluconazolealonegroup.However,thedown-regulationofEFG1 of SAP1 to SAP4 in licofelone alone group shows no obvious expression has no difference between the drug combination differencecomparedtothecontrolgroup. group and the fluconazole group (P > 0.05). In addition, both groups decreased EFG1 expression relative to the growth Effect of Fluconazole-Licofelone on controlgroup. RAS1, CYR1, TPK2, EFG1, BCR1, ALS1, ALS3 and HWP1 Gene Expression Levels Effect of Fluconazole-Licofelone on C. albicans Morphogenesis Tofurtherinvestigatetheeffectsoflicofeloneandfluconazoleon resistantC.albicans(CA10)biofilmdevelopment,theexpression Thehyphalformationwasobservedbyfluorescencemicroscope. levels of 8 biofilm-related genes were analyzed by RT-PCR. The images of resistant C. albicans (CA10) showed that the FrontiersinMicrobiology|www.frontiersin.org 8 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #9 Liuetal. SynergisticEffectofDrugCombination FIGURE8|TheeffectoflicofeloneontheeffluxofRh6Ginresistant C.albicans.TheeffluxofRh6Gintheabsenceandpresenceoflicofelone (16µg/mL)weredeterminedbyaflowcytometer.MFIsrepresentthe intracellularRh6GinC.albicans.∗P>0.05comparedtotheRh6Galone group.Theexperimentwasrepeatedon3independentoccasions. in the combination group. In the control group, there were a large area of hypha gather together, while in the fluconazole or licofelone alone group, there were still some hypha gathered, but it was much lesser than the control group. This may be causedthatthetwo-drugcombinationcandecreasethefilament formation. Effect of Licofelone on Drug Efflux Pumps Activity of Resistant C. albicans The rhodamine 6G assay results showed that the licofelone group and the growth control group have the same decline tendency (Figure 8). No significant differences between groups wereobserved(P>0.05). DISCUSSION Therapies such as the use of novel compounds combined with the azoles could be an effective solution for candida infections, asitcanexpandantibioticspectrum,improveantifungalefficacy, and reduce side effects (Shrestha et al., 2015; Gu et al., 2016). Several in vitro and in vivo studies demonstrated that COX inhibitors, for instance, ibuprofen, aspirin, and indomethacin have certain antifungal activities by suppressing C. albicans PGE production and biofilm formation (Alem and Douglas, 2 2005; Bink et al., 2012; Liu et al., 2014). The combination of ibuprofen and fluconazole can enhance fluconazole susceptibly FIGURE7|Fluorescencemicroscopeexaminedtheyeast-filamenttransition to C. albicans by decreasing the MIC of fluconazole (Costa- ofresistantC.albicans(CA10).After24hofincubation,C.albicanstreated de-Oliveira et al., 2015). mPGES-1, as the terminal enzyme withdifferentdrugsasdescribed.(A,B)Untreatedcontrolsgroup;(C,D) downstreamofCOX-2cancatalyzethePGE biosynthesiswith fluconazoletreated(1µg/mL)group;(E,F)licofelonetreated(16µg/mL) 2 group;(G,H)thecombinationoffluconazole(1µg/mL)andlicofelone fewer side effects, ideally not affecting the formation of other (16µg/mL)treatedgroup.Theexperimentwasrepeatedon3independent housekeeping PGs (Vidal et al., 2007; Koeberle et al., 2010). occasions. Various pharmacodynamic studies have confirmed that a novel dual mPGES-1/LOX inhibitor, licofelone was effective in many types of diseases and has anti-asthmatic and anticonvulsant filamentation was reduced with the presence of licofelone effects (Rotondo et al., 2002; Kulkarni and Singh, 2007). The combined with fluconazole (Figures 7G,H) compared to the simultaneous inhibitions of both enzymes enable licofelone fluconazole treated group (Figures 7C,D) and control group to have a superior anti-inflammatory effect and obviate the (Figures7A,B).Therewereentirelyyeastcellsandnofilaments gastrointestinalside-effectscomparedtotheNSAIDS.Thisbetter FrontiersinMicrobiology|www.frontiersin.org 9 November2017|Volume8|Article2101 fmicb-08-02101 November6,2017 Time:16:42 #10 Liuetal. SynergisticEffectofDrugCombination safety profile has been authenticated in healthy individuals is based on their activity of inhibiting biofilm formation by (CharlierandMichaux,2003;Biasetal.,2004).However,weather decreasing PGE production. Here we investigated the further 2 licofelone has the similar antifungal activity as COX inhibitor antifungalmechanismsoflicofelonecombinedwithfluconazole. has not been elucidated. In this study, we first evaluated the RAS/cAMP/PKApathwayisimportantforC.albicansvirulence effect of fluconazole combined with licofelone against resistant by regulating hyphal growth (Rocha et al., 2001). In this C.albicansinvitro,licofelonealonehasamoderatelygreateffect pathway, the activation of Ras1 can lead to the generation on resistant C. albicans: the MIC of licofelone alone against of a cAMP signal that promotes PKA-mediated activation of resistant C. albicans was 128 µg/mL, and it can synergistically the transcriptional regulator proteins EFG1 and BCR1, which workwithfluconazole:theMIC offluconazolewasdecreased that play an essential role in regulating the morphogenesis and 80 from512to1µg/mLwhencombinedwithlicofelone(16µg/mL), expression level of cell wall-associated adhesion gene such as while the combination has little effect on sensitive C. albicans ALS1,ALS3,andHWP1thatareassociatedwithmorphogenetic and non-albicans strains (Table 1). Secondly, the combined response (Nobile and Mitchell, 2005). Our in vitro result antifungal effect of fluconazole-licofelone against C. albicans showed that licofelone combined with fluconazole has great biofilmcellswasobservedindifferenttimepoint.Thetwo-drug effects on inhibiting biofilm formation in different stages. combinationsignificantlyreducedC.albicansbiofilmformation Regarding how the drug combination influences the biofilm over12h,butafter24h,therewasalmostnoanti-biofilmeffect formation, the results showed that the combination has no observed(Table2).TheC.albicansbiofilmformationhasthree influence on EFG1 expression level, but dramatically down- phases:adherencestage(0–11h)inwhichthefungalpathogens regulated the expression of BCR1 (Figure 6), suggesting that adheretothesurface,developmentstage(12–30h)inwhichthe thepossibilityofdrugcombinationagainstresistantC.albicans carbohydrate-richextracellularmatrix(ECM)areproduced,and biofilm is through the regulation of the central regulator maturationstage(31–72h)inwhichthebiofilmstructureisheld BCR1, not by EFG1. We further examined the adhesion- togetherbyanECM.Theresistanceofbiofilmstoantimicrobial related genes HWP1, ALS1 and ALS3 expression levels in the agentsisoftenattributedtoitsfailuretopassthebiofilmmatrix presence of the combination. We found that the expression (Al-Fattani and Douglas, 2006). This may be the reason why level of those genes was decreased 3-fold, 2-fold, and 8-fold, theantifungaleffectofthecombinationcanonlyinhibitbiofilm respectively, compared to the fluconazole group (P < 0.05) over the process of formation, but has no inhibiting effect on (Figure 6). Mutational analysis indicated that strains lacking the mature biofilm. Further, for the future experiments, we all functional HWP1, ALS1 and ALS3 alleles (hwp1(cid:49)/hwp1(cid:49) will focus on the effect and antifungal mechanisms of resistant als1(cid:49)/als1(cid:49) als3(cid:49)/als3(cid:49)) were not able to generate adherent C.albicans. cells in biofilm models (Nobile et al., 2008). Thus, our results In addition, G. mellonella model recently has been used demonstrated that the fluconazole combined with licofelone to study fungal virulence and antifungal drug activity, since may reduce the biofilm formation of C. albicans by inhibiting it has a similar immune response to mammals (Vilcinskas, adhesion, and this inhibition is partly due to the down- 2011; Favre-Godal et al., 2014), and the correlation between regulation of biofilm cell wall adhesion-related genes ALS1, virulence of C. albicans mutants in mice and G. mellonella ALS3 and HWP1. These results were further confirmed by model has been confirmed (Brennan et al., 2002). It is florescence microscope analysis. The two-drug combination- worth mentioning that, with these advantages and the treatedstrainsproducedabiofilmwithonlyyeastcellsandlittle low cost, lack of ethical concerns and easy manipulation, filament, but the filamentation was more abundant with the G. mellonella model has gained wide acknowledgment. presence of fluconazole group and the growth control group As documented in our in vivo study, the combination of (Figure 7). Moreover, licofelone combined with fluconazole fluconazole with licofelone in G. mellonella infected model decreased the expression level of RAS1, CYR1 and TPK2, significantly improved the survival rate of larvae compared indicating that the inhibition of biofilm-specific related genes to the fluconazole treated group (P < 0.05). The death rate is probably through RAS/cAMP/PKA pathway (Figure 6). of the larvae is a dose dependent (Figure 2), as previously Previousstudieshavealsoshownthatsecretedaspartylproteinase described (Scorzoni et al., 2013). To complement the studies and phospholipases were believed to be important factors in vivo, we further performed the fungal burden determination involved in the pathogenicity and virulence of C. albicans (Figure 3) and microscopic observation (Figure 4) in larvae (Ghannoum,2000;Fengetal.,2016).Thesehydrolyticenzymes after infection. Our results confirmed the correlation of the were secreted by the fungus that lead to the colonization and virulence with the degree of damage on the histological tissue infections (Nailis et al., 2010). Secreted aspartyl proteinases ofG.mellonellawithfewerCFUswasobservedinthecombined are related to the adhesion and hydrolytic activity in mucosal group. tissue (Ramage et al., 2012). The study by Mendes et al. Candida albicans biofilm is a complex three-dimensional has indicated that biofilms of C. albicans secreted more Saps architecturewithanabundantextracellularmatrix.Itisknownto than planktonic cells (Mendes et al., 2007), and Saps are enhancethefungalresistancetomostcommonlyusedantifungal known to be intrinsically associated with the hyphal phase agent and the ability of cells to adhere is indispensable for all of C. albicans (Chen et al., 2002; Felk et al., 2002). We also stages of C. albicans biofilm formation (Chandra et al., 2001; examinedtheSAPgeneexpressionbyRT-PCR.Thecombination Nobile and Johnson, 2015). Earlier evidence only indicated of fluconazole and licofelone significantly down-regulated the that the effectiveness of COX inhibitors against C. albicans SAP1-SAP4 expression levels compared to fluconazole group FrontiersinMicrobiology|www.frontiersin.org 10 November2017|Volume8|Article2101