Evidence for anti-Pseudogymnoascus destructans (Pd) activity of propolis

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Evidence for anti-Pseudogymnoascus destructans (Pd) activity of propolis

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Title:
Evidence for anti-Pseudogymnoascus destructans (Pd) activity of propolis
Series Title:
Antibiotics
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Ghosh, Soumya
McArthur, Robyn
Guo, Zhi Chao
McKerchar, Rory
Donkor, Kingsley
Xu, Jianping
Cheeptham, Naowarat
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MDPI
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White-nose syndrome ( lcsh )
Propolis ( lcsh )
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serial ( sobekcm )

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White-nose syndrome (WNS) in bats, caused by Pseudogymnoascus destructans (Pd), is a cutaneous infection that has devastated North American bat populations since 2007. At present, there is no effective method for controlling this disease. Here, we evaluated the effect of propolis against Pd in vitro. Using Sabouraud dextrose agar (SDA) medium, approximately 1.7 × 107 conidia spores of the Pd strain M3906-2/mL were spread on each plate and grown to form a consistent lawn. A Kirby–Bauer disk diffusion assay was employed using different concentrations of propolis (1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%), in plates incubated at 8 °C and 15 °C. At 8 °C and 15 °C, as the concentration of propolis increased, there was an increasing zone of inhibition (ZOI), reaching the highest degree at 10% and 25% concentrations, respectively. A germule suppression assay showed a similar effect on Pd conidia germination. A MALDI-TOF-MS analysis of propolis revealed multiple constituents with a potential anti-Pd activity, including cinnamic acid, p-coumaric acid, and dihydrochalcones, which could be further tested for their individual effects. Our study suggests that propolis or its individual constituents might be suitable products against Pd.
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Volume 7, Issue 1

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Article EvidenceforAntiPseudogymnoascusdestructans Pd ActivityofPropolis SoumyaGhosh 1, ,RobynMcArthur 1, ,ZhiChaoGuo 1 ,RoryMcKerchar 1 ,KingsleyDonkor 1 JianpingXu 2 ID andNaowaratCheeptham 1, ID 1 DepartmentofBiologicalSciences,ThompsonRiversUniversity,Kamloops,BCV2C0C8,Canada; sghosh@tru.caS.G.;rmcarthur645@gmail.comR.M.;michelle8472@hotmail.comZ.C.G.; rory-mckerchar@hotmail.comR.M.;kdonkor@tru.caK.D. 2 DepartmentofBiology,McMasterUniversity,Hamilton,ONL8S4K1,Canada;jpxu@mcmaster.ca Correspondence:ncheeptham@tru.ca;Tel.:+1-250-371-5891 Equallycontributedauthor. AcademicEditor:LeonardAmaral Received:16October2017;Accepted:20December2017;Published:21December2017 Abstract:White-nosesyndromeWNSinbats,causedbyPseudogymnoascusdestructansPd,isacutaneousinfectionthathasdevastatedNorthAmericanbatpopulationssince2007.Atpresent,thereisnoeffectivemethodforcontrollingthisdisease.Here,weevaluatedtheeffectofpropolisagainstPdinvitro.UsingSabourauddextroseagarSDAmedium,approximately1.7107conidiasporesofthePdstrainM3906-2/mLwerespreadoneachplateandgrowntoformaconsistentlawn.AKirbyBauerdiskdiffusionassaywasemployedusingdifferentconcentrationsofpropolis%,2%,3%,4%,5%,10%,15%,20%,25%,inplatesincubatedat8Cand15C.At8Cand15C,astheconcentrationofpropolisincreased,therewasanincreasingzoneofinhibitionZOI,reachingthehighestdegreeat10%and25%concentrations,respectively.AgermulesuppressionassayshowedasimilareffectonPdconidiagermination.AMALDI-TOF-MSanalysisofpropolisrevealedmultipleconstituentswithapotentialanti-Pdactivity,includingcinnamicacid,p-coumaricacid,anddihydrochalcones,whichcouldbefurthertestedfortheirindividualeffects.Ourstudysuggeststhatpropolisoritsindividualconstituentsmightbesuitableproductsagainst Pd Keywords:propolis;white-nosesyndrome;Pseudogymnoascusdestructans;anti-Pdactivities;anti-fungal;fungalinfectioninbats 1.IntroductionWhite-nosesyndromeWNShasdevastatedmanyeasternNorthAmericanbatpopulationssince2007,killingmorethansixmillionbats[1].SincetherstobservationsofmortalityatacavenearAlbany,NewYorkin2007,WNShasspreadto31USstatesand5easternCanadianprovinces[2],mostrecentlyappearingin2017inthestatesofMississippi,Texas,andWashington,USA[2].TheetiologicalagentofWNSisPseudogymnoascusdestructansPd,apsychrophilicfungusthatgrowsoptimallyonhibernatingbatsattemperaturesbetween12 C[3].PseudogymnoascusdestructansPdpreferentiallyinfectsthinlyhairedregionsontheskinofhibernatingbats,andisabletodegradecollagenandinvadelivingtissues[4].Hibernatingbatslowertheirbodytemperaturetonear-ambienttemperatureduringtorporbouts[5].Duringhibernation,batsgenerallyseekmicroclimatesthatremainabovefreezingandcanbeaswarmas15Cormoreforsomespecies[57],andarerelativelyhighinhumidity.Batsgenerallyadapttothetemperatureoftheirsurroundingstomaximizetheirenergybudget[8].ThesimilaritybetweenthepreferredhibernatingenvironmentofbatsandtheoptimalgrowthconditionofthePdpathogenisamajorcontributortotheWNSepidemic.Pdinfectiondisruptsthenormaltorporandarousalcyclesofhibernatingbats[9],Antibiotics 2018 7 ,2;doi:10.3390/antibiotics7010002www.mdpi.com/journal/antibiotics

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Antibiotics 2018 7 ,2 2of12causingprematuredepletionoffatreserves,inadditiontoelectrolyteimbalancesanddehydration,resultinginmortality[10].Batsareessentialcomponentsofboththenatural,agricultural,andotherhumanecosystems[11].Theyplayimportantrolesinmaintainingecosystemstability,consumeinsectsthatarehumanoranimalpests,andredistributenutrientsthroughtheirguano[11,12].ToreducebatmortalityandeliminatethelikelihoodofspeciesextinctionbyPd,itisessentialtoidentifyeffectivemethodstocontrol Pd .Inregardtoinvestigationsofanti-Pdagents,severalrecentstudieshaveidentiedputativeanti-Pdagents,includingivolatilecompoundsproducedbythebacteriaRhodococcusrhodochrousDAP96253[13]andbyPseudomonasspp.isolatedfrombatwings[14];iicold-pressed,terpenelessorangeoilCPT[15];andiiisesquiterpenetrans,trans-farnesolCandidaalbicansquorum-sensingcompound[13].OurgoalwastoidentifyalternativeoradditionalpotenttreatmentsagainstPdusingsubstanceslikelytobeunharmfultocaveenvironmentsandtobats.Propolisisaresinoussubstanceproducedbyhoneybeesinbeehivesthroughouttheyear[16,17].Stinglessbeesarewidelyspread,especiallyinthetropicalandsubtropicalareasoftheworld.Propolisproducedfromsuchbeespossessestherapeuticproperties[18],includingantimicrobial,antitumor[18],antioxidant[19],anti-stimulant[20],anti-inammatory[2124],antiulcer[2225],andanti-HIVactivities[25].Forinstance,twocompounds,cardanolandcardol,isolatedfromaThaipropolis,possessedantiproliferationandcytotoxicityagainstcarcinomasoriginatedfromthelungs,theliver,andthecolon[26].Khacha-anandaetal.2016[19]foundthatethanolicextractsofpropolisEEPobtainedfromChiangMai,Thailand,exhibitedhigherantioxidantactivitythanEEPfromothersources.In2005,Huetal.[23]showedthatboththeethanolandthewaterextractsofpropolishadanti-inammatoryactivitiesinmiceandrats[21].Signicantanti-HIVactivities[EC<0.1g/mL,TI>186]resultedfrommoronicacidtriterpenoidsisolatedfromaBrazilianpropolis[25].Additionally,becauseofitsantiviral,antibacterial,andantifungalactivities,propolishasbeenusedinhumanhealthcaretotreatcolds,wounds,ulcers,andrheumatism[27,28].PropoliswasfoundtoexhibitantagonisticeffectsagainstanumberofGram-positivecocciandrods[29].ArecentstudybyShimizuetal.2011[30],showedthattheethanolextractofpropolisfromBrazilhadantiviralactivities.Whenadministeredorallyorcutaneouslytoherpessimplexvirustype1HSV-1-infectedmice,theethanolextractofpropolissignicantlyreducedtheherpeticskinlesionsandenhanceddelayed-typehypersensitivity[30].Silicietal.2006[31]reportedthatpropolishadantifungalactivitiesagainst15strainsbelongingtofourspeciesofyeastsisolatedfrompatientswithsupercialmycoses.Traditionally,studiesonthemedicinalbenetsofpropolishaveattributeditseffectstoitscomplexcompositionandtothesynergisticeffectsamongitscomplexchemicalconstituents[32,33].Anotheremergingthemeisthatthechemicalcompositionofpropolisishighlydependentonthegeographicallocation,botanicalorigin[34],andbeespecies[35].Indifferentecosystems,therearedifferentplantspecies,andtheseplantscanvaryintheirsecretionandexudates,andthereforeprovidediversefoodsourcestobees[16].Thus,thevariabilityinchemicalcompositionamongpropolisfromdifferentsourcescanbelarge.Forexample,propolisproducedinthePacicregioncontainsgeranylavanoneswhicharealsoatypicalcomponentfortheAfricanpropolis[17].ThegreenpropolisofBrazilhasprenylatedphenylpropanoidse.g.,artepillinCandditerpenesasmajorcomponents[17],whilethepropolisoftemperateregionsconsistsofavonoidslackingtheB-ringsubstituents,namely,chrysin,galangin,pinocembrin,pinobanksin,caffeicacidphenethylester[36].Theseuniquemixturesofconstituentsfromdifferentsourceslikelycontributetotheobservedmultipleeffectsincludingnotonlythebroadbiologicaleffectsdescribedabove,butalsotheinhibitionofnuclearfactor-B,cellproliferation,cellarrest,andapoptosis[17,36].Inthisstudy,wetestedtheeffectivenessofthecommerciallyavailablepropolispurchasedfromNaturalFactorsinCoquitlam,BritishColumbia,CanadaontheWNSagentPd.WeinvestigatedtheinvitroantagonisticpropertiesofpropolisagainstPd.Wealsotestedtheinhibitionofgermuledevelopmentwhenincubatedwithpropolis.Thisstudyistherstinvestigationoftheanti-Pdactivitiesofpropolis.

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Antibiotics 2018 7 ,2 3of12 2.Results 2.1.KirbyBauerDiffusionAssayPropolisexhibitedanti-Pdactivitiesatalltheconcentrations%wetestedinquadruplets,asrevealedbyaclear`zoneofinhibition'aroundeachoftheimpregnatedpaperdiscs,incomparisontodiscstreatedwithwateroranhydrousethanolFigure1Ai,ii,xii,xiii.ANOVAtestsshowedthatthedifferentconcentrationsofpropolisdifferedsignicantlyintheirinhibitoryeffectsFigure1Aiiixi,xivxxii.PropolissignicantlyinhibitedthegrowthofPdat8Cone-wayANOVA;F=8.309;df=8;p=0.100)]TJ/F178 7.5716 Tf 6.227 0 Td [(4aswellasat15Cone-wayANOVA;F=8.704;df=8;p=0.839)]TJ/F178 7.5716 Tf 6.227 0 Td [(5.Alongtheconcentrationgradient,at8C,thediameteroftheinhibitoryzonesinitiallyincreasedwiththeincreaseinpropolisconcentration,reachingthehighestvalueatthepropolisconcentrationof10%,andshowedsomedeclineathigherpropolisconcentrations.At15C,thoughthereweresomevariations,theinhibitionalsoincreasedwithincreasingpropolisconcentrations,reachingthehighestlevelattheconcentrationof25%ofpropolis.Thepairwisepost-hocT-testresultsareshowninSupplementaryTablesS1andS2. Figure1.Anti-Pdactivityofpropolis.AImagesixiandxiixxiiindicatetheactivityofpropolisat8Cand15C,respectively.TheblackarrowheadsindicatethezoneofinhibitionofPdwhentreatedwithdifferentconcentrationsofpropolisincomparisontowaterandanhydrousethanoltreatments;Bdiameterofthezonesofinhibitionat8Cand15C.Theerrorbarsarestandarddeviationsofthediameters. 2.2.SuppressionofGerminationofPdSporesAsrevealedbythemacroscopicandmicroscopicassayimagesFigure2A,B,therewasacompleteinhibitionofPdsporulationwithallpropolisconcentrationsthroughouttheentireincubationperioddaysatbothincubationtemperaturestested.Ontheseventhdayofincubationat8C,moremycelialextensionsmicroscopicimages,Figure2Axxiii,xxivwereobservedinthesamplestreatedwithwateroranhydrousethanol.ThemycelialgrowthbecamemoreconuentamongstthePdsporestreatedwithwaterandethanolbythe16thdayofincubationFigure2Axxxiv,xxxv.At15C,thePdsporestreatedwithwaterandethanolexhibitedaconuentgrowthfromtheseventhdayofincubationFigure2Bi,ii,xxiii,xxiv.

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Antibiotics 2018 7 ,2 4of12 Figure2.Germulesuppressionassay.A,BrepresentthePdgerminationassayfortreatmentswithwater,anhydrousethanol,andvariousconcentrationsofpropolisat8Cand15C,respectively.ThewhitearrowheadsindicatethemycelialextensionofthePdsporesattwodifferentincubationtemperatures.TheblackarrowheadsindicatetheinhibitionofthePdsporesonexposuretopropolisatdifferentconcentrations.ThegreenarrowheadsindicatetheformationofwhitePdlawnsresultingfromthetreatmentofsporeswithwateroranhydrousethanol. 2.3.MicroscopicExaminationoftheTreatedPdSporesPdsporestreatedwith1%propolisrevealedacompletedeformationMicroscopicimagesat10and40,Figure3iii,ivinvitrowhencomparedtotheuntreatedsporesthatexhibitedellipticalshapes,typicalof Pd spores.Figure3i,ii. Figure3.MicrographsofPdsporesdisplayedat10and40magnication:iiiellipticalshapeofuntreated Pd spores; iii iv deformed Pd sporestreatedwith1%propolis.

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Antibiotics 2018 7 ,2 5of12 2.4.ChemicalCompositionofPropolisMALDIspectraFigure4revealedthatthemajorconstituentsofthepropolisusedinthisstudywerearomaticacids,i.e.,cinnamicacidandp-coumaricacid;dihydrochalcones,i.e.,2,4,6-trihydroxydihydrochalcone;fattyacids,i.e.,stearicacid,palmiticacid;esters,i.e.,benzylmethoxybenzoate. Figure4.AMALDI-TOF-MSofapropolissampleatamassrangeof100Da.Eachofthepeaksonthemassspectrumrepresentsadistinctivecompoundinourpropolissample.ThenumbersabovethepeakscorrespondtothecompoundslistedinTable1;Bmagniedversionofthemassspectrumatamassrangeofthe100Da. Table1.CompositionofpropolisasdeterminedbyMALDI-TOF-MSthepeakscorrespondingtothesevaluescanbeseeninthemassspectruminFigure4. PeakConstituentsIdentiedMass/Charge m / z Intensity 1Benzylalcohol108.140.77 2Hydroquinone110.115.00 3Benzoicacid122.125.93 4Cinnamylalcohol134.1723.81 5Hydroxyacetophenone136.157.82 64-Hydroxybenzoicacid138.1210.98 7Cinnamicacid148.1614.81 8p-coumaricacid164.164.91 93-Phenyl-3-hydroxypropanoicacid166.185.91 10Sesquiterpenes168.3113.90 11Ferulicacid194.183.91 12Benzylbenzoate212.256.85 13Benzylmethoxybenzoate242.27211.88 14Benzyldihydroxybenzoate244.24105.16 15Palmiticacid256.4364.16

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Antibiotics 2018 7 ,2 6of12 Table1. Cont. PeakConstituentsIdentiedMass/Charge m / z Intensity 162,4,6-Trihydroxydihydrochalcone258.275184.70 17Pinostrobinchalcone270.2847.98 182,6-Dihydroxy-4-methoxydihydrochalcone272.25132.69 19Oleicacid282.4728.64 20Stearicacid284.3150.69 21Sakuranetin286.2744.71 222,4,6-Trihydroxy-4-methoxydihydrochalcone288.30247.59 23Cinnamylcaffeate296.3244.87 24Pinobanksin3-O-acetate314.292331.83 3.DiscussionThepopulationsofhibernatingbatsinNorthAmericaaredecliningatunprecedentedratesbecauseofWNS[1,37].Becauseinsectivorousbatsusuallyeatinsects,theyplayimportantrolesintheecosystemandprovidevaluablepestcontrolservicestotheagriculturalandforestrysectorsoftheNorthAmericaneconomy[11].OurresearchhasidentiedanewpotentialtoolforcombattingWNSthatisthreateningmanybatsacrossthecontinent.Wehavediscoveredthatbeepropolismaybeusedasaneffectiveantifungalagentagainst Pd ,thecausativeagentofWNS.Ourstudyhasrevealedthatevenlowconcentration%ofcommerciallyavailablepropolis%tincturecancompletelyinhibitPdsporegerminationatboth8Cand15C.Unfortunately,Pdgrowsoptimallybetween8C[3],andthehibernatingenvironmentcreatesanidealconditionforPdgrowthonhibernatingbats.Theincubationtemperaturesusedinourtestingwerethusrepresentativeoftheeffectivehibernationandfungalinfectionrangethatarealsofoundincaveenvironments[38].Additionally,thebioassayplateswerekeptuntilthe60thdayafterthe22nddayofobservation,andweneverfoundanyencroachmentofgrowthofPdsporesintheobservedZOI.ThismayindicatethatpropoliscancompletelyinhibitPdsporegerminationforupto60dayswhentestedinthelabsetting.Propolishaspreviouslybeenshowntoexhibitantifungal,antibacterial,andantiviralpropertiesandthereforehasbeenwidelyusedinhumanhealthcarefortreatingulcers,wounds,andrheumatisms[27,28].WeemployedtheKirbyBauerdiffusionassaytoidentifyfungicidalactivitiesofpropolis,whilethegermulesuppressionassaywasperformedinordertounderstandthepossibleinhibitorymechanismofthepropolisagainstPdsporesgermination.SimilarstudiesbyCornelisonelal.2014[13,39]haveshownthatbacteria-derivedvolatilecompoundsthatincludedecanal,2-ethyl-hexanol,nonanal,benzothiole,andN,N-dimethyloctylamine,completelyinhibitedthegrowthofconidiaandradialmycelialextensions.Moreover,ourndingsshowadeformationofthePdsporesexposedtopropolis,aresultconsistentwithapossiblefungicidalmechanismofaction.Thedeformationofthefungalconidiabypropolishasneverbeenreportedbefore.ThoughthepropolisusedinthisstudywaspurchasedfromawesternCanadiancompany,NaturalFactors,thecompanyobtainedtherawpropolismaterialsfromavarietyofsourcesandgeographicregions.FurtherexaminationsofourpurchasedpropolistraceditsorigintoMongolia.Unfortunately,nofurtherinformationcouldbeobtained,includingthespecicregionwithinMongolia,ortheplantsthatbeeswerefeedingonpersonalcommunication.However,consistentwithpreviousndings[4042],ourMALDI-TOFanalysisofthetestedpropolisidentiedarangeofconstituents.Theseincludedbenzylbenzoate,benzylmethoxybenzoate,benzyldihydroxybenzoate,hydroxyacetophenone,2,4,6-trihydroxydihydrochalcone,pinostrobinchalcone,2,6-dihydroxy-4-methoxydihydrochalcone,2,4,6-trihydroxy-4-methoxydihydrochalcone,cinnamicacid,andp-coumaricacidasmajorconstituents.ApreviousstudyofCanadianpropolisfromtworegionsshowedthatthepropolisfromVictoriacontainedmainlyp-hydroxyacetophenone,benzylhydroxybenzoate,cinnamicacid,anddihydrochalcones,whilethatfromRichmondhadlargeamountsofcinnamicacidandp-coumaricacid[40].However,bothpropolissamplesshowed

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Antibiotics 2018 7 ,2 7of12signicantantioxidantpropertieswithahighlevelofradicalscavengingactivity.Theotherremainingcompoundsidentiedinourpropolisalsoshowedsomegeographicspecicityinpreviousstudies.Forexample,3,3-dimethylallylcaffeatewasreportedfromEuropeanpoplar-typepropolis[41,42],whileweidentiedcinnamylcaffeateinourpropolis.OthercompoundsfromdiverselocationsincludehydroquinoneBurdocketal.1998[42]andbenzylalcohol[43].Benzylalcoholhasanti-inammatory,antibacterial,antitumour,hepatoprotective,andantioxidantactivities[41].Benzoicacidand4-hydroxybenoicacidfoundinoursampleswerepreviouslyfoundinanIranianpropolis,andhaveshownantibacterialproperties[44].Otherconstituentsrevealedinourpropolisincludedferulicacid,oleicacid,stearicacid,palmiticacid,andpinobanksin3-O-acetate.AnearlieranalysisofAnatolianpropolisalsoidentiedtheabovementionedcompoundsandshowedthattheyexhibitedantibacterialactivitiesagainstGram-positivebacteriasuchasStaphylococcusaureus-P,Streptococcussobrinus,Staphylococcusepidermidis,Streptococcusmutans,Enterococcusfaecalis,andMicrococcusluteus[45].CertainGram-negativebacteria,suchasEscherichiacoli,Salmonellatyphimurium,Pseudomonasaeruginosa,andEnterobacteraerogenesandyeastsuchasCandidaalbicans,C.tropicalis,andC.kruseiwerereportedtobesusceptibletotheAnatolianpropolis[45].Lastly,Sakuranetin,oneoftheavonoidsidentiedinourpropolis,wasreportedtoexhibitantimicrobialactivitiesagainstoralpathogens[46].WehaveconrmedthecompleteinhibitionofPdsporegerminationevenatalowconcentrationofpropolis%.Propolis,alsocalledbeeglue[21,47],issolubleinanhydrousethanol,whicheliminatestheresinousandstickypropertiesofthissubstancemakingitsuitableforapplicationonroostsubstrates.OurstudycontributestoagrowingportfolioofbiologicalandchemicalmeasuresforcontrollingthegrowthofPd[10,12,13,42].Futureapplicationsonbats,andtestsinwildhibernaculaarerequiredtotesttheeffectivenessofpropolisoutsideofalaboratorysetting.TheUSFishandWildlifeServiceUSFWShasalsorecommendedanumberofdecontaminants.Theonlyappropriatemethodsforlaboratoryandelddecontaminationofequipmentandclothingincludeethanol60%,isopropanol60%,isopropylalcoholwipes%,hydrogenperoxidewipes%,Accel,Cloroxbleach,Cloroxwipes,CloroxClean-Upcleaner+bleach,Hibiclens,andLysolICquaternarydisinfectantcleaner.Whetherpropoliswouldbeusefulasadecontaminationsubstanceisyettobeseen,inanycasethesubstanceslistedinthecurrentUSnationalWNSdecontaminationprotocolaremorereadilyavailable.Thetoxicityofmanyoftheselisteddecontaminationsubstancesisofminorconcernforanequipmentdecontaminationprotocol,butitisofutmostconcernwhenthesesubstancesareemployedforthemitigationofadisease.Naturallysourcedanti-Pdsubstanceslikepropoliscouldprovidetreatmentoptionsthataregenerallyconsideredsafeformammals.PreviousstudiesreportedthatGreekandRomanphysiciansprescribedpropolisasamouthdisinfectantandforthetopicaltherapyofcutaneousandmucosalwoundsinhumans[16].Morerecently,astudyshowedthatpropolispasteappliedondogs'cutaneouswoundsresultedinbetterwoundre-epithelization,contraction,andtotalwoundhealingthanaplacebo[48].However,nostudyhasexaminedtheeffectsofpropolisonbats.Overall,ourstudyhasdemonstratedthecompleteinhibitionofPdsporegerminationbypropolis.However,signicantresearchisstillrequired,forexampleaninvestigationonwhetheralongerperiodoftestingtimeinthelaboratorywouldyieldanyadditionalresultsfurtherindicatingarealpotentialofpropolisasoneofthetreatmentoptionsforWNS.Thepotentialactivitiesoftheindividualconstituentsofpropolisandtheircombinations,aswellastheirsynergisticinteractionsagainstPdsporegerminationandmycelialgrowthalsoneedtobeidentied.Atpresent,themedicinalbenetsofpropolishavebeenattributedtoitscomplexcompositionandtothepotentialsynergisticeffectsofitschemicalconstituents.Inaddition,thechemicalcompositionofpropolisishighlydependentonitsgeographicalorigin,onbeespecies,andonthebotanicalfoodsourcesofthebees.Furtherinvestigationsareneededinordertodeterminewhetherpropolis,oritsindividualingredientsorcombinationsthereofmaybe anoptionforthetreatmentof Pd -infectedcaveorbats.

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Antibiotics 2018 7 ,2 8of12 4.MaterialsandMethods 4.1.CultivationofPdSporesTheP.destructansM3906-2strainwasusedinthisstudy.ThisPdstrainwaspreviouslydescribedbyKhankhetetal.[49].ThecultivationandisolationofthePdsporeswereperformedaspreviouslydescribed[37].PdculturesweremaintainedonSabourauddextroseagarSDAplatesat15C.PdsporeswereisolatedfromculturesbysubmergingtheconidiallawninConidiaHarvestingSolutionCHS.05%Tween80,0.9%NaClfor5minfollowedbymechanicalscrappingandltrationthroughglasswoolaspreviouslydescribed[13,14].TheconcentrationofthePdsporeswasquantiedbyahaemocytometerandthesporeswerestoredinphosphatebuffersalinePBSat4Cuntilfurtheruse. 4.2.KirbyBauerDiffusionAssayOnehundredmicroliterofisolatedPdspores107spores/mLweremixedwith250mLofSabourauddextroseagarSDAmediasupplementedwithchloramphenicolmg/Lat50CFisherScientic,Fairlawn,NJ,USAtoavoidbacterialcontamination.Approximately20mLofthemixturewaspouredintoeachofthe85mmpetriplates.Plateswereair-driedinalaminarairowhood.EightmillimeterdiameterpaperdiscsToyoRoshiKaishaLtd.,Tokyo,Japanweresoakedindifferentconcentrations%,2%,3%,4%,5%,10%,15%,20%,and25%ofcommerciallyavailablepropolis%extractNaturalFactors,Coquitlam,BC,Canada,air-dried,andplacedinthecenterofeachseededSDAmediumplatealongwithsterilewaterandanhydrousethanol<0.005%waterSigmaAldrich,St.Louis,MO,USA;thelattertwoconditionswereusedascontrols.Anhydrousethanolwastestedsinceitwasusedasasolventtodissolvepropolisinalldilutionsasperthemanufacturer'sinstructions.Allplateswithdifferentpropolisconcentrationswereincubatedat8Cor15C,includingcontrolplates,induplicate.Anti-Pdactivitieswereidentiedaszonesofinhibitionaroundtheimpregnatedpaperdiscs,andthediametersweremeasuredinmillimeterswithanelectronicVerniercaliperGuilin,Guangxi,China.Notably,themeasurementofthediametersforthezonesofinhibitionwererecordedonthe22ndand15thdayofincubationat8Cand15C,respectively,sincePdsporesgerminatedslowerat8Cthanat15C.Eachtreatmentwasrepeatedfourtimes.Thebioassayplateswerekeptuntilthe60thdayaftertheobservationperformedatthe22ndday.Todeterminewhethertheconcentrationsofpropolisdifferedintheirinhibitoryeffects,weusedasingle-factorANOVAtoanalyzethequantitative-zone-of-inhibitiondata.Ifanoveralldifferencewasfound,allpairwisecomparisonsweremadeusingthetwo-tailedt-test.Sincetherewere36pairwisecomparisons[9concentrations)]TJ/F178 9.9626 Tf 1.02 0 0 1 256.074 280.379 Tm [(1/2=36]ateachofthetwotemperatures,aBonferronicorrectionwasappliedtothetypicalpvalueof<0.05consideredstatisticallysignicant.Thecorrectedp valuewas0.05/36=0.0013888. 4.3.GermuleSuppressionAssaySterilemicroscopicslides.576.2mmwerelayeredwith600LofmoltenSDA%atatemperatureof50Candpremixedwith5LofPdspores107spores/mLand5Lofeachoftheindicatedconcentrationsofpropolis.Quadrupletslideswerepreparedforeachofthepropolisconcentrationsandwereincubatedinduplicateat8Cand15C.ThegerminationofthePdsporeswasmeasuredmacroscopicallybyvisualizingthewhitegrowthconuenceofthePdlawn,andmicroscopicallybythePdhyphalextension.Bothmacroscopicandmicroscopicimageswereacquiredfromslidesonthe7thand16thdayofincubation.ThemicroscopicimagesweretakenusingaDCM130Edigitalcameraformicroscope.3Mpixels,CMOSchipAmScope,Irvine,CA,USA.ThemicroscopicimageswereimportedwiththeScopePhotoSoftwareAmScope,Irvine,CA,USA.

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Antibiotics 2018 7 ,2 9of12 4.4.AnalysisofChemicalConstituentsThechemicalcompositionofpropoliswasdeterminedusingmatrix-assistedlaserdesorptionionizationtime-of-ightmassspectrometryMALDI-TOF-MS.Thepropolissampleconsistedof50%dilutedpropolisinmethanolcoveredwith0.1020Mof-cyano-4-hydroxycinnamicacidHCCAin1:4v/vH2O/acetonitrile.A1.0Ldilutedaliquotofthesamplewasrstspottedontheplateandcoveredwith2.0Lofmatrix.Themassspectrawereobtainedusingabench-topMicroexMALDI-TOFMSfromBrukerDaltonicsBremen,Germanyequippedwithapulsednitrogenlaserat355nmwavelength.Thespectrawererecordedfrom40to2000Dapositivemode,andfrom100to400DapositivemodeusingFlexControl3.3softwareionsource1:19kV;ionsource2:15.5kV;lensvoltage:9.45kV;laserfrequency:60Hz;pulsedionextractionPIEdelay:120ns.OtherparametersareshowninTable2.Massgatesof400m/zpositivemodeweresetforallexperiments.Individualmassspectrafromeachspotwereacquiredbyaveraging350lasershots.Dataacquisitionwassettoautomate,andtherandomwalkmovementwasactivatedat10shotsperrasterduringthesequence.Thepeaklistsandintensitieswerecalculatedusingthepeak-pickingcentroidalgorithminFlexAnalysis3.3software. Table2. ParametersofMALDI-TOF-MS. ParametersValues LaserPulsednitrogen Laserpower20% Peakselectionmassrange40Da Samplerate0.05GS/s MassrangeLowrange ElectronicgainEnhanced100mV RealtimesmoothOff Spectrumsize2069pts Spectrumdelay307pts Laserfrequency60.0Hz Laserattenuatoroffset17% Laserattenuatorrange30% TargetMSP96targetpolishedsteel Matrix -cyano-4-hydroxy-cinnamicacid,HCCA Sample50%dilutedpropolisinMeOHcoveredwith0.1020MofHCCAin1:4 v / v H 2 O/acetonitrile SupplementaryMaterials:Thefollowingareavailableonlineatwww.mdpi.com/2079-6382/7/1/2/s1,TableS1:AnalysisofVarianceANOVAOne-wayCincubationtemperature,TableS2:AnalysisofVarianceANOVAOneway Cincubationtemperature. Acknowledgments:WearethankfultotheUnitedStatesFishandWildlifeServiceCheepthamandLausen'sGrant#F15AS00188forfunding.OursincereappreciationalsogoestoCoriLausenoftheWildlifeConservationSocietyCanadaandMarkRakobowchukofTRUfortheirtimeandassistanceineditingthismanuscript. AuthorContributions:N.C.conceivedtheideatousepropolis.S.G.andR.M.A.designedalltheexperimentsunderthecloseguidanceandsupervisionofN.C.S.G.draftedthemanuscript.R.M.A.conductedtheexperiments.N.C.securedfunding.K.D.designedtheexperimentsusedtodeterminethechemicalcompositionofpropolis.Z.C.G.andR.M.K.conductedtheexperimentsunderthesupervisionofK.D.K.D.draftedthechemistrysectionofthemanuscript.J.P.X.providedtheP.destructansM3906-2strainandcontributedtothestatisticalanalyses.N.C.,S.G.,J.P.X.andK.D.readandeditedthemanuscript. ConictsofInterest: Theauthorsdeclarenoconictofinterest.

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Antibiotics 2018 7 ,2 12of12 41.Bankova,V.Recenttrendsandimportantdevelopmentsinpropolisresearch.Evid.BasedComplement.Altern.Med. 2005 2 ,29.[CrossRef][PubMed] 42.Burdock,G.A.Reviewofthebiologicalpropertiesandtoxicityofbeepropolispropolis.FoodChem.Toxicol.1998 36 ,347.[CrossRef] 43.Bankova,V.;Popova,M.;Trusheva,B.Propolisvolatilecompounds:Chemicaldiversityandbiologicalactivity:Areview. Chem.Cent.J. 2014 8 ,28.[CrossRef][PubMed] 44.Trusheva,B.;Todorov,I.;Ninova,M.;Najdenski,H.;Daneshmand,A.;Bankova,V.Antibacterialmono-andsesquiterpeneestersofbenzoicacidsfromiranianpropolis. Chem.Cent.J. 2010 4 ,8.[CrossRef][PubMed] 45.Uzel,A.;Sorkun,K.;Oncag,O.;Cogulu,D.;Gencay,O.;Salih,B.ChemicalcompositionsandantimicrobialactivitiesoffourdifferentAnatolianpropolissamples.Microbiol.Res.2005,160,189.[CrossRef][PubMed] 46.Koo,H.;Gomes,B.P.;Rosalen,P.L.;Ambrosano,G.M.;Park,Y.K.;Cury,J.A.InvitroantimicrobialactivityofpropolisandArnicamontanaagainstoralpathogens. Arch.OralBiol. 2000 45 ,141.[CrossRef] 47.Santos,F.A.;Bastos,E.M.;Rodrigues,P.H.;deUzeda,M.;deCarvalho,M.A.;FariasLde,M.;Moreira,E.S.SusceptibilityofPrevotellaintermedia/PrevotellanigrescensandPorphyromonasgingivalistopropolisbeeglueandotherantimicrobialagents. Anaerobe 2002 8 ,9.[CrossRef][PubMed] 48.Abu-Seida,A.M.Effectofpropolisonexperimentalcutaneouswoundhealingindogs.Vet.Med.Int.2015,2015 ,1.[CrossRef][PubMed] 49.Khankhet,J.;Vanderwolf,K.J.;McAlpine,D.F.;McBurney,S.;Overy,D.P.;Slavic,D.;Xu,J.ClonalexpansionofthePseudogymnoascusdestructansgenotypeinNorthAmericaisaccompaniedbysignicantvariationinphenotypicexpression. PLoSONE 2014 9 ,e104684.[CrossRef][PubMed] 2017bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttributionCCBYlicensehttp://creativecommons.org/licenses/by/4.0/.


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