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Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay

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Abstract

In this study, a SYBR green quantitative real-time PCR was developed to quantify and detect the Legionella spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and thermal spring area in Taiwan. Legionella was detected in 13.6 % (24/176), and the detection rate for river water, raw drinking water, and thermal spring water was 10, 21.4, and 16.6 %, respectively. Using real-time PCR, concentration of Legionella spp. in detected samples ranged between 9.75 × 104 and 3.47 × 105 cells/L in river water, 6.92 × 104 and 4.29 × 105 cells/L in raw drinking water, and 5.71 × 104 and 2.12 × 106 cells/L for thermal spring water samples. The identified species included Legionella pneumophila (20.8 %), Legionella jordanis (4.2 %), Legionella nautarum (4.2 %), Legionella sp. (4.2 %), and uncultured Legionella sp. (66.6 %). The presence of L. pneumophila in aquatic environments suggested a potential public health threat that must be further examined.

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References

  • Al-Matawah QA, Al-Zenki SF, Qasem JA, Al-Waalan TE, Ben Heji AH (2012) Detection and quantification of Legionella pneumophila from water systems in Kuwait residential facilities. J Pathog 2012(138389):1–5

  • Allegra S, Berger F, Berthelot P, Grattard F, Pozzetto B, Riffard S (2008) Use of flow cytometry to monitor Legionella viability. Appl Environ Microbiol 74:7813–7816

    Article  CAS  Google Scholar 

  • APHA (2005) Standard method for the examination of water and wastewater. APHA, WEF and AWWA, Washington, DC

    Google Scholar 

  • Bangsborg JM (1997) Antigenic and genetic characterization of Legionella proteins: contributions to taxonomy, diagnosis and pathogenesis. APMIS Supplementum 70:1–53

    CAS  Google Scholar 

  • Bej AK (2003) Molecular based methods for the detection of microbial pathogens in the environment. J Microbiol Methods 53:139–140

    Article  Google Scholar 

  • Bonetta S, Bonetta S, Ferretti E, Balocco F, Carraro E (2010) Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real-time PCR and culture methods. J Appl Microbiol 108:1576–1583

    Article  Google Scholar 

  • Costa J, Tiago I, da Costa MS, Verissimo A (2005) Presence and persistence of Legionella spp. in groundwater. Appl Environ Microbiol 71:663–671

    Article  CAS  Google Scholar 

  • Declerck P, Behets J, van Hoef V, Ollevier F (2007) Detection of Legionella spp. and some of their amoeba hosts in floating biofilms from anthropogenic and natural aquatic environments. Water Res 41:3159–3167

    Article  CAS  Google Scholar 

  • DSMZ (2012) Bacterial nomenclature up-to-date. http://old.dsmz.de/microorganisms/bacterial_nomenclature_info.php?genus=Legionella. 7 Jan 2013

  • Edelstein PH (1982) Comparative study of selective media for isolation of Legionella pneumophila from potable water. J Clin Microbiol 16:697–699

    CAS  Google Scholar 

  • Flannery B, Gelling LB, Vugia DJ, Weintraub JM, Salerno JJ, Conroy MJ, Stevens VA, Rose CE, Moore MR, Fields BS, Besser RE (2006) Reducing Legionella colonization in water systems with monochloramine. Emerg Infect Dis 12:588–596

    Article  CAS  Google Scholar 

  • Gomez-Valero L, Rusniok C, Buchrieser C (2009) Legionella pneumophila: population genetics, phylogeny and genomics. Infect Genet Evol 9:727–739

    Article  CAS  Google Scholar 

  • Greub G, Raoult D (2004) Microorganisms resistant to free-living amoebae. Clin Microbiol Rev 17:413–433

    Article  Google Scholar 

  • Herpers BL, de Jongh BM, van der Zwaluw K, van Hannen EJ (2003) Real-time PCR assay targets the 23S-5S spacer for direct detection and differentiation of Legionella spp. and Legionella pneumophila. J Clin Microbiol 41:4815–4816

    Article  CAS  Google Scholar 

  • Hsu BM, Chen CH, Wan MT, Cheng HW (2006) Legionella prevalence in hot spring recreation areas of Taiwan. Water Res 40:3267–3273

    Article  CAS  Google Scholar 

  • Hsu BM, Lin CL, Shih FC (2009) Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area. Water Res 43:2817–2828

    Article  CAS  Google Scholar 

  • Hsu BM, Huang CC, Chen JS, Chen NH, Huang JT (2011) Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments. Water Res 45:5171–5183

    Article  CAS  Google Scholar 

  • Huang SW, Hsu BM, Ma PH, Chien KT (2009) Legionella prevalence in wastewater treatment plants of Taiwan. Water Sci Technol 60:1303–10

    Article  CAS  Google Scholar 

  • Huang SW, Hsu BM, Wu SF, Fan CW, Shih FC, Lin YC, Ji DD (2010) Water quality parameters associated with prevalence of Legionella in hot spring facility water bodies. Water Res 44:4805–4811

    Article  CAS  Google Scholar 

  • Huang SW, Hsu BM, Chen NH, Huang CC, Huang KH, Chen JS, Kao PM (2011a) Isolation and identification of Legionella and their host amoebae from weak alkaline carbonate spring water using a culture method combined with PCR. Parasitol Res 109:1233–1241

    Article  Google Scholar 

  • Huang SW, Hsu BM, Huang CC, Chen JS (2011b) Utilization of polymerase chain reaction and selective media cultivation to identify Legionella in Taiwan spring water samples. Environ Monit Assess 174:427–437

    Article  CAS  Google Scholar 

  • Inoue H, Noda A, Takama T, Ishima T, Agata K (2006) Enhanced antifungal effect of the selective medium for the detection of Legionella species by a combination of cycloheximide, amphotericin B and thiabendazole. Biocontrol Sci 11:69–74

    Article  CAS  Google Scholar 

  • Joly P, Falconnet PA, Andre J, Weill N, Reyrolle M, Vandenesch F, Maurin M, Etienne J, Jarraud S (2006) Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl Environ Microb 72:2801–2808

    Article  CAS  Google Scholar 

  • Kao PM, Hsu BM, Chen NH, Huang KH, Huang CC, Ji DD, Chen JS, Lin WC, Huang SW, Chiu YC (2012) Molecular detection and comparison of Acanthamoeba genotypes in different functions of watersheds in Taiwan. Environ Monit Assess 184:4335–44

    Article  CAS  Google Scholar 

  • Koide M, Saito A, Kusano N, Higa F (1993) Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method. Appl Environ Microb 59:1943–1946

    CAS  Google Scholar 

  • Kuiper MW, Valster RM, Wullings BA, Boonstra H, Smidt H, van der Kooij D (2006) Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR. Appl Environ Microb 72:5750–5756

    Article  CAS  Google Scholar 

  • Lasheras A, Boulestreau H, Rogues AM, Ohayon-Courtes C, Labadie JC, Gachie JP (2006) Influence of amoebae and physical and chemical characteristics of water on presence and proliferation of Legionella species in hospital water systems. Am J Infect Control 34:520–525

    Article  Google Scholar 

  • Lee HK, Shim JI, Kim HE, Yu JY, Kang YH (2010) Distribution of Legionella species from environmental water sources of public facilities and genetic diversity of L. pneumophila serogroup 1 in South Korea. Appl Environ Microbiol 76:6547–6554

    Article  CAS  Google Scholar 

  • Lee TC, Vickers RM, Yu VL, Wagener MM (1993) Growth of 28 Legionella species on selective culture media: a comparative study. J Clin Microbiol 31:2764–2768

    CAS  Google Scholar 

  • Leoni E, Legnani PP, Bucci Sabattini MA, Righi F (2001) Prevalence of Legionella spp. in swimming pool environment. Water Res 35:3749–3753

    Article  CAS  Google Scholar 

  • Mackay IM (2004) Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 10:190–212

    Article  CAS  Google Scholar 

  • Mathys W, Stanke J, Harmuth M, Junge-Mathys E (2008) Occurrence of Legionella in hot water systems of single-family residences in suburbs of two German cities with special reference to solar and district heating. Int J Hyg Environ Health 211:179–185

    Article  Google Scholar 

  • Miyamoto H, Yamamoto H, Arima K, Fujii J, Maruta K, Izu K, Shiomori T, Yoshida S (1997) Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of Legionellae in hospital cooling tower water. Appl Environ Microb 63:2489–2494

    CAS  Google Scholar 

  • Morio F, Corvec S, Caroff N, Le Gallou F, Drugeon H, Reynaud A (2008) Real-time PCR assay for the detection and quantification of Legionella pneumophila in environmental water samples: utility for daily practice. Int J Hyg Environ Health 211:403–411

    Article  Google Scholar 

  • Mouchtouri V, Velonakis E, Tsakalof A, Kapoula C, Goutziana G, Vatopoulos A, Kremastinou J, Hadjichristodoulou C (2007) Risk factors for contamination of hotel water distribution systems by Legionella species. Appl Environ Microbiol 73:1489–1492

    Article  CAS  Google Scholar 

  • Ortiz-Roque CM, Hazen TC (1987) Abundance and distribution of Legionellaceae in Puerto Rican waters. Appl Environ Microb 53:2231–2236

    CAS  Google Scholar 

  • Parthuisot N, West NJ, Lebaron P, Baudart J (2010) High diversity and abundance of Legionella spp. in a pristine river and impact of seasonal and anthropogenic effects. Appl Environ Microbiol 76:8201–8210

    Article  CAS  Google Scholar 

  • Percival SL, Chalmers RM, Embrey M, Hunter PR, Sellwood J, Wyn-Jones P (2004) Legionella. In: Microbiology of waterborne diseases. Elsevier, California, pp. 141–153

  • Qin T, Tian Z, Ren H, Hu G, Zhou H, Lu J, Luo C, Liu Z, Shao Z (2012) Application of EMA-qPCR as a complementary tool for the detection and monitoring of Legionella in different water systems. World J Microbiol Biotechnol 28:1881–1890

    Article  CAS  Google Scholar 

  • Riffard S, Douglass S, Brooks T, Springthorpe S, Filion LG, Sattar SA (2001) Occurrence of Legionella in groundwater: an ecological study. Water Sci Technol 43:99–102

    CAS  Google Scholar 

  • Sanden G, Fields BS, Barbaree JM, Feeley JC (1989) Viability of Legionella pneumophila in chlorine-free waters at elevated temperatures. Curr Microbiol 18:61–65

    Article  Google Scholar 

  • Sheehan KB, Fagg JA, Ferris MJ, Henson JM (2005) Thermophilic amoebae and Legionella in hot springs in Yellowstone and Grand Teton National Parks, Geothermal Biology and Geochemistry in Yellowstone National Park. Montana State University, Bozeman, pp 317–324

    Google Scholar 

  • Su HP, Tseng LR, Tzeng SC, Chou CY, Chung TC (2006) A Legionellosis case due to contaminated spa water and confirmed by genomic identification in Taiwan. Microbiol Immunol 50:371–377

    CAS  Google Scholar 

  • Valster RM, Wullings BA, van den Berg R, van der Kooij D (2011) Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean. Appl Environ Microbiol 77:7321–7328

    Article  CAS  Google Scholar 

  • Wang H, Edwards M, Falkinham JO 3rd, Pruden A (2012) Molecular survey of the occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and amoeba hosts in two chloraminated drinking water distribution systems. Appl Environ Microb 78:6285–6294

    Article  CAS  Google Scholar 

  • WHO (2007) Legionellosis, Legionella and the prevention of legionellosis. World Health Organization, Geneva

    Google Scholar 

  • Wullings BA, van der Kooij D (2006) Occurrence and genetic diversity of uncultured Legionella spp. in drinking water treated at temperatures below 15 degrees C. Appl Environ Microbiol 72:157–166

    Article  CAS  Google Scholar 

  • Yanez MA, Nocker A, Soria-Soria E, Murtula R, Martinez L, Catalan V (2011) Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR. J Microbiol Methods 85:124–130

    Article  CAS  Google Scholar 

  • Yaradou DF, Hallier-Soulier S, Moreau S, Poty F, Hillion Y, Reyrolle M, Andre J, Festoc G, Delabre K, Vandenesch F, Etienne J, Jarraud S (2007) Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems. Appl Environ Microbiol 73:1452–1456

    Article  CAS  Google Scholar 

  • Zanetti F, Stampi S, Luca GD, Fateh-Moghadam P, Sabattini MAB, Checchi L (2000) Water characteristics associated with the occurrence of Legionella pneumophila in dental units. Eur J Oral Sci 108:22–28

    Article  CAS  Google Scholar 

  • Zhou G, Wen S, Liu Y, Li R, Zhong X, Feng L, Wang L, Cao B (2011) Development of a DNA microarray for detection and identification of Legionella pneumophila and ten other pathogens in drinking water. Int J Food Microbiol 145:293–300

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by a research grant from the National Science Council (NSC 100-2116-M-194-004-MY2) and Cheng Hsin General Hospital (101–40) of Taiwan, ROC.

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Authors and Affiliations

  1. Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, Republic of China

    Po-Min Kao, Bing-Mu Hsu, Cheng-Yu She & Shu-Min Shen

  2. Department of Surgery, Tungs’ Taichung MetroHarbor Hospital, Taichung, Taiwan, Republic of China

    Min-Che Tung

  3. General Surgery, Surgical Department, Cheng Hsin General Hospital, Taipei, Taiwan, Republic of China

    Yi-Chou Chiu

  4. Department of Surgery, School of Medicine, National Yang Ming University, Taipei, Taiwan, Republic of China

    Yi-Chou Chiu

  5. Department of Safety Health and Environmental Engineering, National Kaohsiung First University of Science and Technology, Kaohsiung, Taiwan, Republic of China

    Yu-Li Huang

  6. Division of Thoracic Surgery, Department of Surgery, Mackay Memorial Hospital, Mackay Medicine, Nursing, and Management College, Taipei, Taiwan, Republic of China

    Wen-Chien Huang

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  1. Po-Min Kao
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Correspondence to Min-Che Tung or Bing-Mu Hsu.

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Responsible editor: Robert Duran

Authors Yi-Chou Chiu and Wen-Chien Huang have an equal contribution with the first author.

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Kao, PM., Tung, MC., Hsu, BM. et al. Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay. Environ Sci Pollut Res 20, 6128–6137 (2013). https://doi.org/10.1007/s11356-013-1534-z

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  • DOI: https://doi.org/10.1007/s11356-013-1534-z

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