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Applied and Environmental Microbiology, October 2002, p. 4809-4811, Vol. 68, No. 10
0099-2240/02/$04.00+0 DOI: 10.1128/AEM.68.10.4809-4811.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Isolation of Elemental Sulfur as a Self-Growth-Inhibiting Substance Produced by Legionella pneumophila
Hiroaki Inoue,1* Genji Kawano,1 Hiromichi Nagasawa,2 and Shohei Sakuda2*
Tsukuba Research Laboratories, Aquas Corporation, Tsukuba, Ibaraki 300-2646,1
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan2
Received 15 March 2002/
Accepted 25 July 2002
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ABSTRACT
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The addition of HP-20 resin to a medium could enhance the growth of Legionella species. Elemental sulfur was isolated as a self-growth-inhibiting substance produced endogenously by Legionella pneumophila from methanol extracts of the resins used to culture the bacterium. Elemental sulfur shows strong growth-inhibiting activity toward all Legionella species tested.
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INTRODUCTION
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Legionella pneumophila, a ubiquitous gram-negative bacterium, is found in natural or man-made water systems. Its natural hosts are probably various amoebae species. If humans inhale aerosolized water from sources contaminated with L. pneumophila, such as cooling towers or whirlpool spas, the bacterium can invade and replicate within alveolar macrophages, causing a severe form of pneumonia called Legionnaires' disease (4, 9). If the pneumonia is not treated promptly with antibiotics effective against the bacterium, the mortality rate can approach 30 to 40%. Therefore, control and management of Legionella contamination in some water systems are very important.
L. pneumophila requires specialized media for growth (7). It cannot ferment any carbohydrates but can use some amino acids as primary carbon and energy sources. Iron(III) and L-cysteine are nutrients essential for its growth. In addition to these requirements, the addition of activated charcoal to media for the isolation and culture of L. pneumophila is widely practiced, since growth of the bacterium can be strongly enhanced by such an addition. One role of the charcoal is thought to be that of contributing to catalytic decomposition of activated oxygen produced during cultivation (3). On the other hand, activated charcoal has good adsorptive properties. Therefore, it can be easily speculated that charcoal has an important role in absorption and detoxification of an unknown toxic substance which may inhibit the bacterial growth. However, no attempt has been made to clarify whether a growth-inhibiting substance adsorbed on activated charcoal is present. If we assume that the substance is endogenously produced as a growth-inhibiting factor by the bacterium and has a high specificity toward Legionella growth, then such a substance may be very useful as a lead compound in the development of effective drugs to prevent Legionella contamination in water systems. This idea prompted us to study the substance. In our preliminary experiments, we found that activated charcoal can be replaced by Diaion HP-20 resin for the culture of L. pneumophila and that methanol extracts of the Diaion HP-20 resins, which were used to culture L. pneumophila, show strong growth-inhibitory activity toward the bacterium. Thus, we attempted to isolate the active principle from the methanol extracts and have identified it as elemental sulfur.
In this paper, we describe the isolation of elemental sulfur endogenously produced by L. pneumophila and its biological activity as a growth inhibitor of the bacterium.
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MATERIALS AND METHODS
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Bacterial strains.
L. pneumophila serogroup 1 GIFU9134, obtained from the stock culture of Department of Microbiology, School of Medicine, Gifu University, was used throughout this study. This strain was maintained on a buffered charcoal-yeast extract alpha (BCYE
)-agar medium (1) and subcultured monthly. A cell suspension was prepared from a 2-day culture and used as the inoculum. Legionella bozemanii serogroup 1 GIFU9140, obtained from the stock culture of Department of Microbiology, School of Medicine, Gifu University; L. pneumophila serogroup 1 P4202, Legionella dumoffii P4201, and Pseudomonas putida SN2845, isolated from water of cooling towers by us; and Staphylococcus aureus IAM1011, Micrococcus luteus IAM1056, Pseudomonas aeruginosa IAM1514, Escherichia coli IAM12119, and Enterobacter aerogenes IAM1102, obtained from the stock culture of the Microbial and Microalgal Research Center, University of Tokyo, were used as test microorganisms for measuring bactericidal activity of elemental sulfur.
Time course of growth of L. pneumophila.
A cell suspension (100 µl; 105 CFU) of L. pneumophila GIFU9134 was inoculated into a 500-ml Sakaguchi flask containing 100 ml of a medium designated L (0.1% -ketoglutaric acid, 0.5% K2HPO4, 1.5% yeast extract, 0.3% NaCl, 0.1% [6.3 mM] L-cysteine hydrochloride, 0.05% ferric pyrophosphate [pH 6.8]) with or without 5% Diaion HP-20 (Mitsubishi Chemical Corp.). The flask was incubated at 37°C and shaken at 120 rpm, and samples of the culture broth were obtained at 24-h intervals for 7 days. The number of viable cells in each sample was counted on a BCYE agar plate.
Production of a growth inhibitor by L. pneumophila.
A cell suspension of L. pneumophila GIFU9134 was inoculated into a 500-ml Sakaguchi flask containing 100 ml of L medium with 5% Diaion HP-20 and cultured at 37°C and shaken at 120 rpm for 7 days. The culture broth (100 ml) was put into a column with a cotton plug to separate the resins from bacterial cells. By this procedure, the former remained in the column, whereas the latter passed through the column. After washing with water (50 ml), the resins were extracted with methanol (50 ml). The methanol solution was concentrated in vacuo and fractionated between ethyl acetate (5 ml) and water (5 ml). After concentration of each fraction, the residue (3.0 mg from the ethyl acetate fraction and 38.7 mg from the water fraction) was used for the assay to measure bactericidal activity. When the sample from L medium without cultivation of the bacterium was prepared, the same procedure was done without inoculation of the bacterium. In that case, 2.0 and 41.3 mg of samples were obtained from the ethyl acetate and water fractions, respectively.
When a medium without resins was used, the bacterium was cultured in L medium without Diaion HP-20 resins under the same conditions mentioned above. After 7 days of cultivation, the culture broth (100 ml) was centrifuged (6,400 x g, 30 min) to obtain the culture supernatant and Diaion HP-20 resins (5 g) were added to the supernatant. After incubating the mixture for 24 h, the resins were recovered and assay samples (2.0 mg from the ethyl acetate fraction and 36.9 mg from the water fraction) were prepared from the methanol extracts of the resins according to the same procedure mentioned above.
Isolation of elemental sulfur.
Cells of L. pneumophila GIFU9134 maintained on an agar slant were inoculated into a 500-ml Erlenmeyer flask containing 100 ml of L medium with 5% Diaion HP-20 and cultured at 37°C and shaken at 120 rpm for 2 days. This culture (25 ml) was transferred into the same medium (1,000 ml) for the main culture. Incubation was carried out at 37°C with shaking at 120 rpm for 7 days. The culture broth (20 liters) was put into a column with a cotton plug to separate the resins. After washing with water (5 liters), the column with the resins was eluted with methanol (3 liters). The methanol solution was concentrated in vacuo to obtain crude extracts (8.5 g). The extracts were mixed with deionized water (500 ml), and the mixture was extracted three times with 500 ml of ethyl acetate. After evaporation of the ethyl acetate solution, the residue (1.0 g) was applied on a silica gel column (10 g; Silica gel 60 [Merck]). The column was eluted with n-hexane (100 ml), and the n-hexane fraction (12 mg) was further purified by normal-phase high-performance liquid chromatography (HPLC) (column, Senshu pak Pegasil Silica [4.6 by 250 mm]; mobile phase, n-hexane; flow rate, 2.0 ml/min) to afford the active component (retention time, 2.5 min [3.5 mg]). In the matrix-assisted laser desorption ionization-time of flight (mass spectrometry) [MALDI-TOF (MS)] spectrum of the active component (positive, no matrix; Voyager DE-STR [Perkin-Elmer Biosystems]), ions at m/z 160 (41), 192 (8), 224 (33), 256 (100), and 288 (72) were observed. Retention time of the active component by reverse-phase HPLC (column, Capcell-Pak C18, 4.6 by 250 mm [Shiseido]; mobile phase, acetonitrile; flow rate, 1.0 min/ml) is 16.3 min. The MS spectrum and the retention time of authentic elemental sulfur were identical with those of the active component.
Bactericidal activity.
Samples were dissolved in ethanol or dimethyl sulfoxide at appropriate concentrations, and the solution (10 µl) was mixed with phosphate-buffered saline (990 µl) containing cells of L. pneumophila GIFU9134 (104 CFU). After incubating the mixture at 37°C for 24 h, a part of the mixture (100 µl) was spread on a BCYE agar plate, and colony formation on the plate was observed after 6 days of incubation at 37°C. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of the sample at which no colony was observed on the plate.
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RESULTS
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Culture of L. pneumophila with Diaion HP-20 resins.
To study a substance adsorbed on the activated charcoal in a culture broth of L. pneumophila, it is necessary to separate the charcoal from the broth. However, it is not easy to obtain only charcoal particles from the mixture of charcoal and bacterial cells involved in the broth. Therefore, we tested the use of Diaion HP-20 resin instead of activated charcoal to culture the bacterium. Diaion HP-20 resin is a synthetic adsorbent of cross-linked polystyrene, and spherical particles of the resin with diameters of 0.5 mm can be easily recovered by filtration from the culture broth without contamination of the bacterial cells. Figure 1 shows the time course of the growth of L. pneumophila cultured in a medium with or without Diaion HP-20 resins. When the bacteria were cultured with HP-20 resins, the number of viable cells reached maximum 2 days earlier than was the case when the bacteria were cultured without the resin.
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FIG. 1. Time course of growth of L. pneumophila cultured in a medium with ( ) or without (•) Diaion HP-20 resins.
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Production of a growth inhibitor by L. pneumophila.
Next, we investigated whether Diaion HP-20 resins in a medium for the culture of L. pneumophila adsorb a substance which may affect growth of the bacterium itself. To clarify whether the substance is present, we obtained three samples of Diaion HP-20 resins as follows. First, after 7 days of cultivation with a medium containing Diaion HP-20 resins, the resins were recovered from the culture broth. Second, Diaion HP-20 resins were incubated in a liquid medium for 7 days without inoculation of the bacterium and the resins were recovered. Third, the bacterium was cultured in a medium without resins for 7 days and Diaion HP-20 resins were added to the culture supernatant obtained by centrifugation of the culture broth. After incubating the resins in the supernatant for 24 h, we recovered the resins. Each sample of the resins was extracted with methanol, the methanol extracts were partitioned between ethyl acetate and water, and the bactericidal activity of each layer toward L. pneumophila itself was measured. As a result, the activity was detected only in the organic layer fraction of the methanol extracts obtained from Diaion HP-20 resins of the first experiment, i.e., the resins recovered from the culture broth after cultivation in a medium with Diaion HP-20 resins. Analysis of the fraction showed the activity with the MBC to be 25 µg/ml.
Isolation and identification of the growth inhibitor.
Next, we tried to identify the growth inhibitor involved in the methanol extracts of Diaion HP-20 resins used to culture L. pneumophila. The procedure employed for isolation of the substance is summarized in Table 1. Methanol extracts of Diaion HP-20 resins recovered from the culture broth were partitioned between ethyl acetate and water. The ethyl acetate fraction was then applied on a silica gel column, and the active substance was eluted with n-hexane from the column. The n-hexane fraction was further purified by normal-phase HPLC to obtain the active principle.
No significant signal was observed in the 1H and 13C nuclear magnetic resonance spectra, which were measured with 3.5 mg of the sample at 500 and 125 MHz, respectively. A series of ions which differed by 32 mass units was observed at m/z 160, 192, 224, 256, and 288 in the MS spectrum, and elemental analysis showed very high (92.1%) content of sulfur in the sample, suggesting that the active principle was elemental sulfur. The active principle was finally identified as elemental sulfur by comparison of its retention time on HPLC and biological activity with those of authentic elemental sulfur.
Antibacterial activity of elemental sulfur.
Table 2 shows bactericidal activity of elemental sulfur against several Legionella species and other bacteria. Bactericidal activity was measured in a buffer containing cells of each bacterium and elemental sulfur at serial concentrations. All Legionella species tested were killed by addition of elemental sulfur at low concentrations. Elemental sulfur showed strong activity toward Staphylococcus aureus and Enterobacter aerogenes, among other bacteria tested.
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DISCUSSION
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Addition of activated charcoal to a medium is known to enhance the growth of L. pneumophila. In this study, we focused on a substance adsorbed on the charcoal which might inhibit the growth of the bacterium. Since activated charcoal is difficult to separate from the culture broth without contamination of bacterial cells, we developed a method for the culture of L. pneumophila with Diaion HP-20 resins instead of activated charcoal. The growth of the bacterium was strongly accelerated by addition of the resins, and the resins could be easily recovered from the culture broth. In addition, we isolated elemental sulfur as a growth inhibitor from methanol extracts of Diaion HP-20 resins used to culture L. pneumophila.
The fact that methanol extracts of Diaion HP-20 resins showed no growth-inhibitory activity in a medium without cultivation of the bacterium indicates that elemental sulfur is not a constituent of the medium. Therefore, it is produced by L. pneumophila endogenously during cultivation. The medium contains 0.1% L-cysteine, but the biosynthetic origin of elemental sulfur is not clear at present. This production of elemental sulfur by the bacterium was observed in a culture with Diaion HP-20 resins but was not observed in a culture without the resins, suggesting that there are differences not only in growth speed but also in a part of metabolism between the two culture conditions. Some bacteria have been known to produce elemental sulfur. Accumulation of elemental sulfur has been observed in cells of Thiobacillus sp. (8), and elemental sulfur is known as a metabolite of Streptomyces sp. (2, 6). Antibiotic activity of elemental sulfur toward some bacteria has also been known to occur (5).
We isolated 3.5 mg of elemental sulfur from Diaion HP-20 resins recovered from 20 liters of a culture broth of L. pneumophila. Judging from sample loss during the isolation procedure (Table 1) and bactericidal activity of elemental sulfur (Table 2), the amount of elemental sulfur produced by the bacterium is enough to inhibit the growth of the bacterium itself if it is present in solution. At present, it is not clear why L. pneumophila produces such a self-growth-inhibiting substance or whether the requirement of L-cysteine for growth of the bacterium correlates with the production of elemental sulfur. However, our results showed that elemental sulfur might be a good candidate as a drug for preventing Legionella contamination in water systems effectively and cheaply. Work employing elemental sulfur in efforts to prevent contamination in a whirlpool spa system is now in progress.
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ACKNOWLEDGMENTS
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This work was supported by a Grant-in-Aid for Scientific Research (No. 12556016) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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FOOTNOTES
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* Corresponding author. Mailing address for Shohei Sakuda: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81-3-5841-5133. Fax: 81-3-5841-8022. E-mail: asakuda{at}mail.ecc.u-tokyo.ac.jp. 
* Corresponding author. Mailing address for Kiroaki Inoue: Tsukuba Research Laboratories, Aquas Corporation, Tsukuba Techno-park Toyosato, 4-4 Midorigahara, Tsukuba, Ibaraki 300-2646, Japan. Phone: 81-298-47-6000. Fax: 81-298-47-6080. E-mail: h_inoue0417{at}aquas.co.jp.
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REFERENCES
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- Edelstein, P. H. 1981. Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J. Clin. Microbiol. 14:298-303.[Abstract/Free Full Text]
- Hayashi, H., N. Tarui, and S. Murao. 1985. Isolation and identification of cyclooctasulfur, a fruiting-body inducing substance, produced by Streptomyces albus TO447. Agric. Biol. Chem. 49:101-105.
- Hoffman, P. S., L. Pine, and S. Bell. 1983. Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal. Appl. Environ. Microbiol. 45:784-791.[Abstract/Free Full Text]
- Horwitz, M. A., and S. C. Silverstein. 1980. Legionnaires' disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. J. Clin. Investig. 66:441-450.
- Libenson, L., F. P. Hadley, A. P. McIlroy, V. M. Wetzel, and R. R. Mellon. 1953. Antibacterial effect of elemental sulfur. J. Infect. Dis. 93:28-35.
- Nakanishi, S., S. Toki, Y. Saitoh, E. Tsukuda, K. Kawahara, K. Ando, and Y. Matsuda. 1995. Isolation of myosin light chain kinase inhibitors from microorganisms: dehydroaltenusin, altenusin, atrovenetinone, and cyclooctasulfur. Biosci. Biotechnol. Biochem. 59:1333-1335.[Medline]
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Applied and Environmental Microbiology, October 2002, p. 4809-4811, Vol. 68, No. 10
0099-2240/02/$04.00+0 DOI: 10.1128/AEM.68.10.4809-4811.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
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Abstract
Introduction
