Bacteria Isolated from Bats Inhibit the Growth of Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome

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Bacteria Isolated from Bats Inhibit the Growth of Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome

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Bacteria Isolated from Bats Inhibit the Growth of Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome
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PLOS One
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Hoyt, Joseph R.
Cheng, Tina L.
Langwig, Kate E.
Hee, Mallory M.
Frick, Winifred F.
Kilpatrick, A. Marm
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PLOS one
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Bats ( lcsh )
Pseudomonas ( lcsh )
Bacteria ( lcsh )
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serial ( sobekcm )

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Emerging infectious diseases are a key threat to wildlife. Several fungal skin pathogens have recently emerged and caused widespread mortality in several vertebrate groups, including amphibians, bats, rattlesnakes and humans. White-nose syndrome, caused by the fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat species with extinction and there are few effective treatment strategies. The skin microbiome is increasingly understood to play a large role in determining disease outcome. We isolated bacteria from the skin of four bat species, and co-cultured these isolates with P. destructans to identify bacteria that might inhibit or kill P. destructans. We then conducted two reciprocal challenge experiments in vitro with six bacterial isolates (all in the genus Pseudomonas) to quantify the effect of these bacteria on the growth of P. destructans. All six Pseudomonas isolates significantly inhibited growth of P. destructans compared to non-inhibitory control bacteria, and two isolates performed significantly better than others in suppressing P. destructans growth for at least 35 days. In both challenge experiments, the extent of suppression of P. destructans growth was dependent on the initial concentration of P. destructans and the initial concentration of the bacterial isolate. These results show that bacteria found naturally occurring on bats can inhibit the growth of P. destructans in vitro and should be studied further as a possible probiotic to protect bats from white-nose syndrome. In addition, the presence of these bacteria may influence disease outcomes among individuals, populations, and species.

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RESEARCHARTICLEBacteriaIsolatedfromBatsInhibitthe Growthof Pseudogymnoascusdestructans theCausativeAgentofWhite-Nose SyndromeJosephR.Hoyt ,TinaL.Cheng,KateE.Langwig,MalloryM.Hee,WinifredF.Frick,A. MarmKilpatrickDepartmentofEcologyandEvolutionaryBiology,UniversityofCaliforniaSantaCruz,SantaCruz,California, UnitedStatesofAmerica jrhoyt@ucsc.eduAbstractEmerginginfectiousdiseasesareakeythreattowildlife.Severalfungalskinpathogens haverecentlyemergedandcausedwidespreadmortalityinseveralvertebrategroups,includingamphibians,bats,rattlesnakesandhumans.White-nosesyndrome,causedbythe fungalskinpathogen Pseudogymnoascusdestructans ,threatensseveralhibernatingbat specieswithextinctionandtherearefeweffectivetreatmentstrategies.Theskinmicrobiomeisincreasinglyunderstoodtoplayalargeroleindeterminingdiseaseoutcome.We isolatedbacteriafromtheskinoffourbatspecies,andco-culturedtheseisolateswith P destructans toidentifybacteriathatmightinhibitorkill P destructans .Wethenconducted tworeciprocalchallengeexperiments invitro withsixbacterialisolates(allinthegenus Pseudomonas )toquantifytheeffectofthesebacteriaonthegrowthof P destructans .All six Pseudomonas isolatessignificantlyinhibitedgrowthof P destructans comparedtononinhibitorycontrolbacteria,andtwoisolatesperformedsignificantlybetterthanothersinsuppressing P destructans growthforatleast35days.Inbothchallengeexperiments,theextentofsuppressionof P destructans growthwasdependentontheinitialconcentrationof P destructans andtheinitialconcentrationofthebacterialisolate.Theseresultsshowthat bacteriafoundnaturallyoccurringonbatscaninhibitthegrowthof P destructansinvitro andshouldbestudiedfurtherasapossibleprobiotictoprotectbatsfromwhite-nosesyndrome.Inaddition,thepresenceofthesebacteriamayinfluencediseaseoutcomesamong individuals,populations,andspecies.IntroductionEmerginginfectiousdiseasescanhavedevastatingimpactsonwildlife,andtheycurrently threatenmanyspecieswithextinction[ 1 – 4 ].Withanincreaseinanthropogenicdisturbance andriseinglobaltradeandtransportation,thethreatposedbywildlifediseaseislikelyto PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 1/12 OPENACCESS Citation: HoytJR,ChengTL,LangwigKE,HeeMM, FrickWF,KilpatrickAM(2015)BacteriaIsolatedfrom BatsInhibittheGrowthof Pseudogymnoascus destructans ,theCausativeAgentofWhite-Nose Syndrome.PLoSONE10(4):e0121329.doi:10.1371/ journal.pone.0121329 AcademicEditor: R.MarkBrigham,Universityof Regina,CANADA Received: August26,2014 Accepted: January30,2015 Published: April8,2015 Copyright: 2015Hoytetal.Thisisanopen accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense ,whichpermits unrestricteduse,distribution,andreproductioninany medium,providedtheoriginalauthorandsourceare credited. DataAvailabilityStatement: Allrelevantdataare withinthepaperanditsSupportingInformationfiles. Funding: ThisworkwassupportedbyUnitedStates FishandWildlifeService(F12AP01081)toWFF; NationalScienceFoundation(DEB-1115895)toAMK; andBatConservationInternationalStudent ScholarshiptoJRH.Thefundershadnoroleinstudy design,datacollectionandanalysis,decisionto publish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclared thatnocompetinginterestsexist.

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increase[ 5 ].Wildlifediseasescanbeexceedinglychallengingtomanagebecausefreeranging animalsaredifficulttotreatwithdrugsorvaccines,andmanystrategiesrequireconstant humanintervention[ 6 ].Forexample,there-establishmentofBlack-footedferretsintotheirnativerangerequiredvaccinationofadultsandyoungborneachyearforbothplagueandcanine distemper[ 7 ].Newapproachesthatdonotrequirecontinuedinterventionareneededtoreducetheimpactsofthesedevastatingdiseases[ 8 ]. Severalrecentlyemergedwildlifepathogensinfecthostdermaltissue,andinteractionswith hostskinmicrobiotacouldplayanimportantroleindiseaseseverity.Vertebrateskinisanecosystemcomposedofdifferenthabitatswhichharbordiverseassemblagesofmicroorganisms [ 9 ].Previously,studiesofskinmicrobiotaprimarilyexaminedthepathogenicrolesofskinmicrobes,withlittleattentiontothebeneficialfunctionthatmanymicroorganismsmayprovide [ 10 ].However,beneficialbacteriaonskincanprovidevitalfunctionstotheirhosts,including processingofskinproteins,freeingfattyacidstoreduceinvasionoftransientmicroorganisms, andinhibitionofpathogenicmicroorganisms[ 11 ].Somebacteria,termedprobiotics,orbeneficialbacteria[ 12 ],havebeendevelopedtoreducetheimpactofabroadrangeofdiseases. Probioticsthatcanestablishonhostshavethepotentialtoprovidealong-lastingsolution formanagingdiseaseand,unlikechemicalfungicides,maybeabletocoevolvewithapathogen [ 13 ].Probioticsareregularlyusedinthebiologicalcontrolofdiseaseinbothaquacultureand agriculture,buthaveyettobewidelyimplementedincontrollingwildlifedisease,possiblybecauseofperceivedrisksandlackofdemonstratedsuccess[ 14 – 17 ].Risksassociatedwithaugmentingmicro-organismsonahost,whichcaneitherbeineffectiveoraccidentallyharmful, canbeminimizedbyusingbacteriathatnaturallyoccurinthehosts ’ environment[ 18 ].Resistantortolerantspeciesthatarephylogeneticallycloselyrelatedtoaheavilyimpactedspecies mayhostbacteriathatcouldserveasprobiotics,andthesebacteriamaybemorelikelytobe abletocolonizingthetargetspecies ’ skin[ 18 ]. Here,weassesswhetherbacterianaturallyoccurringonbatscanreducethegrowthof Pseudogymnoascusdestructans ,thepathogenthatcauseswhite-nosesyndrome(WNS)[ 19 ].WhitenosesyndromefirstemergedinHowe ’ sCave,NewYork,in2006,andspreadquickly,causing precipitousdeclinesinhibernatingbatsthroughoutEasternNorthAmerica[ 2 20 ].Fourspecies ( Myotisseptentrionalis Myotissodalis Myotislucifugus ,and Perimyotissubflavus )havesuffered > 90%declinesinregionalpopulationsandonespecies, M septentrionalis ,isonatrajectorytowardsextinction[ 2 20 ]. Myotisseptentrionalis hasrecentlybeenproposedbytheU.S. FishandWildlifeServiceforlistingundertheEndangeredSpeciesActandhasbeenlisted undertheCanadianSpeciesatRiskActasEndangered. Pseudogymnoascusdestructans infectsthedermaltissueofbatsandgrowsoptimallybetween10 – 14C[ 21 ],similartothetemperatureofhibernatingbats. Pseudogymnoascusdestructans infectionmaydisruptbats ’ physiologicalprocessesincludingheatandwaterlossandelectrolytebalance[ 22 ],andtypicallyresultsinincreasedarousalfrequencybyhibernating bats[ 19 23 ].Increasedarousalfrequencymayprematurelydepletebats ’ fatstoresresultingin deathapproximately70 – 120daysafterinfection,basedonlaboratoryinfectiontrials[ 19 22 24 ]. Individualsabletosurvivethroughhibernationuntilspringappeartoclearinfectionandfully recover[ 24 25 ].However,thesebatsbecomere-infectedthefollowingfall,inpartbecause Pseudogymnoascusdestructans iscapableofpersistingforlongperiodsoftimeintheabsence ofbats[ 26 27 ]. Currently,therearefewmanagementoptionsthatcanreducemortalityinaffectedregions [ 28 ].Preliminaryinvestigationsoftreatmentstoreducemortalityusingantifungaldrugscaused highermortalityinthetreatedgroupsthancontrolgroups,possiblyduetotoxicity[ 29 ].Other proposedchemicaltreatmentoptionshaveavoidedthetoxiceffectsofthedirectapplicationof chemicals,buthaveyettobevalidated insitu [ 30 ].Thus,treatmentoptionsareurgentlyneeded, BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 2/12

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andaprobioticmaybeaneffectivewaytoreduceWNSimpactsifitcouldatleastpartiallyinhibit P destructans growthanddelaymortalitylongenoughforbatstosurvivehibernation. Weculturedbacteriaisolatedfromtheskinoffourspeciesofhibernatingbatsfromeastern NorthAmericatodeterminewhethernaturallyoccurringbacterialspeciesmightexistwithin theskinmicrobiomeofbatsthatcouldinhibitgrowthof P destructans .Wethenquantifiedthe anti-fungalefficacyofthesebacteriaacrossarangeofconcentrationsintwo challengeexperiments.Methods SamplingandisolatingcutaneousmicrobesWeconductedsamplingforcutaneousbacteriaonhibernatingbatsattwohibernaculainNew YorkandtwoinVirginia(exactlocationsofstudysitesarenotprovidedtoprotectsensitive wildlifehabitat).Werubbedsterilepolyesterswabsdippedinsterilewaterbackandforthfive timesalongeachbat ’ sforearmandmuzzle.Swabswerefrozenin20%glycerolstockforlater culturing.Wecollectedswabsfromtenindividualsfromeachoffourspecies Eptesicusfuscus Myotisleibii M lucifugus ,and M sodalis EpidermalswabsamplecollectionprotocolsforthisstudywereapprovedbytheUniversity ofCalifornia,SantaCruzIACUC(protocol#FrickW1106).Samplecollectionwaspermitted byauthorizedstatebiologistsfromtheNewYorkDepartmentofEnvironmentalConservation andVirginiaDepartmentofGameandInlandFisheries.Handlingandsamplingofendangered species( Myotissodalis) wasconductedundertheappropriatestateandfederalpermits. Eachswabwasplatedontwotypesofgeneralmedia,Reasoner ’ s2Aagar(R2A)andsabourauddextroseagar(SDA),andplateswereincubatedat9Cforthreeweeks.Weclassifiedbacteriaoneachplatebymorphotype,usingcolor,growthform,andgramstainingtechniques.We isolatedonecolonyfromeachsamplebymorphotype(toreducerepeatsamplingofthesame isolate)usingasterileinoculatingloopandre-platedeachisolateonR2Amediaandgrewthem for2 – 5daysat9C.Eachisolatewascryo-bankedbysamplingfromeachofthesecolonieswith asterileinoculatingloop,placingthesamplein30%glycerol,andfreezingitat-80Cfor lateruse.Pre-screenforbacteriawithantiP destructans propertiesWedeterminedwhetherisolatescouldinhibitthegrowthof P destructans usingachallenge protocoladaptedfromtheNationalCommitteeforClinicalLaboratoryStandards.AllculturingwasdoneonSDA.[ 31 ].Asuspensioncontaining1.7x107P destructans conidia/ml(quantifiedusingahemocytometer)waspreparedbyfloodinga3week-oldcultureof P destructans grownonSDAwith20mlof1Xphosphatebufferedsalinewithtween20(PBST20).Colonies weresubmersedfor5minutes,andthengentlyrubbedwithasterileinoculatinglooptofree theconidia.Thesupernatantwasdrawnoffandplacedintoa50mlfalcontubeandvortexedto homogenizethesuspension.Each90mmplatewasinoculatedwith200ulofthe P destructans suspensionandallowedtoairdryfor10minutes.Weaddedbacteriaontheplatealreadyinoculatedwith P destructans fromagrowingcultureusingpinpointinoculationatthreeequally spacedpointsontopofthedried P destructans suspension.Bacteriaculturesweregrownfrom frozenstock24hoursearlieronSDA.Plateswereplacedintoincubatorsat9 – 10C,whichis withintherangethatbatshibernate[ 32 ],andgrowthwasmonitoredeveryotherdayfor14 daysandonday14anybacteriathatproducedazoneofinhibition(avisiblereductionof P destructans growthsurroundingthebacterialcolony)wereincludedinsubsequentchallenge experimentsdescribedbelowInadditiontobacterialisolatesfrombats,a Pseudomonasfluorescens isolate Pf A506commonlyusedinbiocontrolofagriculturalfungalpathogenswasincluded BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 3/12

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asapositivecontrol[ 33 ],andtwotypesofnegativecontrols,1)ashaminoculationwith20% sterileglycerolstock,and2)twobacteriaisolatedfrombatsinthegenera Chryseobacterium and Sphingomonas (bothgram-negativerodshapedbacteria)thatarenotknowntoproduce anti-fungalcompounds[ 31 ].IdentificationofbacterialisolatesWeidentifiedbacterialisolatesusedinthefollowinginhibitionassaysusingPCRamplification andDNAsequencing.DNAforPCRwasobtainedbysuspendingasmallamountofabacterial colonyin100 lofsteriledeionizedwater(SDW)andlysingthecellsat95C(10min).Universalbacterial16SrRNAgeneprimers(16S_F( 5`-ACCGCGATAATACGTCCCGATCG — 3` ) and16S_R( 5`-TGCGGACGTGAAGTGCTAG-3` ))wereusedtoamplifythe~1.5kb16S rRNAgenefragment[ 34 ].ThefollowingwasaddedtoeachPCRtemplate:1 lofcrudelysate DNAtemplate,1.5 lofeach0.6 Mforwardandreverseprimer,and5 lofTaq5XMM (NEB)at1Xconcentration,whichcontains1.5mMMgCl2,2mMdNTPs,andPCRbuffer.Reactionvolumesweremadeupto25 lwithSDW.Thereactionconditionsinvolvedaninitial denaturationat95Cfor3minutes,followedby35cyclesofdenaturationat95Cfor15sec, primerannealingfor15secondsat49C,andextensionfor90secondsat42C.The16SrRNA genesequenceswerecomparedwithknownsequencesintheEMBLdatabaseusingMEGA BLAST(BLASTN2.1.1,[ 35 ])toidentifythemostsimilarsequencealignment. Pseudomonas fluorescens isolate Pf A506wasusedtoassureproperalignmentofsequences.InhibitionAssaysTwoseparateinhibitionassayswereperformed.Inthefirstinhibitionassay,wedeterminedthe abilityofeachbacterialisolatetogrowonlawnsofdifferentstartingconcentrationsof P destructans ,andtoproduceazoneofinhibitioninwhich P destructans growthwaseitherdelayed,haltedadjacenttothebacteriacolony.Inthesecondinhibitionassay,wemeasuredthe growthof P destructans onalawnofdifferentstartingconcentrationsofeachbacterialisolate. Inthefirstinhibitionassay,thegrowthofeachbacterialisolatewasquantifiedinmediainoculatedwithfour P destructans concentrationsestimatedusinghemocytometry(107,106, 105,and104conidia/ml).Afterthe P destructans suspensionwasdryandconidiafixedtothe plate,weusedapipettetoinoculatetheplateswith0.1 lofa108cfu/mlsuspensionofagiven bacterialisolateatthreeevenlyspacedpointsontheplate.Thebacterialsuspensionwaspreparedbysuspendingwholecoloniesin30%glycerol,andusinganinoculatinglooptosuspend thecolony.Eachtreatmentwasreplicatedninetimes,andculturesweregrownat9Cfor37 days.Zonesofinhibitionwerequantifiedbymeasuringthedistancefromtheedgeofthebacterialcolonytotheedgeofthevisible P destructans growtheveryotherday[ 31 ].Wealsomicroscopicallyexaminedthezonesofinhibitiononthefinaldayoftheexperiment(day43)to characterizetheeffectsofbacteriaonthegrowthof P destructans Inthesecondinhibitionassaywedeterminedtheabilityofeachbacterialisolatetoprevent growthof P destructans acrossaseriesofsixbacterialconcentrations.Eachbacterialisolate wasplatedfromcryobankedglycerolstockontoaPetridishwithSDAmediaandallowedto incubatefortwodaysat9Cbeforebeingaddedtoa30%sterileglycerolsuspension.Theconcentrationofeachisolatewasstandardizedbymakingserialten-folddilutionsoftheculturing stockandthencountingthenumberofcolonyformingunits(cfu)perml.Thebacteriaglycerol suspensionwasfrozenat-20Cwhilecalculatingtheconcentration.Eachstockwasstandardizedtothesameconcentrationof7.5x107cfu/mlusing30%glycerol.Weplated50 lofeach bacterialglyceroldilutiononSDAin60mmPetridishes.Weusedthreereplicatespertreatmentforbacterialconcentrations106,102,and101cfu/ml,andfivereplicatesfor BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 4/12

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concentrations105,104,and103cfu/ml.Forthecontrolplate,weadded50 lofsterile30% glycerolsolutiontotheplatesandtheninoculatedwith P destructans usingapinpointinoculation.Thediameterofthe P destructans colonywasmeasuredforatotalof42days.Measurementsweremadeeveryotherdayforthefirst14days,andthenonceeverysevendays thereafteruntiltheendoftheexperiment. Weusedcell-freesupernatantplatedonalawnof P destructans todetermineifanti-fungal compoundswerebeingproducedbythebacteriaintheinitialculture.Weusedculturesof freshbacteriagrowninisolation,andco-culturedwith P destructans ,inlysogenybroth.Cultureswerethencentrifugedfor30minutesandthesupernatantwasdrawnoff.Wetheninoculatedthreeplateswith50 Lofthesupernatantonalawnof P destructans usingthemethods describedabove.BacteriamotilityandchemotaxisBacteriamotilityexperimentswereconductedtoassesswhetherthe Pseudomonas isolatesfrom batspreferentiallymovetowards P destructans .A0.3%agarSDAmediawaspreparedanda sterileinoculatingloopwasdippedintoa7.5x107cfu/mlofbacterialsuspensionandthen stabbed~5cmintothesoftagarforallninebacterialisolates.Todeterminewhetherornotthe bacteriapreferentiallymovedtowards P destructans ,werepeatedthesamemethodsdescribed above,butincludedasmallcolonyof P destructans thatwasstabbedintothemediaonone sideofthetube.Thetubeswereincubatedfor1weekat10C,andthenstabswerevisuallyinspectedforsignsofwhetherbacteriamovedawayfromtheinitialstabormovedtowardsthe P destructans stab.StatisticalanalysisWefitlinearmixedeffectsmodels(functionglmerinpackagelme4[ 36 ]inRv.3.02[ 37 ])with dayasacategoricalrandomeffectandbacteriatypeandconcentrationasfixedeffectstoexaminetheinfluenceofbacteriatypeandserialdilutiononthezoneofinhibition(firstinhibition assay, S2Table )anddiameteroffungalcolony(secondinhibitionassay, S3Table ).Wefitfivea priorimodelsincludingadditiveandinteractiveeffects( S4Table )andcomparedmodelsusing Akaike Â’ sInformationCriterion(AIC)[ 38 ].Results Pre-screenforbacteriawithantiP destructans propertiesWeisolatedatotalof133bacterialmorphotypesfromthe40batsweswabbed.Fourisolates from E fuscus (from3bats)andtwoisolatesfromtwoindividual M lucifugus inhibited P destructans growthinstandardchallengeassays( Table1 ).Thesesixbacteriawereselectedfor furthertestinginthetwoinhibitionassays.Allwereidentifiedasmembersofthegenus Pseudomonas ,withfiveofthesixisolatesmostcloselyrelatedtothe Pseudomonasfluorescens group andtheotherisolate(PA6)beingmostcloselyrelatedto Pseudomonasabietaniphila (HF952541)( S1Table ).InhibitionassaysInthefirstinhibitionassay,bacterialcoloniesinitiallyexpandedquicklyandthenplateauedin size,withgrowthcontinuingforlongeratlowerinitialconcentrationsof P destructans inthe media( S1Fig ., S4Table ).Somebacterialisolatesformedmuchlargercoloniesthanothers, withPF1,PF2,andPF4formingthelargestcolonies( S1Fig .).Thesizeofbacterialcoloniesincreasedwithdecreasinginitialconcentrationsof P destructans ( S1Fig ., S4Table ). BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 5/12

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Zonesofinhibitioncouldnotbevisualizeduntil P destructans growthwasvisibleondays 9 – 11( Fig.1 ).Atthistime,zonesofinhibitionalreadydifferedsignificantlyamongbacterial isolatesandinitialconcentrations( Fig.2 ,S2Fig.,and S5Table ).Twobacterialisolates,PF1 andPF2,generatedlargerzonesofinhibitionacrossmostinitialconcentrationsof P destructans bytheendoftheexperiment( S2Table ).Threeisolates(PF1,PF2,andPF7)established twozones,onewheregrowthof P destructans wassuspendedimmediatelyupongermination ( Fig.1g ),andanotheroutsideofthiszonewheregrowthwasarrested,butonlyafterthemycelialmathadbeguntodevelop( Fig.1f ).Zonesofinhibitiononthelastdayoftheexperiment (day37)increasedwithincreasinginitialconcentrationsof P destructans forthe Pseudomonas isolatesshowingthestrongestinhibition( Fig.2 ;PF1,PF2,andPF4;allconcentrationslopes weresignificantlynegative,allp-values < 0.03).Fortheotherfour Pseudomonas isolates,the zoneofinhibitionwaseithervariableacrossconcentrations( Fig.2 ;PF3,PF5,PA6)orincreased withdecreasinginitial P destructans concentration(PF7;concentrationcoef.1.720.64, p=0.008).Twoisolates,PF1andPF2,out-performedthereference P fluorescens strains(PF7; PfA506 )atallinitialconcentrationswithatleastatwo-folddifferenceinzoneofinhibition ( Fig.2 ).Thetwocontrolbacteria( Chryseobacteriumsp andSphingomonassp ) andtheshaminoculatedcontrolproducednozonesofinhibition( Fig.2 ). Inthesecondinhibitionexperiment, P destructans grewoptimallyintheabsenceofbacteria,andonmediawithlowinitialconcentrationsofthecontrolbacteria( Fig.3 ).Bytheendof theexperiment,thesizeof P destructans coloniesdifferedbetweenbacterialisolatesandinitial concentrationsandtheeffectofbacterialisolatevariedamonginitialconcentrations( S3 and S6 Tables).Atthehighestinitialconcentration(106cells/ml),allbacteria(includingthetwocontrolbacteria)formedlawnsandallreducedgrowthof P destructans .Asthestartingconcentrationofthebacterialawndecreased,fewerisolatessignificantlyreducedthegrowthof P destructans .Atthethreehighestinitialbacterialconcentrations(106– 104cfu/ml),onlyisolates PF1,PF2,andPF5completelysuppressed P destructans growthforthedurationoftheexperiment(day42; S6Table ).Atthethreelowestinitialconcentrationsofthebacteria,wherethere wererelativelyfewcolonies,two Pseudomonas isolates,PF1andPF2performedsignificantly betterthanotherisolatesinreducing P destructans growthandpreventedP. destructans from growingforthefulldurationoftheexperiment( Fig.3 S6Table ).Inbothexperiments,isolates PF1andPF2producedthemaximumreductionofmycelialgrowthacrossallconcentrations, regardlessofthewaytheisolatesand P destructans wereco-cultured. Cell-freesupernatantdrawnfromliquidbacterialcultureshadnoeffectsonthegrowthof P destructans Pseudogymnoascusdestructans grewuniformlyacrossallplates,regardlessof whetherthesupernatantaddedtotheplateswasfrombacteriaco-culturedwith P destructans orculturedalone. Table1.Bacteriaisolatedfrom Myotislucifugus and Eptesicusfuscus usedinchallengeexperiments. GraphIDBatSpeciesCollectionCountyCollectionDatePhylogeneticaf liationMotile CHR1M lucifugus Ulster,NY8-Apr-12 Chryseobacteriumsp .No SPH2E fuscus Bath,VA29-Mar-12 Sphingomonassp .No PF1E fuscus Bath,VA28-Mar-12 Pseudomonassp .Yes PF2E fuscus Bath,VA29-Mar-12 Pseudomonassp .Yes PF3M lucifugus Highland,VA19-Mar-12 Pseudomonassp .Yes PF4M lucifugus Bath,VA29-Mar-12 Pseudomonassp .Yes PF5E fuscus Albany,NYJan-09 Pseudomonassp .Yes PA6E fuscus Bath,VA29-Mar-12 P abietaniphila Yes PF7 NANANA P uorescensA506 Yes doi:10.1371/journal.pone.0121329.t001 BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 6/12

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BacteriamotilityandchemotaxisAllseven Pseudomonas isolatesexhibitedsignsofmotilitybutnochemotaxistowards P destructans colonieswasobserved.Usingmicroscopy,twoofthe Pseudomonas isolates,PF1 andPF2,wereobserveddispersingalong P destructans hyphae.Thetwocontrolbacteria showednosignsofmotility( Table1 ). Fig1.Challengeplatesshowingtheinhibitionof Pseudogymnoascusdestructans Bacteriawereplatedwithaninitialstartingconcentrationof104cfu/ ml(PF2).Theplate(a)showsnovisible P destructans growthonday43,comparedtothe(b)controlplateshowinguninhibited P destructans colonygrowth atday43.(d)Thezonesofinhibitionproducedbyoneofthetopperforming P fluorescens isolates(PF2)comparedtotheshaminoculatedcontrol(c)anda widelyusedstrainof P fluorescens ,(e;PF7: Pf A506).Therearetwodistinctzonesofinhibitionproducedbythetopperformingstrain(asshowninpaneld) indicatedbythegreysolidcircleandthedashedgreycircle.Microscopicimagesoftheinnerandouterzonesareshowninpanels(f)and(g).Weusedgra m stainingtechniquestohelpbettervisualizeconidia(purple)andhyphae(pink)(scalebars,10 m).Withinthefirstzone,indicatedbythedarkring surroundingtheyellowbacteriacolony(PF2),thebacteriaeitherarrestedordelayedconidiagrowth,(g)whichcanbeseenbythesmallhyphaelexten sion fromtheconidia.Outsideofthisfirstzone,thegrowthof P destructans wasmuchmoreextensive(f),producingamycelialnetworkbeforeitsgrowth wasarrested. doi:10.1371/journal.pone.0121329.g001 BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 7/12

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Fig2.Firstinhibitionassaymeasuringthewidthofthezoneofinhibitionproducedbybacteriaonalawnof P destructans Thezonesofinhibition producedbybacterialisolateswheninoculatedonaplatewithfourconcentrationsof P destructans .Linesdenotedbythesameletterdonotdiffer significantlyonthelastdayoftheexperiment( S5Table ).CHRandSPHareisolatesinthegenus Chryseobacterium and Sphingomonas thatarenotknown toproduceantifungalcompounds.Thecontrolisaninoculationof30%glycerolstock.PF1-5,7andPA6areallisolatesinthegenus Pseudomonas doi:10.1371/journal.pone.0121329.g002 Fig3.Secondinhibitionassaymeasuringthediameterof P destructans coloniesgrownonalawnofbacteria. Pseudogymnoascusdestructans was platedwithninebacterialisolatesatsixdifferentconcentrations(highesttolowest,lefttoright).Linesdenotedbythesameletterdidnotdiffe rsignificantlyon thelastdayoftheexperiment.CHRandSPHareisolatesinthegenus Chryseobacterium and Sphingomonas thatarenotknowntoproduceantifungal compounds.TheControlisashaminoculationof30%glycerolstock.PF1-7andPA6areallisolatesinthegenus Pseudomonas doi:10.1371/journal.pone.0121329.g003 BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 8/12

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DiscussionAsthethreatofemerginginfectiousdiseasegrowswithincreasedglobaltravelandtrade[ 5 ], newwaysofmanagingwildlifediseasemustbeconsidered[ 8 ].Traditionally,fungalpathogens havebeenmanagedusingchemicalfungicides[ 39 ],buttoxicityeffectsonnon-targetorganisms,andapplicationchallengesmakesitdifficultforbroad-scaleuseonwildlifefungalpathogens[ 29 ].Theresultsfromourtwosetsofexperimentsdemonstratethat invivo ,bacteria culturedfrombatscaninhibitthegrowthof P destructans .Ourresultssuggestthataugmentationpriorto P destructans exposuremightreducecolonization,whereasbacterialaugmentationafterexposurecoulddisplace P destructans .Ourresultsalsosuggestthatakeychallenge forsuccessfultreatmentisapplyingbacteriasuchthattheywillpersistonbatskinathigh enoughconcentrationstolimit P destructans growthbelowlevelsthatcauselethaldisease. Thebacteriaweisolatedfrombats, Pseudomonasspp .,isubiquitousintheenvironmentand iswellknowntohaveanti-fungalproperties[ 40 ].Thegroupofbacteriathattheseisolateswere mostcloselyrelatedto, Pseudomonasfluorescens ,haspreviouslybeendetectedonseveralmammals(includingbats),aswellasamphibians,fish,andplants[ 41 – 44 ].Membersofthe P fluorescens groupareknowntoproduceasuiteofantifungalcompoundsthatcaninhibitmany plantfungalpathogens[ 45 ]aswellastheamphibianfungalpathogens, Batrachochytriumdendrobatidis [ 46 ].Somestrainsinthe P fluorescens grouparealsocapableofproducingmycolysingenzymesthatcancolonizethemyceliaandconidiaoffungirenderingthemnolonger viable[ 47 ].Allofour Pseudomonasspp .isolatesweremotile,whichmightallowthemtouse themycelialnetworksoffungalcoloniestoaidindispersalandcolonization[ 48 ].Allofthese attributesmake P fluorescens idealasaproposedcandidatetobetestedasabiologicalcontrol agentforreducinginfectionintensityandincreasingsurvivalofbatsexposedto P destructans Whethertheseantifungalbacteriathatnaturallyoccuronbatskincouldpartiallyexplain differencesinmortalityfromWNSamongpopulationsandspeciesiscurrentlyunknown.The isolateswithstrongestinhibitorypropertieswereculturedfrom E fuscus ,whichhaslowermortalityfromWNScomparedtootherspecies[ 2 ].However,wealsoisolatedtwostrainsof P fluorescens (PF3andPF4)thatshowedmoderate P .destructans inhibitionfrom M lucifugus ,a speciesthathassufferedseveremortalityfromWNS[ 2 20 ].Furtherresearchisneededtodeterminetherelativeabundance,distribution,andinhibitoryabilityof P fluorescens onwildbats andwhetherpresenceandabundanceof P fluorescens influencesdiseaseseverity. ThenextstepsindevelopingaprobioticforWNSshouldincludetesting, invivo ,oneor moreofthe P fluorescens strainsthatweisolatedagainst P destructans usingabatspeciesthat suffershighdiseasemortalityfromWNS,suchas M lucifugus M septentrionalis ,or Perimyotis subflavus [ 2 ].Studieswithlivehibernatingbatswilldeterminewhetherinteractionsobserved invitro havefunctionalsignificanceindiseaseoutcomesforbatspeciescurrentlythreatened byWNS.SupportingInformationS1Fig.Bacterialcolonysizeduringfirstinhibitionassay. Colonysizeofninebacterialisolatesgrownonplatesinoculatedwithfourdifferentconcentrationsof Pseudogymnoascus destructans withfungalconcentrationsdecreasingfromlefttoright.CHRandSPHareisolates inthegenus Chryseobacterium and Sphingomonas thatarenotknowntoproduceantifungal compounds.TheControlisashaminoculationof30%glycerolstock.PF1-7andPA6arebacterialisolatesinthegenus Pseudomonas (TIF) BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 9/12

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S1Table.BLASTresultsof16SrRNAsequencefromtheNationalCenterofBiologicalInformationdatabaseforthesixbacterialisolatedinthisstudyfrombats( Myotislucifugus and Eptesicusfuscus ). (DOCX) S2Table.AICvaluesforthefirstinhibitionassaymeasuringthezoneofinhibitionproducedbybacteriaonalawnof P destructans (DOCX) S3Table.AICvaluesforthesecondinhibitionassaymeasuringhowdifferentbacterialisolatesinfluencedthediameterofa P destructans colony. (DOCX) S4Table.AICvaluesforthefirstinhibitionassaymeasuringthediameterofbacteriacoloniesondifferentconcentrationsof P destructans (DOCX) S5Table.Coefficientsforlinearmodelsoftheinfluenceofninebacterialtreatmentsanda controlontheradiusofthezonesofinhibitionof P destructans producedbybacteriaat fourdifferentinitialconcentrationsofP.destructansonday37forthedatashownin Fig.2 (DOCX) S6Table.Coefficientsforlinearmodelsoftheinfluenceofninebacterialisolatesonthediameterof P destructans coloniesforeachbacterialconcentrationonday43forthedata shownin Fig.3 (DOCX)AcknowledgmentsWethankP.Peng,K.GambelandM.McCreeforlabassistance,W.StonefromtheNYSDEC forhelpwithcollectingpreliminarydata.WethankG.Gilbert,M.Brigham,andananonymous reviewerforhelpfulcommentsonthemanuscript.AuthorContributionsConceivedanddesignedtheexperiments:JRHAMKTLCWFFKEL.Performedtheexperiments:JRHTLCMHH.Analyzedthedata:JRHAMKKELWFF.Wrotethepaper:JRHAMK WFFKEL.References1. SkerrattLF,BergerL,SpeareR,CashinsS,McDonaldKR,etal.(2007)Spreadofchytridiomycosis hascausedtherapidglobaldeclineandextinctionoffrogs.Ecohealth4:125 – 134. 2. LangwigKE,FrickWF,BriedJT,HicksAC,KunzTH,etal.(2012)Sociality,density-dependenceand microclimatesdeterminethepersistenceofpopulationssufferingfromanovelfungaldisease,whitenosesyndrome.EcolLett15:1050 – 1057.doi: 10.1111/j.1461-0248.2012.01829.x PMID: 22747672 3. VanRiperCI,vanRiperSG,GoffML,LairdM(1986)Theepizootiologyandecologicalsignificanceof malariainHawaiianlandbirds.EcolMonogr56:327 – 344. 4. McCallumH(2012)Diseaseandthedynamicsofextinction.PhilosTransRSocLondBBiolSci367: 2828 – 2839.doi: 10.1098/rstb.2012.0224 PMID: 22966138 5. DaszakP,CunninghamAA,HyattAD(2000)Emerginginfectiousdiseasesofwildlife-threatstobiodiversityandhumanhealth.Science.287:443 – 449.PMID: 10642539 6. LangwigKE,VoylesJ,WilberMQ,FrickWF,MurrayK,etal.(2015)Contextdependentconservation responsestowildlifedisease.FrontEcolEnviron.doi: 10.1890/140241 BacteriaIsolatedfromBatsInhibits Pseudogymnoascusdestructans PLOSONE|DOI:10.1371/journal.pone.0121329April8,2015 10/12

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