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JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2006, The Society for Biotechnology, Japan

Vol. 101, No. 6, 478–484. 2006

DOI: 10.1263/jbb.101.478

Influence of Temperature on Growth of Legionella pneumophilaBiofilm Determined by Precise Temperature Gradient Incubator

Tadashi Konishi,1* Tetsu Yamashiro,2 Michio Koide,3 and Akira Nishizono1

Faculty of Medicine, Oita University, 1 Idai-gaoka, Yufu, Oita 879-5593, Japan,1 Institute of Tropical Medicine,Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan,2and Faculty of Medicine,

University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan3

Received 27 December 2005/Accepted 22 March 2006

Bacterial growth is influenced by several different culture conditions. Temperature is one of anessential component which regulates bacterial growth and their morphology. The influence oftemperature on the length of bacteria was investigated in broth and on agar in a temperaturerange from 30.0°C to 47.0°C in 0.5°C steps using a newly developed temperature gradient incuba-tor. The incubator is able to reach a set temperature within 2 h and maintain temperature as accu-rate as ±0.1°C of the set temperature. Three Legionella pneumophila serotype 1 strains were incu-bated for 48 h in BCYE-α agar at various temperatures ranging from 30.0°C to 48.0°C and lengthof bacteria grown at each temperature was microscopically measured. Ability of bacteria to mul-tiply at a given temperature was also determined. L. pneumophila serotype 1 strains ATCC 33152,a clinical isolate Okinawa 02-001 were going to elongate to longer than 100 µm when culturedhigher than at 39.5°C and at 41.5°C, respectively. Each strain was unable to multiply when cul-tured higher than at 44.2°C (ATCC 33152) or at 44.0°C (Okinawa 02-001). Those data would pro-vide insights for establishing regulations in terms of maintaining hot water temperature in a facil-ity where a circulating hot water supply-system is available and contamination with Legionellaspp. is likely to happen.

[Key words: Legionella pneumophila, biofilm, temperature gradient incubator, cell elongation]

Bacteria of the genus Legionella are gram-negative, aero-bic, and rod-shaped with one or more polar flagella (1, 2).Legionella pneumophila is considered to be an importantetiologic microbial agent of lower respiratory tract infec-tions in the US or in Western Europe, accounting for 2% to8% of all cases of community-acquired pneumonia, secondonly in importance to Streptococcus pneumonia (3). Thenumber of Legionella spp. associated with pneumonia wasrarely reported in Japan until April 1999, when a new LawConcerning the Prevention of Infectious Diseases and Patientswith Infectious Diseases (New Law on Infectious Diseases)was enacted. The law classified Legionella pneumonia as anationally notifiable infectious disease, with mandatory re-porting of every case of Legionella pneumonia to the Ministryof Public Health and Welfare via a prefectural health center.The number of reported Legionella pneumonia cases hassince increased dramatically: 53 cases in 1999, 151 cases in2000, and 83 cases in 2001. In 2002, an outbreak of legion-ellosis occurred at a public bathhouse in Miyazaki, Japan. Inthe outbreak, 32 cases were laboratory confirmed as Legion-ella pneumonia and 263 cases were suspect.

Legionella spp. are commonly present in natural and manmade aquatic environments (4–6). The bacteria proliferatein stagnant warm water, as found in cooling towers, certain

plumbing systems and evaporative condensers of large air-conditioning systems. Outbreaks of legionellosis have occur-red when people have inhaled aerosols from water sourcescontaminated with Legionella bacteria (7). In these condi-tions, the bacterial count would reach 103–106 cfu ml–1, muchhigher than that seen in the natural environment. In circulat-ing water systems, legionellae are most frequently detectedin biofilms in plumbing fixtures (5, 8–10). Biofilm has a po-tential role as a vehicle to convey large numbers of embed-ded bacteria, causing pneumonia when aerosol droplets con-taining fragments of biofilm are inhaled by humans (11). Inflowing plumbing systems, temperature gradients are formedover a solid wall due to the temperature difference betweenhot water and the ambient environment. As scale (such assilica compounds), debris, biofilm or sediment adhere to thepipe wall, radial temperature gradients are formed inside thebiofilm. Various disinfection measures to prevent legionel-losis have been used (12–15). Legionellae are sensitive totemperature (16–21) and chlorine. Legionellae grow poorlyat temperatures below 20°C or above 50°C and are easilykilled at temperatures above 60°C. In hospital or publicwater systems Legionellae can be killed by raising the tem-perature of the hot water supply to 70°C to 80°C (22, 23).In Japan, government regulations recommend an operatingtemperature ranging from 50°C to 60°C. Legionellae arealso susceptible to chlorine- and bromine-containing com-pounds, ozone, heavy metal ions, and ultraviolet light (21,

* Corresponding author. e-mail: konishi@med.oita-u.ac.jpphone: +81-(0)975-586-5712 fax: +81-(0)975-586-5719

TEMPERATURE ON GROWTH OF LEGIONELLA BIOFILMVOL. 101, 2006 479

23–25). Hyperchlorination (sodium and calcium hypochlo-rite, chlorine dioxide), thermal eradication (superheating andflushing) and copper/silver ionization are widely used in hos-pital or public water systems. However, these measures arenot always efficient because of biofilms and their heteroge-neous features. No previous studies (11, 26, 27) on morpho-logical changes of L. pneumophila by cultivation tempera-ture clarified the quantitative relationship between cultiva-tion temperature and length of the strain. This paper showsthe length of three strains of L. pneumophila (a type strainATCC 33152, two clinical isolates; Okinawa 02-001 andNagasaki 80-045) at temperature ranging from 30.0°C to47.0°C at intervals of 0.5°C determined by newly developedprecise temperature gradient incubator. Viability of bacteriaat particular temperature was also determined. Those dataobtained from a setting with precise temperature regulatingmodule would provide insights for introducing a guidelinefor regulating hot water temperature in a plumbing pipes ofhot water supply systems.

MATERIALS AND METHODS

Design and assembly of the temperature gradient incubator

apparatus (TGI) A bacterial incubator with an accurate tem-perature gradient control was fabricated for this study (Figs. 1 and2). Ten aluminum blocks (80×80×25 mm) with cylindrical cham-bers (17 mm diameter and 85 mm depth) holding a glass test tubefor incubating L. pneumophila were placed side by side in a row togive 10 different culture temperatures. Heat sources were appliedto the aluminum blocks by two constant temperature bath circu-lators (Thermo NESLAB RTE-7; Thermo Electron Corporation,Newington, NH, USA) set at different temperatures using waterjackets placed at both ends of the row of aluminum blocks. Thewater jackets were made of brass, and contained a pectinated brasssheet to provide a higher heat transfer rate. Each aluminum blockwas separated by a silicon sheet that allowed a constant stepped tem-perature gradient to be attained. The module of aluminum blockswith water jackets was wrapped with fiberglass fabric to minimizeheat loss. Temperatures outside and inside a test tube wall weremeasured by standard thermocouples (Chromel-Alumel sheath type,1 mm diameter, THERMIC TMB-KS10/316L-10; Yamato Industry,Tokyo) and a standard thermometer (no. 20907; Japan MeasuringInstrument Industrial Incorporated Company, Tokyo), respectively,and compared. Temperatures measured by the standard thermocou-ples were monitored by a digital multimeter (Keithley model 2001TCSCAN, model 2001; Keithley Instruments, Cleveland, OH, USA)and recorded in a personal computer.

Bacterial strains and inoculation Three strains of L. pneu-mophila serogroup 1 were employed; a type strain and two clinicalisolates. A type strain of L. pneumophila serogroup 1 33152 wasobtained from the American Type of Culture Collection (ATCC).Two clinical isolates of L. pneumophila serogroup 1, Okinawa02-001 (isolated in 2002, male patient) and Nagasaki 80-045 wereisolated from patient with Legionnaires’ Disease (isolated in 1980,male patient). The three strains had been previously frozen at −86°Cin vials containing 10% skim milk (Becton Dickinson and Company,Franklin Lakes, NJ, USA). The vials were thawed at room tem-perature, and the contents were streaked onto buffered charcoal-yeast extract agar (Kanto Kagaku, Tokyo) supplemented with 0.1%α-ketoglutarate (Sigma-Aldrich, St. Louis, MO, USA) (BCYE-α).The plates were incubated for 48 to 72 h at 37.0°C. Growth washarvested from plates with a platinum loop and placed into steriledistilled water in a tube. The tube was mixed in a Vortex mixer.

The culture was then diluted with sterile distilled water to an opti-cal density at 660 nm (OD

660; Bio-Rad Laboratories, Hercules, CA,

USA) of approximately 0.6. To start the experiments, a 500 µl ali-quot of suspensions was transferred into a test glass tube contain-ing 4.5 ml of fresh BCYE-α broth. The final concentration in thetest tube reached approximately 5×108 cfu ml–1. To measure thelength of L. pneumophila biofilm grown on the glass wall in broth,ten identical glass tubes containing broth samples were installed inthe temperature gradient incubator operating at 10 different tem-peratures simultaneously. Sterile slide glass plates, 13 mm wide and76 mm long, were placed in each test tube. For analysis by scan-ning electron microscopy (Hitachi, Tokyo), a sterile cover glass(10 mm square) was used instead of a slide glass. The L. pneumo-phila biofilm adhered to the glass test tube wall and to the slideglass plate. The slide glass plate was readily removed from the testtube without loosening the biofilm. The effect of cultivation tem-perature on bacterial length was verified on BCYE-α agar. The vialswere thawed at room temperature, and the contents were streakedonto BCYE-α agar. The inoculated agar was cut into small cubes(10×10×5 mm), and the agar cubes were then placed into a glasstube and incubated for 48 h at a specified temperature. The agarcube on which L. pneumophila colonies were formed was with-drawn from the glass test tube for analysis. The length of three

FIG. 1. Photograph of temperature gradient incubator (TGI).

FIG. 2. Schematic diagram of the experimental apparatus. 1, Con-stant temperature bath circulator (Thermo electron corporation ThermoNESLAB RTE-7); 2, digital multimeter (Keithley model 2001 TCSCAN,model 200 Multimeter); 3, thermocouples (Clomel-Amumel 1 mmsheath); 4, personal computer (Fujitsu FMV-D5); 5, aluminum blocks(each 80 mm square and 25 mm thick, 10 pieces); 6, brass blocks (each80 mm cubes, two pieces); 7, silicon sheets (each 80 mm square and0.5 mm thick, 11 pieces); 8, glass test tubes (14 mm internal diameterand 105 mm long, 10 pieces).

KONISHI ET AL. J. BIOSCI. BIOENG.,480

strains grown at temperatures ranging from 30.0°C to 47.0°C wasmeasured and evaluated. For the analysis of cell multiplication atdifferent temperatures, three strains were cultured at a temperatureranging from 41.0°C to 45.0°C for 72 h. Two separate experimentswith temperature windows of 41.0°C to 43.0°C and 43.0°C to45.0°C were performed with the TGI apparatus so as to achieve aserial step-wise culture temperature ranging from 41.0°C to 45.0°Cat intervals of 0.2°C. Even one colony of L. pneumophila resultedin a positive finding for cell multiplication. The experiments wererepeated 2 to 3 times.

Scanning electron microscopy For scanning electron mi-croscopy, the biofilm grown on a cover glass was fixed with 5%glutaraldehyde and stained with 1% osmium tetroxide and washedin 0.1 mM phosphate buffer at pH 6.9 for 2 h and then dehydratedthrough an alcohol series. Samples were mounted on 20 mm scan-ning electron microscopy specimen stubs with high-conductivitysilver paint. Specimens were coated with a 20 nm layer of gold in avacuum coating unit and 10 were examined in a Hitachi scanningelectron microscopy operated at 10 kV accelerating voltage.

Bacterial staining and measurement of bacterial length

Biofilms grown on glass slides in broth and colonies on agar cubeswere harvested with a platinum loop and placed into 2 ml volumesof sterile distilled water in a vial. The vial was mixed in a Vortexmixer. We verified that the length of bacteria was not affected bymixing (data not shown). The sample was stained conventionallyon a slide glass by the Gimenez (Muto Pure Chemicals, Tokyo)method and observed with a microscope (Nikon TE2000; Nikon,Tokyo) or with a CCD camera (Sony DXC-390, Sony, Tokyo) andrecorded in a personal computer. After Gimenez staining, photo-graphs including bacterial images were taken and recorded in apersonal computer and processed with image processing software(Adobe Photoshop). Approximately 200 well-isolated bacterialimages were randomly chosen from photos and the length of bac-teria was measured with Scion Image software (Beta 4.02 for Win-dows XP; Scion Corporation Frederick, Maryland, MD, USA) bytracing the image on the computer display with a mouse/pen de-vice.

RESULTS

Status of culture temperatures generated by the tem-perature gradient incubator (TGI) apparatus A step-ped temperature gradient with 10 different temperatures inthe range of 30.0°C (test tube no. 1) to 47.0°C in 2.0°C (testtube no. 10) steps was achieved when the constant tempera-ture bath circulators were set at 30.0°C and 50.0°C in theTGI apparatus. Each test tube reached a stable tempera-ture within 2 h and was able to maintain this temperaturethroughout the experiment with a temperature fluctuation of±0.1°C. The temperature in each test tube and the maxi-mum and minimum temperatures were recorded in the per-sonal computer.

Influence of culture temperature on the length of L.pneumophila Three strains were cultured in broth andon agar at 10 different temperatures for 48 h in the TGI ap-paratus. As no significant difference between two clinicalisolates was observed, the results were only shown for atype strain ATCC 33152 and Okinawa 02-001. The resultsfor ATCC type strain 33152 and a clinical isolate; Okinawa02-001 strain incubated on agar and on broth are shown inFig. 3. The average length of the strain ATCC 33152 ex-ceeded 50 µm when cultured at temperatures ranging from39.0°C to 44.0°C. The bacteria had the longest average

length of approximately 115 µm when cultured at 42.5°C.The average length of the clinical isolate Okinawa 02-001exceeded 50 µm when cultured at temperatures ranging from41.0°C to 43.0°C, with the greatest average length of ap-proximately 70 µm at 42.5°C. Figure 3 shows the differentbacterial lengths formed on agar or in broth media of theOkinawa 02-001 clinical isolate and the ATCC33152 strain.The average length of the strains ATCC 33152 and Okinawa02-001 cultured in test tubes was longer than when culturedon agar at temperatures ranging from 35.0°C to 43.0°C. Asignificant difference of bacterial length was recognized at38.5°C for Okinawa 02-001 and at 43.0°C for ATCC 33152.

Influence of culture temperature on cell multiplicationof L. pneumophila All 36 ATCC 33152 cultures testedwere able to multiply when cultured on BCYE-α agar attemperatures ranging from 41.0°C to 43.1°C. Thirteen outof 22 (59.1%) cultures multiplied when cultured at tempera-tures ranging from 43.1°C to 44.1°C. All nine ATCC 33152cultures were not able to multiply when cultured at tempera-tures higher than 44.1°C (Fig. 4a). Dead cells were deter-mined by plate method on which the strain did not form acolony at 37°C any longer. All 36 Okinawa 02-001 culturestested were able to multiply when cultured on BCYE-α agarat temperatures ranging from 41.0°C to 43.1°C. Fifteen outof 22 (68.2%) cultures multiplied when cultured at the tem-perature ranging from 43.1°C to 44.1°C. Eight out of nine(88.9%) Okinawa 02-001 cultures did not multiply whencultured at the temperature higher than 44.1°C (Fig. 4b).The results of Nagasaki had no meaningful difference toOkinawa.

Growth of L. pneumophila biofilm on glass walls at dif-ferent temperatures The clinical isolate strain Okinawa

FIG. 3. Variation of the averaged length of L. pneumophila withcultivation temperature for ATCC 33152 (a) and Okinawa 02-001 (b).The difference between the bacterial length formed on agar or in brothmedia were also shown. The results are presented as mean±SD (n>100).

TEMPERATURE ON GROWTH OF LEGIONELLA BIOFILMVOL. 101, 2006 481

02-001 was incubated in BCYE-α broth in the glass tube at42.0°C for 72 h. Figure 5 shows L. pneumophila biofilmgrowth on the glass wall. The field of view of the photo-graphs covers the glass tube wall at the air-liquid interface.Three pictures show 0 h (Fig. 5a), 24 h (Fig. 5b), 48 h (Fig.5c) after inoculation. The level of the air-liquid interface inthe broth fell due to evaporation of the medium. We specu-lated that the suspended cells would agglomerate on the sur-face of the broth (Fig. 5a). Thick biofilm developed on theglass wall above the air-liquid interface and granular bio-film adhered to the glass wall below the air-liquid interfaceat 24 h (Fig. 5b). After 48 h of incubation, the biofilm abovethe air-liquid interface extended upwards to a height of 3mm above the interface (Fig. 5c). Below the air-liquid inter-face, a screen mesh pattern was observed with 0.2–0.5 mmpitch. The biofilm sloughed off the glass wall surface andsank to the bottom of the tube after 72 h of incubation.

Figures 6 and 7 show the typical results of biofilm formedon the glass wall surface at 35.0°C and 42.0°C, respectively.At 35.0°C the biofilm adherent to the glass wall surface didnot grow closely together or in large amounts. The biofilmwas granulated but was not easy to detach from the wall; in

other words, it was sticky. We could not observe any differ-ence between the three strains at 35.0°C. Three locations inthe biofilm were magnified: above the air-liquid interface(Fig. 6c), near the air-liquid interface (Fig. 6d), and belowthe air-liquid interface (Fig. 6e). Figures 6f and 6g show amagnified image of Fig. 6e (two different locations). Thebiofilm exhibited a typical short rod morphology that didnot change much with the location in the biofilm. The cellwas 0.2–0.3 µm in diameter and 2–4 µm long. Above the airinterface, the isolated cells rested on the slide glass wall asshown in Fig. 6c. Cells embedded in a sheet of amorphousmaterial on the glass surface appeared near or below the air-liquid interface as shown in Fig. 6d and 6e. Figure 6f showsthe magnified image of the sheet of amorphous material.Figure 6g shows three strands connecting the cells to oneanother. At 42.0°C the biofilm of L. pneumophila developeda heterogeneous structure with three distinct layers. Thelower layer was granulated biofilm approximately 0.2–0.5mm in diameter, and lay approximately 2 mm below the air-liquid interface. The middle layer was a thick biofilm and2–3 mm in height. The upper layer consisted of surface pel-licles 1–3 mm in height. Although the thickness of the threelayers was different in all three strains, no significant differ-

FIG. 4. The effect of cultivation temperature on the viability of L.pneumophila, ATCC 33152 (a) and Okinawa 02-001 (b).

FIG. 5. The growth of L. pneumophila (clinical isolate; Okinawa02-001) on a vertical glass surface observed at 0 h (a), after 25 h (b)and after 48 h (c).

FIG. 6. The L. pneumophila strain (clinical isolate; Okinawa 02-001) was cultured for 72 h in glass tube. (a) Typical results of biofilmformed on the glass wall surface at 35.0°C. Scanning electron micro-graphs were obtained for the entire image (b), over the air-liquid inter-face (c), near the air-liquid interface (d), under the air-liquid interface(e), and magnified images (f, g).

KONISHI ET AL. J. BIOSCI. BIOENG.,482

ences in three-layered structure were recognized at 42.0°C.At 45.0°C no biofilm developed in the three strains. Figures7b and 7c show the middle biofilm developed on the glasswall of Okinawa 02-001 strain at 42.0°C. The air-liquid in-terface seemed to be located in the center of the biofilm.The upper edge of the biofilm was adjacent to the thin upperbiofilm and to the surrounding air in a glass tube. The loweredge was attached to the granulated layer and was soaked inBCYE-α broth. The shape of the border at the upper edgewas straight, but the lower edge was a complex semicircularshape. The degree of border complexity was expressed asthe ratio of the boundary lengths of the liquid side to the airside, and was found to be 3.6. Figures 7d–g show a typicalimage of the middle biofilm of L. pneumophila formed at42.0°C obtained by a scanning electron micrograph of theOkinawa 02-001 strain. Three locations in the biofilm weremagnified: near the air interface (Fig. 7e), in the middle ofthe biofilm (Fig. 7f), and above the liquid (Fig. 7g). Thebiofilm exhibited a typical filamentous morphology that didnot change much with the location in the biofilm. The den-sity of L. pneumophila was greatest near the air interface,and lowest in the middle of the biofilm.

DISCUSSION

The average length of type 1 strain ATCC 33152, a clini-cal isolate Okinawa 02-001 was greatest at a temperature of37.5°C and 39.0°C, respectively. These results indicate thatthe clinical isolates had a potential to retain their lengthwithin a range of 1–10 µm at higher culture temperaturesthan did the type strain. Pine et al. (28) observed markedchanges in cell surface-to-volume ratio, shape, and sizewhich could be related to temperature, nutrients, and aera-tion conditions for both the bacillary and filamentous formsof L. pneumophila. They noted a large mass of filaments orchain of bacilli in the logarithmic phase breaking into shorter

filaments and ultimately forming single and double cigar-shaped cells at 30.0°C and 37.0°C incubated on semisyn-thetic medium (28). Berg et al. (29) investigated the mor-phological changes of L. pneumophila in continuous culture.Filamentous forms approximately 100 µm long were seen at37.0°C and 44.0°C. In the present study, most of the fila-mentous forms approximately 100 µm long were observedover 39.0–41.0°C for all three strains incubated on BCYE-αagar and in broth, but the Okinawa strain incubated at 37.5°Cin broth was an exception. There have been many reportsthat bacterial elongation was closely related to the functionof constitutional enzymes for bacterial binary fission (30).The metabolic activity associated with cell division is likelyto be affected by culture temperature. Werner et al. (31)suggested that when Pseudomonas aeruginosa is exposedto the antibiotic carbenicillin, cell division is blocked andgrowing cells form filaments. The constitutional enzymes ofthe two clinical isolates used in the study probably kept theirfunctional integrity at higher culture temperature (39.0°C)than ATCC 33152 (37.0°C). The average length of type 1strain ATCC 33152, clinical isolate Okinawa 02-001 ex-ceeded 50 µm when cultured at temperatures from 39.5°C to44.0°C (range, 4.5°C) and from 41.0°C to 43.0°C (range,2.0°C), respectively. Since L. pneumophila strains are likelyto form biofilm structures when bacterial length exceeds50 µm, the culture temperature that allows two clinical iso-lates to form biofilm is probably higher than that of the typestrain.

The heterogeneous structure of L. pneumophila biofilmformed on glass walls in BCYE-α broth was observed andanalyzed by scanning electron microscopy. Microscale gra-dients in concentration of nutrients, oxygen and metabolicproducts were assumed to be formed along the glass wall.The environment above an air-liquid interface is oxygen-rich but poor in nutrients, and below the air-liquid interfaceit is oxygen-poor but rich in nutrients. How does L. pneumo-phila transport oxygen and nutrients into biofilm? Porousstructures are known to be liquid carriers by capillary effect.The filamentous form of L. pneumophila biofilm resemblesthis pattern. Nutrients are absorbed by capillary effect fromthe bottom of the biofilm and transported into the upper re-gion and accumulated inside the biofilm. The accumulationof nutrients in the biofilm allows L. pneumophila to survive.The rounded shape of the bottom of the biofilm increasesthe absorption surface area to 3.6 times that of the flat uppersurface of the biofilm. This configuration is well adapted toabsorbing more nutrient into the biofilm. The layer of densepopulation of L. pneumophila at the upper surface seems toprevent the evaporation of nutrients and external contami-nation. Thus, the heterogeneous structures of the L. pneu-mophila biofilm formed on the glass wall were found to beunique.

In terms of bacterial multiplication, culture temperaturewas categorized into three ranges: range I (<43.1°C), a cul-ture temperature in which almost all bacterial clones testedmultiplied; range II (43.1°C to 44.1°C), a culture tempera-ture in which 9.1% to 40.9% of bacterial clones tested did notmultiply; and range III (>44.1°C) in which most (more than88.9%) bacterial clones tested did not multiply. Kusnetsovet al. (20) demonstrated that the culture temperature at which

FIG. 7. The L. pneumophila strain (clinical isolate; Okinawa 02-001) was cultured for 72 h. Typical results of biofilm formed on theglass wall surface at 42.0°C (a) and magnified images (b, c). Scanningelectron micrographs were obtained for entire image (d), near the airinterface (e), in the middle of the sediment (f), and at the liquid inter-face (g).

TEMPERATURE ON GROWTH OF LEGIONELLA BIOFILMVOL. 101, 2006 483

no cell multiplication of strain ATCC 33152 was observedwas 48.4°C when the bacteria was grown in BCYE-α brothwithout charcoal for 69 h. This difference in critical temper-ature was probably due to: (i) agar being used rather thanbroth, (ii) more accurate temperature control with the TGIapparatus used in our experiment. It could be extrapolatedthat when the water temperature in a recirculating water sup-ply system is kept higher than at 44.1°C, more than 88.9%of wild strains would not survive, and that when the temper-ature is kept higher than at 44.5°C, no strains would survive.However, this extrapolation would not apply to bacteria em-bedded in a biofilm structure, because the biofilm wouldprotect the bacteria from unfavorable growth conditions,and a much higher temperature would be needed to kill anybacteria embedded in the biofilm. In this work, bacteriawere grown in a rich medium (BCYE-α) whereas the real-world system of a hot water distribution system is expectedto be oligotrophic, in which the nutrient concentrations areperhaps two orders of magnitude smaller. In the next futurework, we would like to verify the heterogeneous structure ofLegionella biofilm by using biofilm flow cell system, inwhich oligotrophic environment can be demonstrated.

ACKNOWLEDGMENTS

This research was sponsored in part by a grant-in-aid (nos.15500337 and 17500324) for Science Research from the Ministryof Education, Culture, Sports, Science and Technology of Japan.

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