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482 KONISHIETAL.
1.BroscI.BrOENG.，
filamentsandultimatelyformingsingleanddoublecigar- shaped cel ls at 30.0oC and 37.0oC incubated on semisyn- theticmedium(28).Bergetal.(29)investigatedthemor- phologicalchangesofL.pneumophilaincontinuousculture. Filamentousformsaproxim剖 ely100μmlongweresenat 37.00 C and 4 4.00 C. In the present study，most of the fila- mentousformsaproximately100μmlongwereobserved
over39.0-41.0oCforalthrestrainsincubatedonBCYEα
，-
ag訂 andinbroth，buttheOkinawastrainincubatedat37.50C inbrothwasanexception.Therehavebeenmanyreports thatbacterialelongation.wascloselyrelatedtothefunction ofconstitutionalenzymesforbacterialbinaryfision(30). Themetabolicactivityasociatedwithceldivisionislikely tobeafectedbyculturetemperature.Werneretal.(31) sugestedthatwhenPseudomonasaeruginosαisexposed totheantibioticcarbenicilin，celdivisionisblockedand growingcelsformfilaments.Theconstitutionalenzymesof thetwoclinicalisolatesusedinthes旬dyprobablykepttheir functionalintegriザ athigherculturetemperature(39.00C) thanATCC33152(37.00C).Theaveragelengthoftype1 strainATCC33152，clinicalisolateOkinawa02-01ex- ceded50μmwhenculturedattemperaturesfrom39.50Cto 4 4.00 C (range，4.50 C) and from 41.0oC to 43.0oC (range， 2.00C)，respectively.SinceL.pneumophilastrainsarelikely toformbiofilmstructureswhenbacteriallengthexceds 50J.lm，theculturetemperaturethatal10wstwoc1inicaliso- latestoformbiofilmisprobablyhigherthanthatofthetype stram.
Theheterogeneou~ structureofL.pneumophilabiofilm formedonglaswalsinBCYE-αbrothwasobservedand analyzedbyscaningele:ctronmicroscopy.Microscalegra- dientsinconcentrationofnutrients，oxygenandmetabolic productswereassumedtobeformedalongtheglaswal.l Theenvironmentaboveanair-liquidinterfaceisoxygen- richbutporinnutrients，andbelowtheair-liquidinterface itisoxygen-porbutrichinnutrients.HowdoesL.pneumQ-
philαtransportoxygenandnutrientsintobiofilm?Porous structuresareknowntobeliquidcariersbycapi1laryefec.t ThefilamentousformofL.pneumophilabiofilmresembles thispatem.Nutrientsareabsorbedbycapilaryefectfrom thebotomofthebiofilmartdtransportedintotheuperre- gionandacumulatedinsidethebiofilm.Theacumulation o
FIG 7. TheL.pneumophilastrain(clinicalisolate;Okinawa02- 01)wasculturedfor72h.Typicalresultsofbiofilmformedonthe glaswalsurfaceat42.0oC(a)andmagnifiedimages(b，c).Scaning electronmicrographswereobtainedforentireimage(d)，neartheair interface(e)，inthemidleofthesediment(f)，andattheliquidinter- face(g).
encesinthre-layeredstructurewererecognizedat42.0oC. At45.0oChobiofilmdevelopedinthethrestrains.Figures 7band1cshowthemidlebiofilmdevelopedontheglas walofOkinawa02-01strainat42.0oC.Theair-liquidin- terfaceseemedtobelocatedinthecenterofthebiofilm. Theuperedgeofthebiofilmwasadjacenttothethinuper biofilm姐 dtothesuroundingairinaglastube.Thelower edgewasatachedtothegranulatedlayerandwassoakedin BCYE白 αbroth.Theshapeoftheborderattheuperedge wasstraight，buttheloweredgewasacomplexsemicircular shape.Thedegreofbordercomplexitywasexpresedas thetatiooftheboundaη，lengthsoftheliquidsidetotheair side，andwasfound.tobe3.6.Figures7d-gshowatypical imageofthemidlebiofilmofL.pneumophilafoned拭 42.0oC obtained by a scan ning electron micrograph ofthe Okinawa02-01strain.Threlocationsinthebiofilmwere magnified:neartheairinterface(Fig.7e)，inthemidleof thebiofihn(Fig.7，)f andabovetheliquid(Fig.7g).The bioflmexhibitedatypicalfilamentousmorphology出atdid notchangemuchwiththelocationinthebiofilm.Theden- sityofL.pneumophilawasgreatestneartheairinterface， andlowestirtthemidleofthebiofilm.
DISCUSSION
Theaveragelengthoftype1strainATCC33152，ac1ini- calisolateOkinawa02-01wasgreatestatatempera旬 reof 37.50Cand39.0oC，respectively.Theseresultsindicatethat theclinicalisolateshadapotentialtoretaintheirlength withinarangeofト 10ドm athigherculturetemperatures thandidthetypestrain.Pineetal.(28)observedmarked changesincelsurface四 to-volumeratio，shape，andsize whiehcouldberelatedtotemperature，nutrients，andaera- tiOnconditionsforboththebacilaryandfilamentousfoロm ofL.pneumophila.Theynotedalargemassoffilamentsor chainofbaciliinthelogarithmicphasebreakingintoshorter
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