JOURNALOFCLINICALMICROBIOLOGY,Sept.1989,p.1961-19640095-1137/89!091961 - PDF document

JOURNALOFCLINICALMICROBIOLOGY,Sept.1989,p.1961-19640095-1137/89!091961-04$02.O0/0Copyright(O1989,AmericanSocietyforMicrobiologyPlaqueAssayforVirulentLegionellapneumophilatRACHELC.FERNANDEZ,SPENCERH.S.LEE,*DAVIDHALDANE,ROBERTSUMARAH,ANDKENNETHR.ROZEEDepartmentofMicrobiology,DalhousieUniversity,VictoriaGeneralHospital,Halifax,NovaScotiaB3H4H7,CanadaReceived6March1989/Accepted9May1989MethodsofassessingvirulenceofLegionellapneumophila,theetiologicagentofLegionnairesdisease,includetheinfectionofguineapigs,fertilechickeneggs,andmammalianandprotozoancellcultures.Guineapigassays,inparticular,areexpensive,laborious,orunsuitableforroutinescreeningofLegionellaisolates.WehavedevelopedavirulenceassaythatrequirestheenumerationofviruslikeplaqueswhicharetheresultofvirulentL.pneumophilainfectingmouseL929cells.EachplaqueistheconsequenceoftheinitialinfectionofanLcellwithasinglebacterium.AnonvirulentmutantderivedfromtheserialpassageofvirulentL.pneumophilaonMueller-HintonagarfailstosurvivewithinLcellsandconsequentlyfailstoproduceplaques.AlthoughLegionellapneumophila,theetiologicagentofLegionnairesdisease(14),isubiquitousinwater,studieshaveshownthatvariousenvironmentalisolatesdifferedinvirulence.IsolatesofL.pneumophilafromhospitalpotablewaterhavebeenassociatedwithdifferencesinattackratesinpatients(18),in50%lethaldosesand50%infectivedosesinguineapigs(1),andintheabilitytosurvivethekillingeffectsofserum(17).Furthermore,virulenceofclinicalisolateshasbeenlostbycultivationonbacteriologicalmedia(6,13)orhasbeenregainedinlaboratoryanimalsandcellsinvitro(6,19).However,arecentstudy(2)hassuggestedthattheconversionofL.pneumophilafromvirulenttoavirulentformsafterprolonged_ultivationonsupplementedMueller-Hintonagarisaone-wayphenomenon.Inassessingthevirulenceofteststrains,itisconventionaltouseguineapigassaystodeterminethe50%lethaldose(1,5,6,7,13,14),buttheseareexpensive,timeconsuming,andoftennotfeasibleinahospitaldiagnosticlaboratorysetting.ComparisonsofvirulenceofLegionellastrainsalsohavebeenmadebyusingfertilechickeneggs(3,6,13),monocyte-macrophages(8,10),andTetrahymenapyriformis(7).How-ever,thesesystemsdonoteasilylendthemselvestotheroutinescreeningoflargenumbersofstrains.Wehavedevelopedaninvitrovirulenceassaythatiscomparativelyrapid,relativelyinexpensive,andeasilyamenabletoroutinetesting.ThisassayutilizesthedevelopmentanddetectionofPFUafter4daysofinfectionofmouseL929cellswithL.pneumophila.MATERIALSANDMETHODSOrganisms.Aclinicalisolate(2064)fromthesputumofapatientwithLegionnairesdiseasewasusedforthevirulencestudies.ThisstrainwasserologicallyidentifiedasL.pneu-mophilaserogroup1(OLDA).Itwaspassagedtwiceonbufferedcharcoal-yeastextract(BCYE)agarandstoredat-70°Cin20%glycerol.Forexperimentalpurposes,strain2064wasseriallypassagedfivetimesonMueller-HintonagarsupplementedwithhemoglobinandIsoVitaleX(BBLMicro-biologySystems,Cockeysville,Md.)(3),atwhichtimeacolony(designated2064M)waspickedandastockculturewaspreparedonBCYEagar.Thiswasstoredat-70°Cas*Correspondingauthor.tWededicatethispapertothememoryofC.E.vanRooyen,formerHead,DepartmentofMicrobiology,andProfessorEmeritus.describedabove.Whenneeded,theorganismsweregrownfor2daysonBCYEagarat37°CinahumidifiedC02incubator.Celllines.MouseL929cells(ATCCcloneCCLI)weregrownandmaintainedinminimumessentialmediumsupple-mentedwith10%heat-inactivatedfetalbovineserum(FlowLaboratories,Mississauga,Ontario,Canada),100Uofpen-icillinperml,and50p.gofstreptomycinperml.HumanA549cells(lungcarcinomacellsobtainedfromF.Jay,DepartmentofMedicalMicrobiology,UniversityofManitoba,Winnipeg,Canada)weregrownandmaintainedundersimilarconditionsbutwithRPMI1640medium.Virulencedetermination.Unlessotherwiseindicated,10-folddilutionsof2064and2064Mrangingfrom109to103organismspermlwereusedtoinoculatetissueculturecells,fertilechickeneggs,andguineapigsasdescribedbelow.Ineverycase,furtherdilutionsweremade,andaliquotsofeachdilutionwereplatedonBCYEagartodeterminetheinitialsizeoftheinoculum.(i)Cellculture.A549orL929cellswereseededin96-wellplates(Costar,Cambridge,Mass.)ataconcentrationof105cellsperwellin0.1mlofculturemedium(CM)consistingofantibiotic-freeRPMI1640orminimumessentialmedium,respectively,withadded1%fetalbovineserum.After3hofincubation,duplicatemonolayerswereinoculatedwith2064or2064Min0.1mlofCM.Threedayslaterthemonolayerswereexaminedunderaninvertedmicroscopeforanydis-cernablecytopathiceffect.Theywerethenstainedfor2hat37°Cwith0.01%neutralred(50,ulperwell),adyeordinarilytakenuponlybyviablecells(12).Neutralred-stainedcellswerewashedtwiceinphosphate-bufferedsaline,andthedyewasthenelutedintothewellwith100,ulofa1:1solutionofethanoland0.1MNaH2PO4(12).ThecolorintheplateswassubsequentlyassayedinaMicroelisareaderat570nm,andtheresultingopticaldensityreadingswereplottedagainstthenumberoforganisms.Fromthis,thenumberoforgan-ismscausinga50%reductionofneutralreduptakebycontrol(uninfected)cellswasdetermined.(ài)Embryonatedeggs.Yolksacsof8-day-oldembryo-natedWhiteLeghornheneggswereinfected(6eggsperdilution)with0.2mloftheorganisms(14).Theeggswerecandleddailyfor8daystodeterminethenumberofembryoskilled.The50%egginfectivitydosewascalculatedaccord-ingtothemethodofKarber(11).(iii)Guineapigs.MaleHartleyguineapigs(HighOak1961Vol.27,No.9 1962FERNANDEZETAL.TABLE1.ComparisonofvirulenceofL.pneumophilastrains2064and2064Mincellculture,eggs,andguineapigsCFU/mlL.pneumophilaCellstrainculture,EID50bLDpsoLD50020643x1051x1061x1062064M�6x1081x108�ix109aThenumberoforganismspermillilitercausinga50%reductioninneutralreduptakecomparedwithuptakebyuninfectedA549cells.LD50,50%Lethaldose.bAssayedbytheinoculationofyolksacsof8-day-oldchickeneggs.EID50,50%Egginfectivitydose.cAssayedbyintraperitonealinoculationofmaleguineapigsweighing250to300g.LD50,50%Lethaldose.Ranch,Goodwood,Ontario,Canada),eachweighing250to300g,wereinfectedintraperitoneally(4animalsperdilution)with1mloftheorganisminsaline(7),andthe50%lethaldosewasdeterminedasdescribedabove.Plaqueassay.MouseL929cellswereplatedin24-wellplates(Costar,Cambridge,Mass.)inCMataconcentrationof5xi05perwell.Afterovernightincubation,oralterna-tively,afterallowingthecellstoadherefor2to3h,theresultingconfluentmonolayerswereinfectedinduplicatewith,perwell,0.5mloftheorganismsinCM.Aftera60-minadsorptionperiodat37°C,theinoculumwasremovedbyaspiration,andthemonolayerswerewashedthreetimeswithCMcontaining50,ugofgentamicinsulfateperml(washmedium)(3,9).Thecultureswerethenincubatedforanadditional60minin,perwell,1mlofgentamicinwashmedium,afterwhichthemonolayerswerewashedthreetimeswithCMalonepriortoaddinga1mlperwelloverlayof0.6%agaroseinantibiotic-freeminimalessentialmediumcontaining1%fetalbovineserum.Themonolayerswereincubatedfor4daysat37°Candthenfixedwith10%Formalin.TheagaroseoverlaywasAremoved,andthefixedmonolayerswerestainedwith1%crystalvioletin20%ethanol.Theresultingplaquesweremacroscopicallyenumerated.Immunofluorescence.DilutionsoftheorganismsinCMwereaddedtoL929cellsinLabTekchamber/slides(Can-Lab,Mississauga,Ontario,Canada).After60minat37°C,bacteriawerewashedandthemonolayerswerefixedwithFormalinandstainedbyindirectimmunofluorescence(5)witharabbitanti-Legionellaantibodypreparedasprevi-ouslydescribed(15)andafluoresceinisothiocyanate-conjugatedswineanti-rabbitantibody(Dako,DimensionLaboratories,Mississauga,Ontario,Canada).CultureswereobservedmicroscopicallybyusingaNikonepifluorescencemicroscope.RESULTSANDDISCUSSIONTable1showsacomparisonofthevirulenceof2064and2064Minguineapigs,eggs,andtissueculture.Onthebasisoftheseassays,2064Mwasdeemedtobeavirulent.Atamultiplicityofinfection(MOI)greaterthan10,2064producedacompletedestructionofA549andL929cells.AtlowerMOIs,thecytopathiceffectonA549cellspresentedasageneralizedformofcelldestruction,whereasthecyto-pathiceffectproducedby2064onL929cellswasfocalandresembledthatofviralplaques(Fig.1).Therewasnoovertdamagetothemonolayerinfectedwith2064MevenatanMOIof1,000.Sincegentamicinkillsallbacteriaunabletopenetratemammaliancells(3,9),onlythosebacteriawhichhadsuccessfullyinfectedtheL929cellseventuallycausedthisfocaldestructionoftheL-cellmonolayer.Confirmingpreviousreports,itshouldbenotedthatL.pneumophiladoesnotgrowinthiscell-freemedium(16).Todeterminethenumberofbacterianecessarytoproduceasingleplaque,serialtwofolddilutionsoforganismsatastartingMOIof0.01wereplatedontheL929cellsinamannercomparabletotheDulbecco(4)dose-responsecurveofvirusplaqueassays.Theresultingnumberofplaques,BFIG.1.(A)L.pneumophilaplaquesseenonL929monolayers.L929monolayerswereinfectedwithL.pneumophilafor60min.Theinoculumwasremoved,washed,andreplacedwithgentamicinsulfateforanadditional60min.Afterthegentamicinwaswashedaway,themonolayerswereoverlaidwithminimalessentialmediumcontainingagaroseandwereincubatedfor4days.ThemonolayerswerethenfixedwithFormalin;theagaroseoverlaywasremoved,andthecellsweresubsequentlystainedwithcrystalviolet.(B)Mock-infectedL929monolayers.J.CLIN.MICROBIOL. PLAQUEASSAYFORVIRULENTL.PNEUMOPHILA1963z300z°200O'o-100-z00.040.070.130.250.51.0RELATIVEL.PNEUMOPHILACONCENTRATIONFIG.2.PlaqueformationonL929monolayers.L929cellswereinfectedwithtwofolddilutionsofL.pneumophila,startingwithaconcentrationof104organismspermlasdescribedinthelegendtoFig.1.whenplottedagainstthedilutions,renderedalinearrelation-ship(Fig.2),indicatingthattheinitialinfectionwithasinglebacteriumwassufficienttoproduceasingleplaque.Toinvestigatewhetheranenhancedcapacitytobindtohostcellswasrelatedtoplaqueformation,dilutionsof2064and2064MwereincubatedwithL929cells.Indirectimmu-nofluorescenceshowedthat,atsimilarconcentrations,bothstrainswereequallycapableofbindingtotheL929cells(Fig.3).Todeterminethefateof2064Mafterbinding,L929cellswereinfectedwith2064Mfor60minatanMOIof0.1,accordingtothemethodoutlinedfortheplaqueassay,exceptthattheagaroseoverlaywasomitted.Instead,0.5mloftheculturemediumwasaddedtotheinfectedcells.Toestablishtheinitialnumberof2064Mthathadpenetratedthemonolayers,theculturemediumwascollectedandreplacedwith0.5mlof0.1%TritonX-100inphosphate-bufferedsaline(10).Afterlysisofthemonolayers,thelysatewasaddedtothepreviouslycollectedculturemediumand0.1mlofthismixturewasplatedonBCYEagarplates.Thisgaveriseto500CFU/ml.Theinfectedmonolayerswerethenlysedasdescribedaboveat24,48,72,and96hpostinfec-tion.TheresultingnumberofCFUpermldroppedfrom500to10by24handremainedassuch,indicatingthat2064MwasrenderednonviableintheL929cells.Thus,theplaquesrepresentboththeabilityoftheorganismtobindandpenetratethehostcellsanditscapacitytosurviveandmultiplyintracellularly.ThelackofPFUinaLegionellaplaqueassaycannotbyitselfdiscriminatebetweentheinabilityoftheorganismtoeitherbindortosurviveandmultiplyintracellularly.However,thisassaycaneffectivelyseparatevirulentandnonvirulentorganisms.Recently,Dreyfus(3)describedaningestionassayforL.pneumophilawhichusedHeLacellsthatwereinfectedandoverlaidwithmoltenBCYEagar.OrganismswhichhadpenetratedtheHeLacellswereidentifiedascolonieswhichgrewbeneaththeBCYEagar.WhenwecomparedourplaqueassayonL929cellswiththeassaydescribedbyDreyfusonHeLacells,weobservedthatserialtwofolddilutionsyielded79,41,and19plaques,asopposedto42,20,and9coloniesatanequivalentMOI.Thus,theplaqueassaywasamoresensitiveindicatorofvirulencebyafactorof2.ThisappearedtoderivefromtheclarityandeaseofenumerationofplaquesinL929cells,ascomparedwithcountingthevarious-sizedbacterialcolonieswhichwerefoundrandomlyonthesurfaceofandbeneaththeBCYEagarintheDreyfusassay.Forreasonsyettobedetermined,thefocalnatureofthecytopathologyidentifiableasplaquesinL929cellsisuniqueandquiteunlikethegeneralizeddestructioncausedbyLegionellainfectionofVero,MRC-5,HEp-2,andHeLacells(16)orA549cells.IIFIG.3.ImmunofluorescencestainingofL.pneumophilashowingthebindingof2064(A)and2064M(B)toL929cells.L929monolayerswereinfectedwithequalnumbers(MOIofapproximately100)of2064or2064Mfor60min.Aftertheinoculawereremoved,thecellswerewashed,fixedwithFormalin,andstainedbyindirectimmunofluorescence.VOL.27,1989 1964FERNANDEZETAL.Ifvirulencemaybedescribedastheabilitytoinvadeintracellularly,thisL929-plaqueassayrepresentsarepro-ducible,simple,yetsensitiveassayforvirulenceinL.pneumophila.Thisphenotypicmeasurecorrelateswellwithotherassaysofvirulence,suchastheabilitytokillguineapigsorinfectembryonatedeggs,butcomparedwiththeuseofguineapigs,theplaqueassayismuchlessexpensiveandlesslaborintensive.Additionally,whiletheplaqueassaymaynotcompletelyreplaceconventionalassays,itdoesprovideameansforrapidlyscreeningforvirulence,aswellasofferingamethodfortheselectionandpurificationofvirulentL.pneumophila.ACKNOWLEDGMENTThisworkwassupportedinpartbyagrant(MT-1615)fromtheMedicalResearchCouncil,Canada.LITERATURECITED1.Bollin,G.E.,J.F.Plouffe,M.F.Para,andR.B.Prior.1985.DifferenceinvirulenceofenvironmentalisolatesofLegionellapneumophila.J.Clin.Microbiol.21:674-677.2.Catrenich,C.E.,andW.Johnson.1988.VirulenceconversionofLegionellapneumophila:aone-wayphenomenon.Infect.Immun.56:3121-3125.3.Dreyfus,L.A.1987.VirulenceassociatedingestionofLegion-ellapneumophilabyHeLacells.Microb.Pathog.3:45-52.4.Dulbecco,R.1952.Productionofplaquesinmonolayertissueculturesbysingleparticlesofananimalvirus.Proc.Natl.Acad.Sci.USA38:747-752.5.Edelstein,P.H.,K.B.Beer,andE.D.DeBoynton.1987.InfluenceofgrowthtemperatureonvirulenceofLegionellapneumophila.Infect.Immun.55:2701-2705.6.Elliott,J.A.,andW.Johnson.1982.VirulenceconversionofLegionellapneumophilaserogroup1bypassageinguineapigsandembryonatedeggs.Infect.Immun.35:943-946.7.Fields,B.S.,J.M.Barbaree,E.B.Shotts,Jr.,J.C.Feeley,W.E.Morrill,G.N.Sanden,andM.J.Dykstra.1986.Compar-isonofguineapigandprotozoanmodelsfordeterminingviru-lenceofLegionellaspecies.Infect.Immun.53:553-559.8.Horwitz,M.A.1987.CharacterizationofavirulentmutantLegionellapneumophilathatsurvivebutdonotmultiplywithinhumanmonocytes.J.Exp.Med.166:1310-1328.9.Isberg,R.R.,andS.Falkow.1985.AsinglegeneticlocusencodedbyYersiniapseudotuberculosispermitsinvasionofculturedanimalcellsbyEscherichiacoliK-12.Nature(London)317:262-264.10.Jacobs,R.F.,R.M.Locksley,C.B.Wilson,J.E.Haas,andS.J.Klebanoif.1984.Interactionofprimatealveolarmacro-phagesandLegionellapneumophila.J.Clin.Invest.73:1515-1523.11.Karber,G.1931.BeitragzurkollektivenBehandlungpharma-kologischerReihenversuche.Arch.Exp.Pathol.Pharmakol.162:480-487.12.Katz,L.J.,S.H.S.Lee,andK.R.Rozee.1974.Interferon-mediatedlysisofL-cellsbyvesicularstomatitisvirus.Can.J.Microbiol.20:1077-1083.13.McDade,J.E.,andC.C.Shepard.1979.VirulenttoavirulentconversionofLegionnaires'diseasebacterium(Legionellapneumophila)-itseffectonisolationtechniques.J.Infect.Dis.139:707-711.14.McDade,J.E.,C.C.Shepard,D.W.Fraser,T.R.Tsai,M.A.Redus,W.R.Dowdle,andtheLaboratoryInvestigationTeam.1977.Isolationofabacteriumanddemonstrationofitsroleinotherrespiratorydisease.N.Engl.J.Med.297:1197-1203.15.McKinney,R.M.,B.M.Thomason,P.P.Harris,L.Thacker,K.R.Lewallen,H.W.Wilkinson,G.A.Herbert,andC.W.Moss.1979.RecognitionofanewserogroupofLegionnairesdiseasebacterium.J.Clin.Microbiol.9:103-107.16.Oldham,L.J.,andF.G.Rodgers.1985.Adhesion,penetrationandintracellularreplicationofLegionellapneumophila:aninvitromodelofpathogenesis.J.Gen.Microbiol.131:697-706.17.Plouffe,J.F.,M.F.Para,andK.A.Fuller.1985.SerumbactericidalactivityagainstLegionellapneumophila.J.Clin.Microbiol.22:863-864.18.Plouffe,J.E.,M.F.Para,W.E.Maher,B.Hackman,andL.Webster.1983.SubtypesofLegionellapneumophilaserogroup1associatedwithdifferentattackrates.Lancetii:649-650.19.Wong,M.C.,W.L.Peacock,Jr.,R.M.McKinney,andK.-H.Wong.1981.Legionellapneumophila:avirulenttovirulentconversionthroughpassageinculturedhumanembryoniclungfibroblasts.Curr.Microbiol.5:31-34.J.CLIN.MICROBIOL.