Factors Stimulating Propagation of Legionellae in Cooling Tower Water

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1992, p. 1394-1397

Vol. 58, No. 4

0099-2240/92/041394-04$02.00/0 Copyright © 1992, American Society for Microbiology

Factors Stimulating Propagation of Legionellae in Cooling Tower Water HIROYUKI YAMAMOTO,* MINORU SUGIURA, SHINJI KUSUNOKI, TAKAYUKI EZAKI, MASANARI IKEDO, AND EIKO YABUUCHIt Department of Microbiology, Gifu University School of Medicine, Tsukasa-Machi 40, Gifu 500, Japan Received 12 November 1991/Accepted 14 January 1992

Our survey of cooling tower water demonstrated that the highest density of legionellae, 104 CFU/100 ml, appeared in water containing protozoa, >102 MPN/100 ml, and heterotrophic bacteria, >10' CFU/100 ml, at water temperatures between 25 and 35°C. Viable counts of legionellae were detected even in the winter samples, and propagation, up to 10S CFU/100 ml, occurs in summer. The counts of legionellae correlated positively with increases in water temperature, pH, and protozoan counts, but not with heterotrophic bacterial counts. The water temperature of cooling towers may promote increases in the viable counts of legionellae, and certain microbes, e.g., protozoa or some heterotrophic bacteria, may be a factor stimulating the propagation of legionellae.

Legionella species have been found in various aquatic environments, e.g., rivers, lakes, ponds (8), and cooling tower or water distribution systems (9, 10, 12). Viable counts of Legionella pneumophila of 15 to 30 CFU/liter have been reported in water from the Allegheny River (15), while counts of 10 to 10' CFU/100 ml have been found in cooling tower water (9). The reason why the legionella population in cooling tower water can be larger than that in natural habitats has not yet been clearly elucidated. Several investigations (15-17, 21) have suggested a relationship between legionella multiplication and certain abiotic factors such as pH, temperature, organic matter, and certain metals, factors considered to play an important role as growth enhancers or inhibitors of legionellae in the environment. On the other hand, legionellae have been shown to be symbiotic bacteria. Cyanobacteria (2), heterotrophic bacteria (17, 22), ciliates (1, 7), and amoebae (11, 14, 19, 20) are thought to be influential in the survival or multiplication of legionellae in natural and man-made aquatic environments. However, there is a dearth of quantitative information regarding populations of protozoan and other heterotrophic bacteria in microbial communities in the research on legionellae in environmental water. In this study, we determined viable populations of legionellae, other heterotrophic bacteria, and bacterivorous protozoa in cooling tower water, and, from the results, we assessed the factors possibly contributing to increases in the viable counts of legionellae in a cooling tower system. The water samples were collected in sterile plastic bottles (1 liter) from the basins of cooling towers from 10:00 a.m. to 12:00 noon. The bottles, placed in an ice box, were carried to the laboratory, where water samples were tested within 4 h after collection. The pH and temperature of the water in the tower basins were measured by a pH-temperature meter (model pH 51; Yokogawa Electric Co., Tokyo, Japan) just before sample collection. A total of 82 samples of cooling tower water were collected from 40 towers, located in Nara * Corresponding author. t Present address: Department of Bacteriology, Osaka City University Medical School, Osaka 545, Japan.

(4 towers), Gifu (25 towers), Aichi (7 towers), and Shizuoka (4 towers) prefectures. Sample collections were performed one to five times at each tower between April 1986 and July 1987. Sixty-three of the 82 samples were collected from July to September, since most of the cooling towers were being used for air-conditioning during the period. The water samples collected in winter (December to January) were from the four cooling towers operating throughout the year. In the basins of these four towers, many deposits or aggregates of sedimentary matter were observed in autumn and winter. The viable counts of heterotrophic bacteria other than legionellae in cooling tower water were determined on nutrient agar plates (Eiken Chemical Co., Ltd., Tokyo, Japan). After incubation at 20°C for 7 days, all colonies on the plates were counted as heterotrophic bacteria. The viable counts of legionellae were determined by the following method. After centrifugation (7,000 x g, 20 min) of 200 ml of the sample water, the sediment was resuspended in 2 ml of sterile distilled water, and 0.5 ml of the suspension was mixed with 0.5 ml of 0.2 M KCl-HCl buffer (pH 2.2). The mixture was maintained for 20 min at 35°C, and each 0.1-ml portion was spread in duplicate on buffered charcoal yeast extract (BCYE) agar and Wadowsky-Yee-Okuda agar (9). These plates were incubated at 35°C for 10 days, and suspicious colonies were counted and then inoculated on BCYE and blood agar medium. If the isolate could grow only on BCYE and the Gram stain was negative, it was determined to be a legionella. Several colonies isolated from each positive sample were used for the identification. Serological identification of legionella isolates was carried out by slide agglutination tests with commercial antiserum (L. pneumophila serogroup 1 to 6, Legionella bozemanii serogroup 1, Legionella dumoffii, Legionella gormanii serogroup 1, and Legionella miedadeii; Denka Seiken Co. Ltd., Tokyo, Japan). Species identification of serologically unidentified strains was carried out by the microplate hybridization method of Ezaki et al. (4). DNA microplates for 26 species of the genus Legionella were obtained from the Kobayashi Pharmaceutical Co., Osaka, Japan. The viable counts of bacterivorous protozoa were estimated by the most-probable-number (MPN) method (24). 1394

VOL. 58, 1992

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FIG. 1. Profiles of abiotic and biotic parameters of cooling tower water by month. Maximum determinable MPN is 2.4 x 103.

Escherichia coli GIFU 1424 (type strain) was suspended in autoclaved river water to a concentration of 108 CFU/ml, and the suspension was used as a medium to culture the protozoa. Briefly, 10-, 1-, and 0.1-ml volumes of the sample water were inoculated separately into three flasks (Falcon 3013; Becton Dickinson, Oxnard, Calif.), each containing 0.1 ml of E. coli suspension. The flasks were incubated at

20°C for 6 days, and the growth of the protozoa was observed every day by use of an inverted phase-contrast microscope at a magnification of 200x. Protozoa were tentatively identified as ciliates, flagellates, or amoebae by their morphological traits (5). The highest determinable number of bacterivorous protozoa was 1.1 x 103 cells per 100 ml. Determination of bacterivorous protozoa could not be made for seven samples collected from Aichi prefecture in September 1986. Statistical analyses were carried out by using a personal computer,

Macintosh II (Apple Computer, Inc., Cupertino,

Calif.), and a statistical program, SYSTAT (SYSTAT Inc., Evanston, Ill.). As shown in Fig. 1, water temperatures in the basin of the cooling tower ranged between 8.3 and 35.2°C, but 87% (71 of 82 samples) were above 20°C. Legionellae were recovered from 73% (29 of 40) of the cooling towers. In spring (April and May), legionellae were detected in 33% (4 of 12) of the samples, with viable counts of 10 CFU/100 ml (Fig. 1), the lowest detectable count obtained by the method employed.

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Heterotrophic bacteria showed the same viable counts (105 to 108 CFU/100 ml) in spring as they did in summer, but viable counts of protozoa were lower in spring than they were in other seasons. Eight of the towers surveyed in spring also showed increases in legionella counts in summer. In summer (July and August), legionellae were detected in 68% (28 of 41) of the samples (Fig. 1), and maximum viable counts reaching 105 CFU/100 ml appeared in August 1986 and July 1987 (Table 1). In autumn (September), legionellae were detected in 59% (13 of 22) of the samples (Fig. 1), but the average of viable counts of bacteria was lower than it was in summer (Table 1). In winter (December and January), legionellae were detected in 71% (5 of 7) of the samples from cooling towers operating throughout the year, with viable counts of 102 to 103 CFU/100 ml (Fig. 1). These towers retained the average numbers of viable heterotrophic bacteria (105 CFU/100 ml) and protozoa (10 to 103 MPN/100 ml), and maximum water temperatures of 28.40C were recorded (Table 1). The detection rate of legionellae increased with the temperature of basin water, e.g., 45% (5 of 11) of the samples at 250C. Water temperature positively correlated with viable counts of legionellae, ciliates, and amoebae (Table 2). Low viable counts of legionellae in the spring may have resulted from the fact that some towers in the survey area were not yet working continuously at an average atmospheric temperature of 10 to 22°C. Of the 359 isolates from the cooling tower water, 325 strains (90%) were L. pneumophila, including serogroup 1 (206 strains), serogroup 3 (18 strains), serogroup 4 (2 strains), serogroup 5 (8 strains), serogroup 6 (26 strains), and those serologically unidentified (65 strains). L. pneumophila serogroup 1 was the dominant legionella population in the cooling tower water, as it was in previous investigations of water from cooling towers (9, 10) and natural environments (8). The remaining 34 strains were L. bozemanii serogroup 1 (21 strains), Legionella anisa (11 strains), and one each of Legionella feeleii and Legionella sainthelensi. All of the L. anisa strains reacted to antiserum for L. bozemanii and/or L. micdadeii. Strains of L. pneumophila that were serologically unidentified also included 18 strains that were cross-reactive with another species or complex serogroups. These serologically unidentified strains, including strains of L. feeleii and L. sainthelensi, were identified by quantitative DNA-DNA hybridization by the microplate method (4). As shown in the results of Pearson's correlation analysis (Table 2), the pH of the water positively correlated with viable counts of legionellae. The maximum viable counts of legionellae (105 CFU/100 ml) appeared in water samples of pH 8.4 to 9.1 at 26.3 to 29.9°C. We have found, in contrast to previous results, that multiplication of L. pneumophila was less apparent in water samples collected from the cooling system at pH 8.2 to 8.5 than at pH 6.9 to 7.3 (16). However, no water in acidic pH range was found in the cooling towers in our survey or in a previous report (9). A sequential survey from the beginning of the cooling tower operation (24) demonstrated that the pH of tower water usually stabilized in an alkaline range, around pH 8.2, during operation. One reason for the occurrence of legionella propagation in alkaline water may be the coexistence of legionellae with biofilms or aggregates, which provides an environment of chemical and physical factors more favorable than those of the ambient environment (13). The physical appearance of the tower basin and water, e.g., biofilm from algae, deposits, or aggregates, seemed to relate to the occurrence of legionel-

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APPL. ENVIRON. MICROBIOL.

NOTES TABLE 1. Summary of statistics on survey of cooling tower water by season Spring Parameter

Mean

SD

35.2 9.10 8.6

22.1 7.98 6.0

2.4 0.41 0.6

18.5 7.45 5.2

26.5 9.10 7.6

23.6 7.97 5.6

29.4 9.10 8.2

27.9 8.40 7.0

3.1 0.38 0.8

14.7 7.17 4.7

8.3 7.67 5.6

Legionellae (CFU/100 ml) Ciliates (MPN/100 ml) Flagellates

SD

Mean

Mean

21.4 7.1 Temp (°C) 8.34 0.48 pH Heterotrophic bacteria 7.1 0.8 (CFU/100 ml)

Mean

Maximum

Maximum

Maximum

Winter'

Autumnd MiniSD mum

Summer' MiniSD mum

Minimum

Minimum

Maximum

4.7 0.12 1.0

15.6 7.85 4.7

28.4 8.18 7.2

0.3

0.5

0.0

1.0

2.4

1.8

0.0

5.5

1.2

1.2

0.0

3.3

2.4

1.7

0.0

3.7

0.7 1.8

0.6 0.5

0.0 1.0

1.4 2.4

1.7 3.2

0.9 0.6

0.0 0.0

3.4 3.4

1.1 3.2

0.6 0.2

0.0 2.6

2.3 3.3

1.0 3.1

0.6 0.5

0.0 2.4

3.4

1.6

0.6

0.0

2.4

2.9

0.8

0.0

3.4

2.2

0.7

0.9

3.3

2.8

0.5

2.3

3.4

1.9

(MPN/100 ml) Amoebae

(MPN/100 ml) a Numbers of viable counts (heterotrophic bacteria, legionellae, ciliates, flagellates, and amoebae) are converted to log. Maximum determinable MPN is 3.4 in log. b April and May; 12 cases. c July and August; 41 cases. d September; 22 cases. e December and January, 7 cases.

lae, since the viable count was frequently observed in cooling towers with biofilms and deposits (24). Another reason may be that protozoan cells shield the intracellular legionellae from ambient water and sustain their growth under alkaline conditions. Bacterivorous protozoa were found in 98% (74 of 75) of the water samples from cooling towers (Fig. 1). The detection rates of the protozoa were 82% (62 of 75 samples) for ciliates, 98% (74 of 75 samples) for flagellates, and 97% (73 of 75 samples) for amoebae. Ciliates showed viable counts in the range of 103 MPN/100 ml in summer (Table 1). Flagellates and amoebae showed viable counts of 103 MPN/100 ml throughout the year, except in April and May (Table 1). In this study, we found many protozoa belonging to several taxa (5) in water samples of the cooling tower (e.g., ciliates of the families Aspidiscidae, Colpodidae, Pleuronematidae, Tetrahymenidae, and Vorticellidae; amoebae of the families Acanthamoebidae, Amoebidae, and Hartmannellidae; and flagellates of the family Bodonidae. As symbiotic and pathogenic bacteria, legionellae must grow within protozoan or mammalian phagocytes. Several protozoa of the ciliate Tetrahymena pyriformis (1, 7) and the amoebae Acanthamoeba spp., Echinamoebae sp., Naegleria spp., and Hartmannella spp. (6, 11, 14, 19, 20) have been reported to serve as the host cells of L. pneumophila. Bacterivorous protozoa, including a host group of legionellae, are very common organisms in the microbial community of cooling tower water. The counts of these protozoa closely correlated with viable counts of legionellae (Table 2). In TABLE 2. Matrix of Pearson's correlation coefficients among the parameters in cooling tower water" Parameter

Heterotrophic bacteria Legionellae Ciliates Flagellates Amoebae

Water temp 0.190 0.311 0.332 0.122 0.328

pH pH

Heterotrophic bacteria

Legionellae Leiela

0.331 0.319 0.388 0.042 0.208

0.104 0.146 -0.079 0.002

0.300 0.383 0.300

a Underlined numbers indicate a significant value at P < 0.01 (99%

confidence).

cooling tower water, legionellae may utilize some protozoan cells as their growth habitats, e.g., Hartmannella vermiformis in tapwater culture with Legionella spp. (20) and in the microbial community of hot water tanks (6). Several isolates of heterotrophic bacteria from potable water have been reported as bacteria supporting the growth of L. pneumophila on BCYE agar without added L-cysteine (17, 22). We isolated similar supporting bacteria, namely, six strains from 500 isolates. These were gram-negative nonfermenters, two strains of Sphingomonaspaucimobilis (23) and four strains with G+C contents of 69 or 71% (3). However, the number of growth-supporting bacteria was fewer than expected, in spite of the fact that the highest density of legionellae (.104 CFU/100 ml) appeared in water with high numbers (.106 CFU/100 ml) of heterotrophic bacteria. The correlation between the occurrence of legionellae and that of heterotrophic bacteria was not significant in our results (Table 2). The biological function of heterotrophic bacteria in the legionella environment in the cooling tower is probably dependent on indirect or synergistic relationships in the microbial community rather than on a direct relationship to specific bacteria. A cooling tower appears to afford a suitable environment for sustaining a stable microbial community. The water is supplied automatically in the tower, maintaining constant water level and volume during operation. The substrata for microbial growth, present in the water or introduced from the atmosphere, may accumulate by the recycling of the water. After the cooling tower is cleansed with detergent, an increase in viable legionellae, up to 104 CFU/100 ml, appears after increases in heterotrophic bacteria and protozoan populations during operation in summer (24). Sustaining growth of bacterivorous protozoa is an important ecological function of heterotrophic bacteria in the microbial community. Some of the protozoan population may serve as host cells for the multiplication of legionellae. Our results demonstrated that the highest density of legionellae, >104 CFU/100 ml, in cooling tower water at >250C occurs in summer, with their population remaining high (102 to 103 CFU/100 ml) in the seasons that follow, including winter. Thus, the water temperature of the cooling tower could act as an important factor in promoting an

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increase in the viable count of legionellae, and the existence of other organisms in the microbial community, e.g., protozoa or some heterotrophic bacteria, may be a factor stimulating the propagation of the legionella population.

pneumophila. Infect. Immun. 50:449-452. 12. Paszko-Kolva, C., H. Yamamoto, M. Shahamat, T. K. Sawyer, G. Morris, and R. R. Colwell. 1991. Isolation of amoebae and Pseudomonas and Legionella spp. from eyewash station. Appl.

We thank Y. Ido for technical assistance and C. Paszko-Kolva for advice. We also thank A. Honda, Shizuoka General Hospital, and M. Aihara, Tenri Hospital, for authorizing the sample collection. DNA microplates for Legionella identification were kindly donated by the Kobayashi Pharmaceutical Co., Osaka, Japan. This research was supported by a grant from the Nippon Life Insurance Foundation to E.Y.

13. Revsbech, N. P., B. B. Jorgensen, T. H. Blackburn, and Y. Cohen. 1983. Microelectrode studies of the photosynthesis and 02, H2S, and pH profiles of a microbial mat. Limnol. Oceanogr.

REFERENCES 1. Barbaree, J. M., B. S. Fields, J. C. Feely, G. W. Gorman, and W. T. Martin. 1986. Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila. Appl. Environ. Microbiol. 51:422-424. 2. Bohach, G. A., and I. S. Snyder. 1983. Cyanobacterial stimulation of growth and oxygen uptake by Legionella pneumophila. Appl. Environ. Microbiol. 46:528-531. 3. Ezaki, T., Y. Hashimoto, and E. Yabuuchi. 1989. Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine generic relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39:224-229. 4. Ezaki, T., Y. Hashimoto, H. Yamamoto, S. Liu, S. Kusunoki, K. Asano, and E. Yabuuchi. 1990. Evaluation of the microplate hybridization methods for rapid identification of Legionella species. Eur. J. Clin. Microbiol. Infect. Dis. 9:213-217. 5. Farmer, J. M. 1980. The protozoa, introduction to protozoology. The C.V. Mosby Co., St. Louis. 6. Fields, B. S., G. N. Sanden, J. M. Barbaree, W. E. Morrill, R. M. Wadowsky, E. H. White, and J. C. Feeley. 1989. Intracellular multiplication of Legionella pneumophila in amoebae isolated from hospital hot water tanks. Curr. Microbiol. 18:131137. 7. Fields, B. S., E. B. Shotts, Jr., J. C. Feeley, G. W. Gorman, and W. T. Martin. 1984. Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis. Appl. Environ. Microbiol. 47:467-471. 8. Fliermans, C. B., W. B. Cherry, L. H. Orrison, S. J. Smith, D. L. Tison, and D. H. Pope. 1981. Ecological distribution of Legionella pneumophila. Appl. Environ. Microbiol. 41:9-16. 9. Ikedo, M., and E. Yabuuchi. 1986. Ecological studies of Legionella species. I. Viable counts of Legionella pneumophila in cooling tower water. Microbiol. Immunol. 30:413-423. 10. Negron-Alvira, A., I. Perez-Suarez, and T. C. Hazen. 1989. Legionella spp. in Puerto Rico cooling towers. Appl. Environ. Microbiol. 54:2331-2334. 11. Newsome, A. L., R. L. Baker, R. D. Miller, and R. R. Arnold. 1985. Interactions between Naegleria fowlen and Legionella

Environ. Microbiol. 57:163-167.

28:1062-1074. 14. Rowbotham, T. 1980. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J.

Clin. Pathol. 33:1179-1183. 15. States, S. J., L. F. Conley, J. M. Kuchta, B. M. Oleck, M. J. Lipovich, R. S. Wolford, R. M. Wadowsky, A. M. McNamara, J. L. Sykora, G. Keleti, and R. B. Yee. 1987. Survival and multiplication of Legionella pneumophila in municipal drinking water systems. Appl. Environ. Microbiol. 53:979-986. 16. States, S. J., L. F. Conley, S. G. Towner, R. S. Wolford, T. E. Stephenson, A. M. McNamara, R. M. Wadowsky, and R. B. Yee. 1987. An alkaline approach treating cooling towers for control of Legionella pneumophila. Appl. Environ. Microbiol. 53:17751779. 17. Stout, J. E., V. L. Yu, and M. G. Best. 1985. Ecology of Legionella pneumophila within water distribution systems. Appl. Environ. Microbiol. 49:221-228. 18. Tamaoka, J., and K. Komagata. 1984. Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25:125-128. 19. Wadowsky, R. M., L. J. Butler, M. K. Cook, S. M. Verma, M. A. Paul, B. S. Fields, G. Keleti, J. L. Sykora, and R. B. Yee. 1988. Growth-supporting activity for Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors. Appl. Environ. Microbiol. 54:2677-2682. 20. Wadowsky, R. M., T. M. Wilson, N. J. Kapp, A. J. West, J. M. Kuchta, S. J. States, J. N. Dowling, and R. B. Yee. 1991. Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis. Appl. Environ. Microbiol. 57:1950-1955. 21. Wadowsky, R. M., R. Wolford, A. M. McNamara, and R. B. Yee. 1985. Effect of temperature, pH, and oxygen level on the multiplication of naturally occurring Legionella pneumophila in potable water. Appl. Environ. Microbiol. 49:1197-1205. 22. Wadowsky, R. M., and R. B. Yee. 1985. Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water. Appl. Environ. Microbiol. 49:1206-1210. 23. Yabuuchi, E., I. Yano, H. Oyaizu, Y. Hashimoto, T. Ezaki, and H. Yamamoto. 1990. Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonasparapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbiol. Immunol. 34:99-119. 24. Yamamoto, H., T. Ezaki, M. Ikedo, and E. Yabuuchi. 1991. Effects of biocidal treatments to inhibit the growth of legionellae and other microorganisms in cooling tower. Microbiol. Immunol. 35:795-802.