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JOURNALOFBroSCIENCEANDBroENGINEERlNG 。206，TheSocietyforBiotechnology，Japan Vo.I101， ~0. 6，478-484.206
DOI:10.1263/jb.101.478
Inf1uenceofTemperatureonGrowthofLegionelapneumophila BiofilmDeterminedbyPreciseTemperatureGradientIncubator
TadashiKonishV*TetsuYamashiro，2MichioKoide，3andAkiraNishizono1
FacultyofM診dicine，OitaUniversity，1dai-gaoka，Yufu，Oita879-593，Japan，IlnstituteofTropicalMedicine， NagasakiUniversi初 1-12-4Sakamoto，Nagasaki852-8523，Japan2andFacultyofMedicine，
UniversityoftheRyuわJUS，1Senbaru，Nishihara，Okinawa903-0213，Japan3 Received27December205/Acepted2March206
Bacterialgrowthisinfluencedbyseveraldiferentcultureconditions.Temperatureisoneofan esentialcomponentwhichregulatesbacterialgrowthandtheirmorphology.Theinfluenceof temperatureonthelengthofbacteriawasinvestigatedinbrothandonagarinatemperature
0 rangefrom30.0oCto47.0oCin0.5Cstepsusinganewlydevelopedtemperaturegradientincuba-
to工 Theincubatorisabletoreachasettemperaturewithin2handmaintaintemperatureasacu-
ratel;ls:f:0.1oCofthesettemperature.ThreeLegionelαpneumophilaserotype1strainswereincu-
batedfor48hinBCYE-αagaratvariouste~peratures rangingfrom30.0oCto48.0oCandlength
ofbacteriagrownateachtemperaturewasmicroscopicalymeasured.Abilityofbacteriatomul-
tiplyatagiventemperaturewasalsodete;r:mined.L.pneumophilaserotype1strainsATCC33152，
aclinicalisolateOkinawa02・01weregoingtoelongatetolongerthan10J1m whencultured
higherthanat39.50C andat41.50C，respectively.Eachstrainwasunabletomultiplywhencul- o
turedhigherthanat4.20C(ATCC3152)orat4.0C(Okinawa02-01).Thosedatawouldpro- videinsightsforestablishingregulationsintermsofmaintaininghotwatertemperatureinafacil- itywhereacirculatinghotwatersuply-systemisavailableandcontaminationwithLegionela sp.islikelytohappen.
[Keywords:Legionelapneumophila，biofilm，temperaturegradientincubator，celelongation]
BacteriaofthegenusLegionelaaregram-negative，aero目 bic，androd-shapedwithoneormorepolarf1agela(1，2). Legionelapneumophilaisconsideredtobeanimportant etiologicmicrobialagentoflowerrespiratorytractinfec- tionsintheUSorinWestemEurope，acountingfor2%to 8%ofalcasesofcomunity-acquiredpneumonia，second onlyinimportancetoStreptococuspneumonia(3).The numberofLegionelasp.asociatedwithpneumoniawas rarelyreportedinJapanuntilApril19，whenanewLaw ConcemingthePreventionofInfectiousDiseasesandPatients withInfectiousDiseases(NewLawonInfectiousDiseases) wasenacted.ThelawclasifiedLegionelapneumoniaasa nationalynotifiableinfectiousdisease，withmandatoryre- portingofeverycaseofLegioneUαpneumoniatotheMinistry ofPublicHealthandWelfareviaaprefecturalhea1thcenter. ThenumberofreportedLegionelap:rieumoniacaseshas sinceincreaseddramaticaly:53casesin19，151casesin 2000，and83casesin2001.In2002，anoutbreakoflegion- elosisocuredatapublicbathouseinMiyazaki，Japan.In theoutbreak，32caseswerelaboratoryconfirmedasLegion- elapneumoniaand263casesweresuspect.
Legionela.sp.arecommonlypresentinnaturalandman madeaquaticenvironments(4-6).Thebacteriaproliferate instagnantwarmwater，asfoundincolingtowers，certain
*Corespondingauthor.e-mail:konishi@med.oita-u.ac.jp phone:+81-(0)975-586-5712fax:+81-(0)975-586-5719
plumbingsystemsandevaporativecondensersoflargeair- conditioningsystems.Outbreaksoflegionelosishaveocur- redwhenpeoplehaveinhaledaerosolsfromwatersources contaminatedwithLegionelabacteria(7).Inthesecondi- tions，thebacterialcountwouldreach103-106cfuml-1，much higherthanthatseninthenaturalenvironmen.tIncirculat- ingwatersystems，legionelaearemost企equentlydetected inbiofilmsinplumbingfixtures(5，8-10).Biofilmhasapo- tentialroleasavehic1etoconveylargenumbersofembed- dedbacteria，causingpneumoniawhenaerosoldropletscon- tainingfragmeptsofbiofilmareinhaledbyhumans(1).In f10wingplumbingsystems，temperaturegradientsareformed overasolidwalduetothetemperaturediferencebetwen hotwaterandtheambienteI1vironmen.tAsscale(suchas silicacompounds)，debris，biofilmorsedimentadheretothe pipewal，radialtemperaturegradientsareformedinsidethe biofilm.Variousdisinfectionmeasurestopreventlegionel- losishavebeenused(12-15).Legionelaearesensitiveto tempera加 re(16-21)andchlorine.Legionel laegrowpo orly attemperaturesbelow200C orabove500C andareeasily kil1edattemperaturesabove60oC.Inhospitalorpublic watersystemsLegionelaecanbekiledbyraisingthetem- peratureofthehotwatersuplyto700Cto800C(2，23).
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InJapan，governmentregulationsrecommendanoperating o
temperaturerangingfrom500Cto60C.Legionel加 佐e alsosusceptibletochlorine-andbromine-containingcom- pounds，ozone，heavymetalions，andultravioletlight(21，
