Legionella Pneumophila, Escherichia Coli, and Staphylococcus Aureus

[Environmental Health and Preventive Medicine 10, 233–238, September 2005]

Original Article

Effects of Japan Sea Proper Water on the Growth of Legionella pneumophila, Escherichia coli, and Staphylococcus aureus

Yasuo TSUCHIYA1, Michihiro TERAO2, Takanori FUJIMOTO1, Kazutoshi NAKAMURA1 and Masaharu YAMAMOTO1

1Department of Community Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 2Department of Medical Technology, School of Health Sciences Faculty of Medicine, Niigata University, Niigata, Japan

Abstract

Objective: To assess whether Legionella pneumophila serogroup 1 and serogroup 6, Escherichia coli, and Staphylococcus aureus can survive in Japan Sea Proper Water (JSPW). Methods: The inhibitory effects of JSPW, surface seawater (SSW), phosphate buffer solution with 3.5% NaCl of pH 7.0 (3.5%NaClPBS), and the 102- and 104-fold dilute solutions with purified water or phosphate buffer solution of pH 7.0, and purified water were investigated. Survival cells were counted immediately after the water and the bacteria were mixed, and at 1, 3, 5, and 7 days after incubation at 37°C. If the number of surviving cells was decreased more than 2 log units compared with the starting value, we judged the medium to have had an inhibitory effect on the growth of the bacteria. Results: The survival cells of the bacteria in JSPW had decreased more than 2 log units compared with the starting value at 1 day after incubation. After 1 day of incubation, the cells of Legionella pneumophila serogroup 6 and Staphylococcus aureus were found to have decreased more than 2 log units in purified water (PW) used as a control. Furthermore, Legionella pneumophila serogroup 1 in the 102-fold dilute solution of JSPW was only 1.04 log units lower than the starting value at 7 days after incubation. In the 102- and 104-fold dilute solutions of JSPW, Escherichia coli survived for 7 days after incubation. These results were almost similar to the results in SSW and 3.5%NaClPBS. Conclusions: The present findings demonstrate that Legionella pneumophila serogroup 1 and Escherichia coli cannot survive in undiluted JSPW for over a day at 37°C, suggesting the inhibitory effects may be due to the sodium chloride contained in JSPW.

Key words: bath water, inhibitory effect, surface seawater, Legionella pneumophila, Escherichia coli, Staphylococcus aureus

Introduction improving blood circulation, and mitigating muscular fatigue and pain because of the water’s warm temperature, pressure, Japan Sea Proper Water (JSPW) has been collected from and buoyancy effect (1). In addition, it has been demonstrated the Sado offing of the Japan Sea of Niigata Prefecture as a new that the salt in hot spring water has the effect of raising body resource. Recently, the use of JSPW in thalassotherapy has temperature for a longer period. Previous studies regarding the begun. Japanese people traditionally make a practice of daily effects of balneotherapy in the treatment of some diseases at the bathing and may prefer to bathe in hot springs, getting away Dead Sea have been published (2–5). Furthermore, we previ- whenever they can for a relaxing stay at their favorite resorts. In ously demonstrated that hot JSPW bathing had a beneficial general, hot spring bathing is useful for maintaining hygiene, influence on human health (6, 7). Therefore, it is expected that use of JSPW will be promoted for human health. Many bacteria exist in our environment. Of these bacteria, Received Jan. 24, 2005/Accepted Jul. 5, 2005 Legionella pneumophila (L. pneumophila) has been detected in Reprint requests to: Yasuo TSUCHIYA environmental water and soil, and Legionnaires’ disease, which Division of Social and Environmental Medicine, Department of Community is caused by the bacteria, is a growing public health concern. L. Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata 951-8510, Japan pneumophila can survive under a wide range of environmental TEL: +81(25)227-2121, FAX: +81(25)227-0764 conditions: temperatures from 0° to 63°C; pH from 5.0 to 8.5; E-mail: [email protected] dissolved oxygen level from 0.2 to 15.0 mg/L (8). When

233 Environ. Health Prev. Med. Characteristics of Japan Sea Proper Water

JSPW is used as bath water, the water is contaminated, after 4. Surviving cell count people have bathed in it, with not only L. pneumophila but To assess the inhibitory effects of JSPW on the growth of also Escherichia coli (E. coli) and Staphylococcus aureus (S. L. pneumophila, E. coli, and S. aureus, bacteria suspensions that aureus). E. coli is isolated from human feces, and S. aureus is were 0.5 ml in volume were added to 4.5 ml of each of the one of the indigenous microbiota on human skin. Therefore, it following 11 kinds of water: JSPW, SSW, 3.5%NaClPBS, and is important to determine the antibacterial activities of JSPW their 102- or 104-fold dilute solutions with PW, PBS, and PW, that inhibit the growth of the bacteria in order to prevent the respectively. PBS was purchased from Wako Pure Chemicals, infections that may be caused by taking a JSPW bath. Co. Ltd. (Osaka, Japan), and PW was made using Millipore Previously, several authors have reported that Legionella ELIX 5 (Japan Millipore Corporation, Tokyo). All the testing species can survive in sea water (9–11). Nevertheless, some water samples were sterilized by being passed through a 0.45 trials demonstrated an inhibitory effect of sodium chloride μm syringe filter (Milex-HV, Millipore Corporation, Tokyo, (NaCl) on growth of Legionella species (12–14). Thus, previ- Japan). ous reports on inhibitory effects of sea water on growth of Immediately after the water and the bacteria suspension Legionella species have been conflicting. were mixed, each 0.1 ml of 2 Legionella suspensions was Because JSPW contains about 3.5% NaCl (15), a lot of inoculated on WYOα and buffered charcoal yeast extract bacteria may not be capable of growing in JSPW. However, to medium supplemented with 0.1% α-ketoglutarate (BCYEα) our knowledge, no study has investigated the effect of JSPW on agars. Then the bacteria were incubated for 5 days at 37°C. the survival of bacteria. Meanwhile, each 0.1 ml of the suspensions of E. coli and S. The aim of the present study was to determine the ability aureus were inoculated on Nutrient agar medium, and the of L. pneumophila, E. coli, and S. aureus to survive in JSPW. medium was stored overnight in an incubator at 37°C. Two The results of JSPW were compared with those of surface plates per one water sample were used in this study. After the seawater (SSW) and other media including phosphate buffer incubation, the surviving cells on the agars were counted and solution with 3.5% NaCl of pH 7.0 (3.5%NaClPBS), phosphate then the mean value of the cells on duplicate plates was calcu- buffer solution of pH 7.0 (PBS), and purified water (PW). lated. The value was used as the starting value in each water sample. Materials and Methods The remaining suspension was incubated at 37°C in a shaking incubator operating at 50 times per min. After incuba- 1. Collection of JSPW and SSW tion for 1, 3, 5, and 7 days, the bacteria were incubated by the JSPW and SSW were collected from depths of about methods described above, and the mean values of the surviving 300 meters and 0 meters in the Sea of Japan (37.50–38.00°N cells were calculated. latitude; 138.30–138.45°E longitude), respectively, using the We examined the inhibitory effects of JSPW and PW on JSPW-drawing system developed jointly by HONMA Corpora- the growth of L. pneumophila SG1 and SG6 in 3 different tion (Niigata, Japan) and KITAC Corporation (Niigata). Water concentrations of bacteria: 2×102 (Concentration 1), 4×104 was collected in a one-liter plastic container and sent immedi- (Concentration 2), and 4×106 (Concentration 3) cfu/ml. Each ately, under refrigeration, to our laboratory. The water was bacteria suspension was added to JSPW and PW, and the stored in a refrigerator and used within 7 days. mixtures were incubated in a shaking incubator for 1 day at 37°C. Both before and after incubation, the bacteria were 2. Bacterial strains inoculated on BCYEα agars and incubated for 5 days at 37°C. Two strains of L. pneumophila were used in this study. After 1 day of incubation, the surviving cells on the agars were One strain (serogroup 1; SG1) was obtained from Denka Seiken counted. Co., Ltd. (Tokyo, Japan). The other (serogroup 6, strain 25K; We judged that the water had an inhibitory effect on the SG6) was isolated from environmental water and identified. E. growth of the bacteria when the number of surviving cells was coli (IID 5208) and S. aureus (IID 980) were also used in this decreased more than 2 log units compared with the starting study. value.

3. Preparation of bacteria Results To obtain inocula for the test, L. pneumophila SG1 and SG6 were cultured on glycine-vancomycin-polymyxin B- 1. Inhibitory effects of the tested water samples on the growth amphotericin B supplemented with α-ketoglutarate (WYOα, of L. pneumophila Eiken Chemical Co. Ltd., Tokyo) agar, which is a selecting agar L. pneumophila SG1 and SG6 were used to assess the for Legionella species, for 5 days at 37°C. E. coli and S. aureus inhibitory effects of JSPW, SSW, 3.5%NaClPBS, PBS, and PW. were cultured overnight on Nutrient agar medium (Eiken The experiment was performed 5 times within 7 days (i.e., Chemicals Co. Ltd.) at 37°C. After the incubation, each bacteria immediately after the mixture of the water and bacteria, and at suspension was prepared in sterilized purified water, and the 1, 3, 5, and 7 days after incubation), and the surviving cells concentration of the bacteria was adjusted to approximately 104 were counted. As shown in Table 1, the starting value of L. colony-forming units per milliliter (cfu/ml). pneumophila SG1 on BCYEα agar was between 3.94 and 5.43 log units. After 1 day of incubation on BCYEα agar, the number of surviving cells in JSPW, SSW, 3.5%NaClPBS, and

234 Environ. Health Prev. Med. Characteristics of Japan Sea Proper Water

Table 1 Survival of Legionella pneumophila SG1 in water Table 2 Survival of Legionella pneumophila SG6 in water samples samples

(a) BCYEα agar (a) BCYEα agar

Incubation time (days) Incubation time (days) Testing water samples 01357 Testing water samples 0 1 3 5 7

JSPW 4.48 <1 <1 <1 <1 JSPW 4.23 <1 <1 <1 <1 JSPW×10−2 3.94 3.76 2.90 2.78 2.90 JSPW×10−2 4.23 <1 <1 <1 <1 JSPW×10−4 4.00 2.60 <1 <1 <1 JSPW×10−4 4.18 <1 <1 <1 <1 SSW 5.11<1<1<1<1 SSW 4.04 <1 <1 <1 <1 SSW×10−2 4.72 4.26 2.60 2.04 <1 SSW×10−2 4.23 <1 <1 <1 <1 SSW×10−4 5.20 3.80 <1 <1 <1 SSW×10−4 4.43 <1 <1 <1 <1 3.5%NaClPBS 4.70 2.78 <1 <1 <1 3.5%NaClPBS 4.00 2.00 <1 <1 <1 3.5%NaClPBS×10−2 4.89 3.72 2.60 <1 <1 3.5%NaClPBS×10−2 4.08 2.30 <1 <1 <1 3.5%NaClPBS×10−4 5.15 <1 <1 <1 <1 3.5%NaClPBS×10−4 4.11 <1 <1 <1 <1 Purified water 4.74 4.73 4.11 2.48 2.00 Purified water 4.26 2.00 <1 <1 <1 PBS 5.43 4.83 4.99 3.52 4.04 PBS 3.45 2.95 2.00 <1 <1

(b) WYOα agar (b) WYOα agar

Incubation time (days) Incubation time (days) Testing water samples 01357 Testing water samples 0 1 3 5 7

JSPW 4.36 2.00 <1 <1 <1 JSPW 3.64 <1 <1 <1 <1 JSPW×10−2 3.69 3.68 3.18 2.30 3.08 JSPW×10−2 3.45 <1 <1 <1 <1 JSPW×10−4 4.61 2.30 <1 <1 <1 JSPW×10−4 3.84 <1 <1 <1 <1 SSW 4.89<1<1<1<1 SSW 3.57 <1 <1 <1 <1 SSW×10−2 3.86 3.23 2.48 <1 <1 SSW×10−2 3.91 <1 <1 <1 <1 SSW×10−4 4.57 2.48 2.00 <1 <1 SSW×10−4 3.54 <1 <1 <1 <1 3.5%NaClPBS 4.62 2.68 <1 <1 <1 3.5%NaClPBS 3.04 2.30 <1 <1 <1 3.5%NaClPBS×10−2 4.23 3.61 2.00 <1 <1 3.5%NaClPBS×10−2 3.70 <1 <1 <1 <1 3.5%NaClPBS×10−4 4.84 2.85 <1 <1 <1 3.5%NaClPBS×10−4 2.78 <1 <1 <1 <1 Purified water 5.18 3.36 3.18 <1 2.00 Purified water 3.43 <1 <1 <1 <1 PBS 4.96 4.18 3.71 3.23 3.83 PBS 3.30 2.00 2.30 <1 <1 JSPW; Japan Sea Proper Water, SSW; surface seawater, 3.5%NaClPBS; JSPW; Japan Sea Proper Water, SSW; surface seawater, 3.5%NaClPBS; phosphate buffer solution with 3.5% NaCl of pH 7.0, PBS; phosphate phosphate buffer solution with 3.5% NaCl of pH 7.0, PBS; phosphate buffer solution of pH 7.0. buffer solution of pH 7.0. DSW, SSW, and 3.5%NaClPBS were diluted with purified water to 102- DSW, SSW, and 3.5%NaClPBS were diluted with purified water to 102- and 104-fold dilute solutions. and 104-fold dilute solutions. Values show logarithmic colony forming unit per milliliter (log cfu/ml) Values show logarithmic colony forming unit per milliliter (log cfu/ml) after incubation at 37°C. after incubation at 37°C. the 104-fold dilute solution of 3.5%NaClPBS had decreased more than 2 log units from the starting values. In the 102-fold more than 2 log units compared with the starting value. In the dilute solution of 3.5% NaClPBS, the cell number was 1.78 log 102-fold dilute solutions of SSW and 3.5%NaClPBS, and the units lower at 1 day after incubation than the starting value. In 104-fold dilute solutions of JSPW and SSW, the cells had PBS, the cell numbers had decreased more than 2 log units at decreased more than 2 log units compared with the starting 5 days after incubation than the starting value. Although the value at 3 days after incubation. Additionally, the cells in PW starting values on WYOα agar were slightly lower than those had decreased more than 2 log units compared with the starting on BCYEα agar, the changes in the numbers of surviving cells value at 5 days after incubation. The cell numbers in the 102- after incubation showed tendencies quite similar to those on fold dilute solution of JSPW or PBS were 1.04 or 1.39 log units BCYEα agar. lower at 7 days after incubation than the starting value. The We also examined the inhibitory effects of JSPW and PW changes in the number of surviving cells on WYOα agar were on the growth of L. pneumophila SG1 and SG6 in the different quite similar to the results obtained on BCYEα agar. bacteria concentrations. In JSPW, the cells of L. pneumophila Table 2 shows the inhibitory effects of the tested water SG1 and SG6 in concentrations 1 and 2 had decreased more samples on the growth of L. pneumophila SG6. The starting than 2 log units at 1 day after incubation. In PW, however, the value was between 3.45 and 4.43 log units on BCYEα agar. cells of L. pneumophila SG1 and SG6 had decreased more than After 1 day of incubation on BCYEα agar, the number of 2 log units at 1 day after only in concentration 1. Inhibitory surviving cells in all tested water samples except for the 102- effects of JSPW on the growth of L. pneumophila SG1 and SG6 fold dilute solution of 3.5% NaClPBS and PW had decreased in concentrations 1 and 2 were found at 1 day after incubation.

235 Environ. Health Prev. Med. Characteristics of Japan Sea Proper Water

2. Inhibitory effects of the tested water samples on the growth Discussion of E. coli and S. aureus The inhibitory effects of the tested water samples on the In the present study, we found that the surviving cells of growth of E. coli and S. aureus are shown at the top and bottom L. pneumophila SG1 and SG6, E. coli, and S. aureus had of Table 3, respectively. The starting values of E. coli were decreased more than 2 log units in undiluted JSPW at 1 day between 4.46 and 4.59 log units. E. coli in JSPW and SSW had after incubation at 37°C. In the control PW, however, the decreased more than 2 log units at 1 day after incubation than surviving cells of L. pneumophila SG6 and S. aureus had the starting value. In 3.5%NaClPBS, the cell numbers had decreased more than 2 log units at 1 day after incubation, as in decreased more than 2 log units at 3 days after incubation JSPW. This provides evidence that undiluted JSPW has ability compared with the starting value. However, E. coli in the 102- to inhibit the growth of L. pneumophila SG1 and E. coli under and 104-fold dilute solutions of JSPW, SSW, and 3.5% the present experimental conditions. NaClPBS, and that in PBS and PW, survived for 7 days after Before making efficient use of JSPW as bath water, it is incubation. On the other hand, the starting values of S. aureus very important to assess the inhibitory effects of JSPW on the were between 4.63 and 4.94 log units, and the cell numbers in growth of bacteria in order to prevent microbial infections that all of the tested water samples had decreased more than 2 log can be caused by JSPW bathing. L. pneumophila is a facultative units compared with the starting value at 1 day after incubation. bacterium, and Legionnaires’ disease is normally caused by the S. aureus could not survive 3 days after incubation in any of the inhalation or aspiration of Legionella from contaminated tested water samples. aquatic bodies including rivers, lakes, streams, and thermally polluted waters (8). Legionella usually does not harm healthy people, but can seriously infect immunocompromised patients Table 3 Survival of Escherichia coli and Staphylococcus aureus in in hospitals or elderly people. Legionnaires’ disease has been water samples recognized in many countries throughout the world (16–19). In (a) Escherichia coli addition, a large outbreak of Legionnaires’ disease caused by an inadequate circulating and filtration system for communal bath Incubation time (days) water has been reported in Japan (20). In bathhouses or hot Testing water samples 0 1 3 5 7 springs used by the general public, it is necessary to confirm JSPW 4.46 <1 <1 <1 <1 not only the extent of bacterial contaminations such as L. JSPW×10−2 4.48 5.04 5.63 5.41 5.38 pneumophila, E. coli, or S. aureus but also the inhibitory effects JSPW×10−4 4.56 4.57 5.68 5.60 5.73 of bath water on the growth of the bacteria for the purpose of SSW 4.48<1<1<1<1 preventing transmission. We conducted this study to clarify SSW×10−2 4.59 4.08 5.20 5.40 5.38 whether JSPW has an inhibitory effect on the growth of L. SSW×10−4 4.48 4.15 3.26 4.49 3.52 pneumophila SG1 and SG6, E. coli, or S. aureus. 3.5%NaClPBS 4.53 2.78 <1 <1 <1 The activities of bacteria are greatly affected by the −2 3.5%NaClPBS×10 4.49 5.36 5.98 5.99 5.72 chemical and physical conditions of their environment, such −4 3.5%NaClPBS×10 4.57 4.54 5.73 5.98 5.97 as temperature, pH, ionic strength or osmosis, oxygen, carbon Purified water 4.52 4.11 4.62 4.60 5.49 dioxide, and humidity. Although temperature is one of the PBS 4.54 4.71 6.08 5.77 5.76 most important environmental factors influencing the growth and survival of bacteria, pH and osmosis are also important Staphylococcus aureus (b) factors. Because each bacterium usually has a well-defined Incubation time (days) optimum pH for growth (near the neutral pH range, pH 6–7.5), Testing water samples 0 1 3 5 7 we used a pH buffer solution of 7.0 (1/15 mol PBS) as one of the tested waters. On the other hand, JSPW and SSW contain JSPW 4.69 2.63 <1 <1 <1 −2 about 3.5% NaCl plus small amounts of many other minerals JSPW×10 4.79 <1 <1 <1 <1 and elements (15). As most bacteria require about 0.9% NaCl JSPW×10−4 4.90 <1 <1 <1 <1 for growth, many bacteria appeared to be unable to cope with SSW 4.86<1<1<1<1 SSW×10−2 4.91 <1 <1 <1 <1 environments of 3.5% NaCl. We used a 3.5%NaClPBS to SSW×10−4 4.93 <1 <1 <1 <1 assess the effect of the salt in seawater on the growth of the 3.5%NaClPBS 4.85 <1 <1 <1 <1 bacteria. A total of 5 kinds of water (JSPW, SSW, 3.5% 3.5%NaClPBS×10−2 4.71 <1 <1 <1 <1 NaClPBS, PBS, and PW) were used in the present study. 3.5%NaClPBS×10−4 4.63 <1 <1 <1 <1 Our data have demonstrated that undiluted JSPW had an Purified water 4.73<1<1<1<1 inhibitory effect on the growth of L. pneumophila SG1 and PBS 4.94 2.48 <1 <1 <1 SG6, E. coli, and S. aureus. The results obtained in L. JSPW; Japan Sea Proper Water, SSW; surface seawater, 3.5%NaClPBS; pnuemophila corresponded to the previously reported finding phosphate buffer solution with 3.5% NaCl of pH 7.0, PBS; phosphate that salt concentrations of 1.5% NaCl or more had a great buffer solution of pH 7.0. 2 influence on the survival of L. pneumophila serogroup 3 (21). DSW, SSW, and 3.5%NaClPBS were diluted with purified water to 10 - However, the 102-fold dilute solution of JSPW showed no and 104-fold dilute solutions. Values show logarithmic colony forming unit per milliliter (log cfu/ml) inhibitory effect on the growth of L. pneumophila SG1, or E. after incubation at 37°C. coli. Previous studies have demonstrated that virulent cell

236 Environ. Health Prev. Med. Characteristics of Japan Sea Proper Water growth of L. pneumophila was susceptible to inhibition by 103–104 cfu/ml for L. pneumophila SG6, and 104 cfu/ml for S. relatively low NaCl concentrations and appeared to be aureus. Since PW showed inhibitory effects on the growth of L. completely inhibited by NaCl concentrations higher than 0.4% pneumophila SG6 and S. aureus after 1 day of incubation, these (22), and the ability of L. pneumophila to survive in various concentrations might be too low to assess the inhibitory effects concentrations of NaCl and sea water was shown to be of PW on the growth of the bacteria. Further study is needed to enhanced by the addition of small amounts of NaCl (0.1–0.5%), determine whether PW also shows these inhibitory effects when suggesting a protective effect of NaCl (21). The 102-fold dilute higher concentrations of bacteria are present. In addition, no solution of JSPW among the 3 concentrations we used in this nutrients for the growth of the bacteria were added to the test study was the concentration at which L. pneumophila SG1 had tubes. Under environmental stress, such as the low nutrients and its optimal survival rate. JSPW contains higher concentrations high NaCl concentrations used in the present study, the bacteria of nitrite-nitrogen and phosphate-phosphorus, which are inor- may be in a viable but non-culturable (VBNC) state. It is known ganic nutrient salts, than SSW (14). This may be one reason the that, in low-nutrient environments, L. pneumophila cells are 102-fold dilute solution of JSPW showed no inhibitory effect on able to enter the VBNC state (24). Therefore, an examination of the growth of L. pneumophila SG1 whereas the solution of the VBNC state of these bacteria under the conditions tested in SSW did. the present study is essential to prevent the spread of infection. The possibility that the NaCl concentration is associated Moreover, bath water becomes contaminated with soaps, with the survival of E. coli was suggested based on the fact that shampoos, skin-scales, sweat, and other body secretions that the results obtained from L. pneumophila SG1 and E. coli may act as nutrients for bacterial invaders after bathing. Further showed similar tendencies (Tables 1–3). Moreover, an inverse monitoring tests of the bacterial pollution in bath water is association between the calcium and magnesium concentrations necessary to support our findings. Consequently, our next trial of the water and contamination with L. pneumophila has been will be the survival test of the bacteria in water samples previous reported (23). JSPW and SSW are richer in calcium supplied with nutrients for the bacteria. and magnesium than PW (15), and they are susceptible to We have previously reported the following: hot JSPW pollution with L. pneumophila. However, the salt contained in bathing might have the ability to change human immune cell seawater is bound to inhibit the growth of L. pneumophila. distribution, whereas SSW bathing showed no such effect (6). On the other hand, all of the tested water samples showed Also, among JSPW, SSW, and PW, JSPW was shown to be inhibitory effects on the survival of S. aureus at 1 day after mildest to the human body (7). In addition, subjects reported a incubation or later, although S. aureus is relatively resistant to sticky feeling after SSW bathing, whereas no sticky feeling was high salt content and also requires many macronutrients and reported after JSPW bathing (6, 7). These findings suggest that micronutrients for growth. The inhibitory effects may be due to JSPW is better bath water than SSW. a lack of the nutrients required for growth in the tested water In conclusion, the current study indicates that the growth samples, as no nutrients were added to the test tubes. of L. pneumophila SG1 and SG6, E. coli, and S. aureus were The pH levels of JSPW and SSW have been examined inhibited in undiluted JSPW for 7 days after incubation at 37°C, using a glass electrode method with the following results: just as they are in SSW and 3.5%NaClPBS. In the control PW, JSPW pH, 7.87; SSW pH, 8.25 (15). Despite the different pH however, the surviving cells of L. pneumophila SG6 and S. values, the results of surviving cells obtained for JSPW were aureus had also decreased more than 2 log units after 1 day of quite similar to those for SSW and 3.5%NaClPBS (pH 7.0). incubation. These findings suggest that undiluted JSPW has These results suggest that the inhibitory effect of pH on the inhibitory effects on the growth of L. pneumophila SG1 and growth of L. pneumophila SG1 and E. coli may be smaller than E. coli, and that the inhibitory effects may be due to the high that of NaCl concentration. concentration of salt in JSPW. In PW and PBS, no inhibitory effects on the growth of L. pneumophila SG1 and E. coli were found. Therefore, it is Acknowledgements possible that these bacteria can survive in environmental water under typical environmental temperature conditions, and so This study was supported by a Grant-in-Aid from Niigata environmental water can be a source of contagion. Prefecture as a part of the Japan Sea Proper Water Utilization Potential limitations of our study warrant discussion. For Technology Research and Development Program in 2003. example, we used bacteria concentrations of approximately

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