Antimicrobial activity of endophytic and rhizospheric fungi associated with soft fern (Christella sp.) and cinderella weed (Synedrella nodiflora) inhabiting a hot spring in Los Baños, Laguna, Philippines

@#<p style="text-align: justify;"><strong>Background.</strong> The growing resistance of microorganisms to antimicrobial agents is a pressing public health issue. Bioprospecting efforts have mainly focused on well-known environments such as soil and animal gut in search for microorganisms with antibiotic production or antimicrobial activity, or terrestrial ecosystems for endemic plants with bioactive compounds. However, microbial communities thriving in stressed environments such as hot springs, are potential sources of metabolites that can be screened for antimicrobial activity. There is a need for research on bioprospecting of fungi as potential sources of antimicrobials.</p><p style="text-align: justify;"><strong>Objectives.</strong> The study aimed to test the antimicrobial activity of endophytic and rhizospheric fungi associated with soft fern (Christella spp.) and Cinderella weed (Synederella nodiflora) inhabiting a hot spring in Los Baños, Laguna, Philippines.</p><p style="text-align: justify;"><strong>Methods.</strong> A total of 23 endophytic and rhizospheric fungi isolated from soft fern and Cinderella weed were purified and phenotypically identified. These isolates were subjected to agar well diffusion and agar plug diffusion methods as preliminary assays for antimicrobial activity against Bacillus subtilis var. spizizenii (ATCC® 6633™), Staphylococcus  aureus (ATCC® 25923™), four multi-antibiotic resistant Escherichia coli (OT11, OT16, OT18, OT22), and Cladosporium  cladosporioides. Based on the results of the preliminary screening, ethyl-acetate extracts of selected fungal isolates were subjected to broth microdilution assay to determine the minimum inhibitory concentrations (MICs) for antibacterial activity, as well as poisoned food technique to determine the percent mycelial inhibition for antifungal activity. The nearest phylogenetic affiliations of fungal isolates with higher antimicrobial activities were determined.</p><p style="text-align: justify;"><strong>Results.</strong> Ten rhizospheric fungal isolates from Cinderella weed and seven rhizospheric and six endophytic fungal isolates from soft fern were phenotypically identified as Aspergillus, Coniothyrium, Fusarium, Penicillium, Talaromyces,  and Trichoderma species. Ethyl acetate extracts from endophytic fungal isolates UL1 (Trichoderma sp.) and UL2 (Trichoderma sp.) and rhizospheric fungal isolates UR1 (Trichoderma sp.) and UR3 (Trichoderma sp.) showed activity against the test bacteria at 128-256 ?g/mL concentrations. Isolates UL1, UL2, and UR3, which exhibited higher antibacterial activities, were sequenced and confirmed to be most phylogenetically related to Trichoderma  virens. Eleven fungal isolates belonging to Aspergillus spp., Coniothyrium spp., Fusarium spp., Penicillium spp., and Talaromyces spp. demonstrated antagonism against C. cladosporioides. The rhizospheric fungal isolate FCRU4 (Talaromyces sp.), from where ethyl acetate extracts were recovered for testing mycelial inhibition, was confirmed to be most phylogenetically related to Talaromyces islandicus.</p><p style="text-align: justify;"><strong>Conclusion.</strong> Endophytic and rhizospheric fungi asso ciated with Cinderella weed (Synedrella nodiflora) and soft fern (Christella sp.) from a hot spring in Los Baños, Laguna, Philippines have antimicrobial activity.</p>

Screening and Characterization of Bacillus Strains Producing Highly Thermostable Amylase from Various Hot Springs of Algeria

HIGHLIGHTS Screening extremophile Bacillus strains from various Hot Springs Characterization Of Bacillus Strains Producing Highly Thermostable Amylase Genetic identification of the best strains

Abstract Currently thermostable Amylase represents a broad biotechnological interest and desired by a various industries. In this study, forty-six bacterial strains belonging to the genus Bacillus were isolated from various hot springs in the North West of Algeria based on their ability to degrade starch and produce amylase in Starch Agar medium. The majority of isolates showed a positive amylolytic activity. In order to select the most thermostables amylase the effect of temperature on enzymes was estimated, therefore the study of amylase thermostability was culminated by the selection of Four Strains having an interesting optimum of activity and range of stability, reaching 75 °C for the strains HBH1-2, HBH1-3, HBH3-1and 85 °C for the strain HC-2, This indicates that the Enzyme produced by retained strains have optimum activity at high temperature. The identity of the selected strains was established on the basis of the morphological, biochemical characteristics and phylogenetic position as determined by 16S Ribosomal DNA gene sequencing. The whole strains belonged to the genus Bacillus and their phylogeny were also reported in this study.

Toxicidad a dosis repetidas (28 días) por vía oral del agua termomineral de San Antonio de Putina, Puno / Repeated dose 28-day oral toxicity study of hot springs water from San Antonio de Putina, Puno

Objetivo. Determinar la toxicidad a dosis repetidas durante 28 días, de la ingestión por vía oral del agua termal de San Antonio de Putina-SAP-(Puno), en un modelo murino. Materiales y métodos. Diseño experimental, se utilizaron diez ratas de experimentación de cepa Holtzman, los que fueron divididos en dos grupos de cinco cada uno: un grupo de estudio, al que se le administró en forma repetida, agua termal traída de SAP, en una dosis de 1000 mg/kg en un volumen de 2 mL/100g, y un grupo control con agua potable esterilizada en el mismo volumen. Se realizaron observaciones clínicas diarias, determinación semanal del peso corporal, y después de 28 días se procedió con el estudio histopatológico de órganos (corazón, riñón, hígado y pulmón) y la determinación de parámetros hematológicos y bioquímicos; previo sacrificio de los animales de experimentación. Resultados. No se produjeron muertes (DL50> 1000 mg/kg) ni alteraciones permanentes de signos clínicos. Se observó aumento de peso, sin diferencias estadísticas entre los grupos. Los resultados hematológicos y bioquímicos reflejaron ligeras variaciones no significativas entre grupos, pero dentro del rango de la normalidad. No se observaron alteraciones histopatológicas. Conclusiones. No se encontraron signos de toxicidad aguda ante la administración de agua termomineral de San Antonio de Putina (Puno) en dosis repetidas por un periodo de 28 días.

Objective. To determine 28-day oral acute toxicity at repeated doses of hot springs water from San Antonio de Putina ­SAP- (Puno) in rats. Materials and methods. Experimental design, ten Holtzman rats were used, which were divided into two groups of five each: a study group that was exposed to repeated administration of SAP hot spring water in a dose of 1000 mg / kg in a volume of 2 mL/100g; and a control group, which sterilized water was given in the same volume. Daily clinical observations were made, weekly determination of body weight; and after 28 days; previous sacrifice of animals, the histopathological study of organs (heart, kidney, liver and lung) and determination of hematological and biochemical parameters were made. Results. No deaths (LD50> 1000 mg/kg) or permanent alterations of clinical signs were observed. Weight gain without statistical differences between the groups was observed. The hematological and biochemical results showed slight nonsignificant variations between groups, but in normality levels. No histopathological alterations were observed. Conclusions. No signs of acute toxicity were found after 28-days repeated administration of hot spring water from San Antonio de Putina (Puno).

Investigation of Immune-Regulatory Effects of Mageumsan Hot Spring via Protein Microarray In Vitro

BACKGROUND: Empirical evidences for efficacy of hot spring (HS) water in inflammatory skin disorders have not been substantiated with sufficient, immunological “hard evidence”. Mageumsan HS water, characterized by its weakly-alkaline properties and low total dissolved solids content, has been known to alleviate various immune-inflammatory skin diseases, including atopic dermatitis (AD). OBJECTIVE: The trial attempted to quantitatively analyze in vitro expression levels of chemical mediators in cutaneous inflammation from HaCaT cell line treated with Mageumsan HS, and suggest the likely mode of action through which it exerts the apparent anti-inflammatory effects in AD. METHODS: Using membrane-based human antibody array kit, customized to include 30 different, keratinocyte-derived mediator proteins, their expression levels (including interleukin [IL]-1, IL-6, IL-8, thymic stromal lymphopoietin, thymus and activation-regulated chemokine, and granulocyte macrophage colony-stimulating factor) were assessed in vitro. Selected key proteins were further quantified with enzyme-linked immunosorbent assay. RESULTS: There was a clear pattern of overall suppression of the mediators, especially those noted for their pro-inflammatory role in AD (monocyte chemoattractant protein [MCP]-1, regulated on activation, normal T cell expressed and secreted, cutaneous T-cell-attracting chemokine, Eotaxin, and macrophage inflammatory protein-1α, etc.). Also, reduced expression of involucrin and cytokeratin 1 was also reduced in the HS-treated group. CONCLUSION: The present study has shown that Mageumsan HS water may exert its effects on inflammatory skin disorders through regulation of proinflammatory cytokines. These evidences are to be supported with further future investigations to elucidate immunological mechanism behind these beneficial effects of HS water in the chronically inflamed skin of AD.

Characterization of a hyperthermophilic sulphur-oxidizing biofilm produced by archaea isolated from a hot spring

Background: Sulphur-oxidizing microorganisms are widely used in the biofiltration of total reduced sulphur compounds (odorous and neurotoxic) produced by industries such as the cellulose and petrochemical industries, which include high-temperature process steps. Some hyperthermophilic microorganisms have the capability to oxidize these compounds at high temperatures (N60°C), and archaea of this group, for example, Sulfolobus metallicus, are commonly used in biofiltration technology. Results: In this study, a hyperthermophilic sulphur-oxidizing strain of archaea was isolated from a hot spring (Chillán, Chile) and designated as M1. It was identified as archaea of the genus Sulfolobus (99% homology with S. solfataricus 16S rDNA). Biofilms of this culture grown on polyethylene rings showed an elemental sulphur oxidation rate of 95.15 ± 15.39 mg S l-1 d-1, higher than the rate exhibited by the biofilm of the sulphur-oxidizing archaea S. metallicus (56.8 ± 10.91 mg l-1 d-1). Conclusions: The results suggest that the culture M1 is useful for the biofiltration of total reduced sulphur gases at high temperatures and for other biotechnological applications.

Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils

The diversity of thermophilic bacteria was investigated in four hot springs, three salt marshes and 12 desert sites in Morocco. Two hundred and forty (240) thermophilic bacteria were recovered, identified and characterized. All isolates were Gram positive, rod-shaped, spore forming and halotolerant. Based on BOXA1R-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by the genus Bacillus (97.5%) represented by B. licheniformis (119), B. aerius (44), B. sonorensis (33), B. subtilis (subsp. spizizenii (2) and subsp. inaquosurum (6)), B. amyloliquefaciens (subsp. amyloliquefaciens (4) and subsp. plantarum (4)), B. tequilensis (3), B. pumilus (3) and Bacillus sp. (19). Only six isolates (2.5%) belonged to the genus Aeribacillus represented by A. pallidus (4) and Aeribacillus sp. (2). In this study, B. aerius and B. tequilensis are described for the first time as thermophilic bacteria. Moreover, 71.25%, 50.41% and 5.41% of total strains exhibited high amylolytic, proteolytic or cellulolytic activity respectively.

Immunomodulatory Effects of Balneotherapy with Hae-Un-Dae Thermal Water on Imiquimod-Induced Psoriasis-Like Murine Model

BACKGROUND: Balneotherapy, although not a well-established dermatological treatment, is thought to have therapeutic properties for psoriasis and is used as an alternative treatment modality throughout the world. OBJECTIVE: To evaluate the mechanism underlying the therapeutic immunologic effects of thermomineral water. METHODS: A murine model of imiquimod-induced psoriasis-like skin inflammation was used for evaluating the therapeutic effects of balneotherapy with Hae-Un-Dae hot spring mineral water. The clinical improvements were evaluated by a dermatologist. Lesional cytokines, including interleukin (IL)-17A, IL-23, and IL-22, were quantitatively measured by real-time reverse transcriptase polymerase chain reaction. Serum levels of interferon-gamma, IL-4, IL-5, and IL-17A were measured by enzyme-linked immunosorbent assay. T cell proportions in the spleen were evaluated by flow cytometry, and histopathological evaluation of the skin was also performed. RESULTS: The mineral water balneotherapy group showed faster improvement in skin erythema and scales than the distilled water bathing group. A substantial reduction was observed in the lesional mRNA levels of IL-17A and IL-23 in the mineral water group. Serum levels of IL-4 and IL-5 were significantly decreased in the mineral water group but not in the distilled water group. Normalized T cell proportions were observed after bathing. CONCLUSION: Balneotherapy showed immunomodulatory effects in a psoriasis-like murine model. Balneotherapy suppressed lesional IL-23 and IL-17A, which are important cytokines in the pathogenesis of psoriasis. These results suggest that balneotherapy can be used as an effective and safe treatment for psoriasis.

Three quantitative methods to continuously monitor Legionella in spring water / 中华预防医学杂志

<p><b>OBJECTIVE</b>To compare the detection effect of Legionella pollution in spring water by three methods, namely traditional plating method, fluorescent quantitation PCR method and ethidium monoazide (EMA) fluorescent quantitation PCR method.</p><p><b>METHODS</b>Every month (except May), we collected 11 water samples from the 5 selected hot spring pools in one hot spring resort in Beijing in 2011. A total of 121 water samples were collected, and then were detected by the above three methods qualitatively and quantitatively.</p><p><b>RESULTS</b>In our study, the Legionella pollution rate was separately 74.4% (90/121), 100.0% (121/121) and 100.0% (121/121) by the above three methods. The quantitative value of Legionella in the 121 water samples detected by the three methods were around 0.10-216.00 colony-forming units (CFU)/ml, 1.47-1557.75 gene units (GU)/ml and 0.20-301.69 GU/ml, respectively. The median (25th and 75th percentiles) was 75.30 (32.51-192.10) GU/ml, 36.46 (16.08-91.21) GU/ml and 5.30 (0.00-33.70) CFU/ml, respectively. The difference in the quantitative value of Legionella detected by the three methods showed statistical significance (χ(2) = 187.900, P < 0.01). The quantitative value of Legionella detected by fluorescent quantitation PCR method was the highest, followed by the value Legionella detected by EMA-fluorescent quantitation PCR method and traditional plating method.</p><p><b>CONCLUSION</b>The sensitivity of the PCR methods was higher than traditional plating method, in detecting Legionella pollution in spring water, especially the EMA- fluorescent quantitation PCR method, which was more suitable for detecting Legionella in water.</p>

Therapeutic Effects and Immunomodulation of Suanbo Mineral Water Therapy in a Murine Model of Atopic Dermatitis

BACKGROUND: Balneotherapy is widely used as an alternative treatment modality for AD. Although the clinical benefit of some mineral waters has been established, their mechanisms of action in alleviating AD are only partly understood. OBJECTIVE: The clinical modification and immunomodulatory or anti-inflammatory effects of mineral water from the Suanbo hot springs on the differentiation and cytokine production of Th1, Th2, and regulatory T cells (Treg) were investigated using spleen, skin tissue, and serum from NC/Nga mice. METHODS: The therapeutic effects of bathing in mineral water in a Dermatophagoides farinae body extract ointment (Dfb ointment)-induced AD mouse model were assessed by measuring the modified Scoring atopic dermatitis (SCORAD) index scores, transepidermal water loss (TEWL), histological and immunohistochemical changes of the skin lesion, serum levels of interferon (IFN)-gamma, interleukin (IL)-4, IL-5 and immunoglobulin E, mRNA expression of IFN-gamma, IL-4 and IL-5 of dorsal skin, and helper T cell differentiation in the spleen. RESULTS: Bathing in mineral water significantly reduced the modified SCORAD index scores, TEWL, epidermal hyperplasia, and inflammatory cell infiltration. IL-4 production and Th2 cell differentiation showed a decreasing tendency with mineral water bathing, but the Th1 cells did not. On the contrary, differentiation to Treg cells was promoted with mineral water bathing. CONCLUSION: Balneotherapy not only has anti-inflammatory activity, but also shows positive effects on cutaneous barrier homeostasis. These results suggest that the favorable effects of balneotherapy may be mediated by modifying the Th2 response, and possibly in part by inducing Treg cell differentiation.

Occurrence of thermotolerant hartmannella vermiformis and naegleria spp. in hot springs of Ardebil province, northwest Iran

Geothermal waters could be suitable niches for thermophilic free living amoebae including Naegleria and Hartmannella. Ardebil Province, northwest Iran is popular for having many hot springs for recreational and health purposes activity. The present research is the first molecular based investigation regarding the presence of Naegleria and Hartmannella in the hot springs of Ardebil Province in Iran. Overall, 30 water samples were taken from waters of thermal hot springs in Ardebil Province, Iran during 2010-2011. All collected samples were transferred to Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Cultivation of concentrated water samples was performed using culture-enrichment method. Cloning of the target amoebae was obtained and morphological and molecular analysis was done using page key combined with two sets of primers, respectively. Sequence analysis and homology search was used for strains identification. Of 30 water samples, 8 [26.7%] were positive for thermotolerant Vahlkampfiids and Hartmannella based on morphological characteristics of vegetative form and double walled cysts. Cloning of the target amoebae were done successfully. Sequencing of the positive isolates revealed that the strains belonged to Naegleria [N. carteri and N. spp] and H. vermiformis. The result highlights a need for improved filtration and disinfection and periodic monitoring of recreational thermal waters in order to prevent disease related to free- living amoebae. This is the first comprehensive molecular study of thermophilic Naegleria and Hartmannella in hot springs of Iran

The Anti-inflammatory and Antimicrobial Effects of Selenium-rich Hot Spring Water on a Chronic Bacterial Prostatitis Rat Model / 대한남성과학회지

PURPOSE: The conventional primary therapy for chronic bacterial prostatitis (CBP) is the use of antibiotics. However, the therapy has a low cure rate and long-term use of antibiotics can lead to adverse effects including bacterial resistance. For these reasons, a new therapy for CBP is strongly needed. MATERIALS AND METHODS: To evaluate the anti-inflammatory and antimicrobial effects of selenium-rich hot spring water on CBP, rats were divided into four groups and treatment was administered for four weeks as follows: (1) control (n=8), (2) ciprofloxacin (n=8), (3) selenium-rich hot spring water (n=8), and (4) selenium-rich hot spring water with ciprofloxacin (n=8). Drip infusion of bacterial suspension (E. coli Z17 O2:K1;H-) into Spargue-Dowley rats was then conducted to induce CBP. Four weeks later, the results of prostate tissue and urine culture and histological analysis on the prostate were analyzed in each group. RESULTS: The use of ciprofloxacin, and selenium-rich hot spring water with ciprofloxacin showed statistically significant decreases in bacterial growth and improvements in prostatic inflammation compared with the control group (p<0.05). The selenium-rich hot spring water with ciprofloxacin group showed a statistically significantly lower rate of bacterial growth and and greater improvements in prostatic inflammation than the ciprofloxacin group (p<0.05). CONCLUSIONS: These results suggest that spring water may be an effective material in the treatment of CBP. Notably, the combination treatment of selenium-rich hot spring water and ciprofloxacin has synergistic effects. Therefore, we can suggest that the combination of selenium-rich hot spring water and ciprofloxacin may be effective in the treatment of CBP, and with a higher success rate than ciprofloxacin alone.

[Effective dose rate of radon gas in Jooshan hot spring of Kerman Province]

Human beings are constantly exposed to different radiations that have always been recognized as a health hazard. Radon-222 and its daughter products are major sources of natural radiations and a significant total inhalation dose is related to them. Hence, the measurement of radon activity in the environment has gained an increasing importance. In this study, with the measurement of radon concentration, the indoor radon activity level and radon effective dose rate in Jooshan hot spring were determined. Concentration of radon gas inside the pool of Jooshan hot spring was measured using the active detector Rad7 type and the annual effective dose was estimated for individuals inhaling the indoor air of this pool for a specified period of time. Concentration of indoor air radon gas was 98.3 +/- 4.9 Bq/m3 and for a person staying in the pool twice a week and each time for two hours, the annual effective dose rate, due to the inhalation of radon, equals to 0.06 +/- 0.003 mSv/y. Comparison of the obtained effective dose with the standards of Environmental protection Agency and Health physics Society, it can be concluded that for individuals that normally use Jooshan hot spring pool, the resulted dose is in the recommended range

Isolation of free-living amoebae from Sarein hot springs in Ardebil province, Iran

Free-living amoebae [FLA] are a group of ubiquitous protozoan, which are distributed in the natural and artificial environment sources. The main aim of the current study was to identify the presence of FLA in the recreational hot springs of Sarein in Ardebil Province of Iran. Seven recreational hot springs were selected in Sarein City and 28 water samples [four from each hot spring] were collected using 500 ml sterile plastic bottles during three month. Filtration of water samples was performed, and culture was done in non-nutrient agar medium enriched with Escherichia coli. Identification of the FLA was based on morphological criteria of cysts and trophozoites. Genotype identification of Acanthamoeba positive samples were also performed using sequencing based method. Overall, 12 out of 28 [42.9%] samples were positive for FLA which Acanthamoeba and Vahlkampfiid amoebaewere found in one [3.6%] and 11 [39.3%] samples, respectively. Sequence analysis of the single isolate of Acanthamoeba revealed potentially pathogenic T[4] genotype corresponding to A. castellanii. Contamination of hot springs to FLA, such as Acanthamoeba T[4] genotype [A. castellanii] and Vahlkampfiid amoebae, could present a sanitary risk for high risk people, and health authorities must be aware of FLA presence

The Effect of Selenium-rich Hot Spring Water on Serum Leptin and IGF-1 in a Rat Model / 대한남성과학회지

PURPOSE: There have been reports that a higher serum level of selenium is related to a lower incidence of prostate cancer. Serum leptin and IGF-1 are factors related to metabolic syndromes which are also known to be related to prostate cancer, and the higher their levels, the higher the possibility of prostate carcinogenesis. Thus, we measured the serum levels of leptin and IGF-1 in rats that were given selenium-rich hot spring water to drink in an attempt to elucidate the role of selenium on prostate cancer prevention. MATERIALS AND METHODS: A total of twelve white SD rats was included in the study, and they were further divided into a control group (Group 1, n=6) induced to drink normal saline, and those rats that drank selenium-rich hot spring water for 4 weeks (Group 2, n=6). The hot spring water that we used was from the Kum-jin region of Korea, which contains a rich supply of the minerals selenium, vanadium, calcium and magnesium. The concentration of selenium in the hot spring water was 480 microgram/l. The two groups were each given 50 ml/day of normal saline or selenium-rich hot spring water. After the period of the experiment, we drew blood samples from both groups and measured their serum leptin and IGF-1 levels using ELISA. RESULTS: The serum leptin level (pg/ml) was 964.04+/-127.77 in Group 1, and 531.92+/-151.04 in Group 2, allowing us to confirm that it was significantly less in those that drank the selenium-rich hot spring water (p<0.05). Also, the serum IGF-1 levels (pg/ml) were 1,247.72+/-53.85 and 933.03+/-113.69 in Groups 1 and 2, respectively, which again, illustrated the significantly lower level in those rats that were given selenium-rich hot spring water (p<0.05). CONCLUSIONS: Significantly decreased serum levels of leptin and IGF-1 were observed in rats that drank selenium-rich hot spring water. A study of the long-term effect of selenium-rich hot spring water on prostate cancer is necessary; if it is indeed proven to have long-term effects, we believe it will become valuable in clinical practice.

Study on the status of pollution by Legionella species in hot spring vacation center and the related factors / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the pollution status of Legionella species in hot spring vacation center and the related factors.</p><p><b>METHODS</b>Field surveys were performed in four big hot spring vacation centers of Changping district. Uniform questionnaires was used and colony count was made together with the isolation of Legionella species from hot spring water based on mip gene typing.</p><p><b>RESULTS</b>47 isolates of Legionella pneumophila (Lp) from 87 samples showed 4 serotypes as Lp1, Lp6, Lp12, Lp5 with percent of 57.45%, 21.28%, 14.89%, 6.38% respectively. The hot spring centers controlled the temperature of recycled water between 34-47 degrees C by hot water heating and filtrating system. All the isolates were cultured from the hot water with temperature between 34-44 degrees C: 56.75% (21/37) in high temperature (40-47 degrees C) and 61.90% (26/42) in low temperature (34-39.9 degrees C). There were no statistically significant difference between the high and the low temperature samples (P > 0.05). In the four hot spring vacation centers, the pH value was under control at 6.4-7.3 and the ambient temperature was under control between 26-28 degrees C. The humidity was controlled between 56% -69% relative humidity, which were the best growing conditions for the Legionella species. Disinfectors as chlorine deviratives was used in the four hot spring vacation centers. Though the concentration of chlorine in the water was 0.3-0.5 mg/L, 14.29%-48.00% of the samples were still positive of having Legionella species.</p><p><b>CONCLUSION</b>The pollution of Legionella species was considered to be quite serious in the four hot spring vacation centers and the predominant serotype was Lp1. The pH and temperature of the hot spring water, ambient temperature and humidity and the way of heating up the water were the best conditions for the growth of Legionella species in these centers. Because of the high temperature of the hot spring water, chlorine of the disinfector volatilized quickly, affecting the effect of disinfection. The result revealed that water temperature achieving 44 degrees C could have had the effect of prevention.</p>

Spa, springs and safety.

Natural mineral water has long been used worldwide for bathing and health purposes. At present, Thailand is famous for health spas and natural hot springs among local people and tourists. Due to possible risks of exposure to harmful agents, we studied hazardous pollutants at 57 natural hot springs from 11 provinces in northern, central, eastern and southern Thailand. Pathogenic, free-living amebae of the genera Naegleria and Acanthamoeba, which can cause central nervous system infection, were found in 26.3% (15/57) and 15.8% (9/ 57), respectively. Dissolved radon, a soil gas with carcinogenic properties, was present in nearly all hot springs sites, with concentration ranging from 0.87-76,527 Becquerels/m3. There were 5 water samples in which radon concentration exceeded the safety limit for drinking. Legionella pneumoniphila (serogroups 1, 3, 5, 6, 7 10 and 13) were found in samples from 71.9% (41/57) of studied sites. Because spas and natural springs are popular tourist attractions, health authorities should be aware of possible hazards and provide tactful measures and guidelines to ensure safety without causing undue alarm to foreign and Thai tourists.

Free-living ameba contamination in natural hot springs in Thailand.

Thermo tolerant free-living ameba, Naegleria spp and Acanthamoeba spp contamination in natural hot springs in Thailand were carried out from 13 provinces. The temperature of hot springs water varied from 28 degrees-65 degrees C and pH from 6-8. We found that 38.2 % (26/68) of water samples were positive, Acanthamoeba was 13.2% (9/68) whilst Naegleria was 35.3% (24/68). Contamination by free-living ameba in natural hot springs may pose a significant health risk to people who use such water for recreation activities.