Simultaneous detection of Legionella species and Legionella pneumophila by duplex PCR (dPCR) assay in cooling tower water samples from Jakarta, Indonesia

Andi Yasmon; Yusmaniar Yusmaniar; Anis Anis; Budiman Bela

doi:10.13181/mji.v19i4.408

Authors

Andi Yasmon
Yusmaniar Yusmaniar
Anis Anis
Budiman Bela

DOI:

https://doi.org/10.13181/mji.v19i4.408

Keywords:

BCYE media, mip gene, 16S-rRNA gene

Abstract

Aim: Since culture method is time-consuming and has low sensitivity, we developed a duplex PCR (dPCR) assay for the detection of Legionella sp. and L. pneumophila in cooling tower samples. We used culture method as a gold standard.

Methods: Optimization of dPCR method was performed to obtain an assay with high sensitivity and specifi city. The optimized method was used to detect Legionella sp. dan L. pneumophila in 9 samples obtained from 9 buildings in Jakarta. For culture method, the bacteria were grown or isolated on selective growth factor supplemented-buffered charcoal yeast extract (BCYE) media.

Results: Of 9 samples tested by dPCR assay, 6 were positive for Legionella species,1 was positive for L. pneumophila, and 2 showed negative results. For the same samples, no Legionella sp. was detected by the culture method.

Conclusion: dPCR assay was much more sensitive than the culture method and was potentially used as a rapid, specifi c and sensitive test for routine detection of Legionella sp. dan for L. pneumophila in water samples. (Med J Indones 2010; 19:223-7)

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References

Pedro-Botet ML, Stout JE, Yu VL. Legionnaires' disease contracted from patient homes: the coming of the third plague? Eur J Clin Microbiol Infect Dis. 2002;21:699-705. https://doi.org/10.1007/s10096-002-0813-2

Palusinska-Szysz M, Cendrowska-Pinkosz M. Pathogenicity of the family Legionellaceae. Arch Immunol Ther Exp (Warsz). 2009;57:279-90. https://doi.org/10.1007/s00005-009-0035-8

Diederen BM. Legionella spp. and Legionnaires' disease. J Infect. 2008;56:1-12. https://doi.org/10.1016/j.jinf.2007.09.010

Fields BS, Benson RF, Besser RE. Legionella and Legionnaires' disease: 25 years of investigation. Clin Microbiol Rev. 2002;15:506-26. https://doi.org/10.1128/CMR.15.3.506-526.2002

Devos L, Boon N, Verstraete W. Legionella pneumophila in the environment: The occurrence of a fastidious bacterium in oligotrophic conditions. Rev Environ Sci Biotechnol. 2005;4:61-74. https://doi.org/10.1007/s11157-004-8174-1

Gobin I, Susa M, Begic G, Hartland EL, Doric M. Experimental Legionella longbeachae infection in intratracheally inoculated mice. J Med Microbiol. 2009;58:723-30. https://doi.org/10.1099/jmm.0.007476-0

Bollin GE, Plouffe JF, Para MF, Hackman B. Aerosols containing Legionella pneumophila generated by shower heads and hot-water faucets. Appl Environ Microbiol. 1985;50:1128-31. https://doi.org/10.1128/AEM.50.5.1128-1131.1985

Hsu BM, Chen CH, Wan MT, Cheng HW. Legionella prevalence in hot spring recreation areas of Taiwan. Water Res. 2006;40:3267-73. https://doi.org/10.1016/j.watres.2006.07.007

Ragull S, Garcia-Nunez M, Pedro-Botet ML, Sopena N, Esteve M, Montenegro R, et al. Legionella pneumophila in cooling towers: fl uctuations in counts, determination of genetic variability by pulsed-fi eld gel electrophoresis (PFGE), and persistence of PFGE patterns. Appl Environ Microbiol. 2007;73:5382-4. https://doi.org/10.1128/AEM.00066-07

Levi K, Smedley J, Towner KJ. Evaluation of a real-time PCR hybridization assay for rapid detection of Legionella pneumophila in hospital and environmental water samples. Clin Microbiol Infect. 2003;9:754-8. https://doi.org/10.1046/j.1469-0691.2003.00666.x

Koide M, Saito A, Kusano N, Higa F. Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method. Appl Environ Microbiol. 1993;59:1943-6. https://doi.org/10.1128/AEM.59.6.1943-1946.1993

Nintasen R, Utrarachkij F, Siripanichgon K, Bhumiratana A, Suzuki Y, Suthienkul O. Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specifi c nested polymerase chain reaction. Microbiol Immunol. 2007;51:777-85. https://doi.org/10.1111/j.1348-0421.2007.tb03967.x

Joly P, Falconnet PA, Andre J, Weill N, Reyrolle M, Vandenesch F, et al. Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl Environ Microbiol. 2006;72:2801-8. https://doi.org/10.1128/AEM.72.4.2801-2808.2006

Devos L, Clymans K, Boon N, Verstraete W. Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples. J Appl Microbiol. 2005;99:916-25. https://doi.org/10.1111/j.1365-2672.2005.02668.x

Templeton KE, Scheltinga SA, Sillekens P, Crielaard JW, van Dam AP, Goossens H, et al. Development and clinical evaluation of an internally controlled, single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species. J Clin Microbiol. 2003;41:4016-21. https://doi.org/10.1128/JCM.41.9.4016-4021.2003

England AC, Fraser DW, Mallison GF, Mackel DC, Skaliy P, Gorman GW. Failure of Legionella pneumophila sensitivities to predict culture results from disinfectant-treated air-conditioning cooling towers. Appl Environ Microbiol. 1982;43:240-4. https://doi.org/10.1128/AEM.43.1.240-244.1982

Ohno A, Kato N, Yamada K, Yamaguchi K. Factors infl uencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water. Appl Environ Microbiol. 2003;69:2540-7. https://doi.org/10.1128/AEM.69.5.2540-2547.2003

Declerck P, Verelst L, Duvivier L, Van Damme A, Ollevier F. PCR as a test for the presence or absence of Legionella in (cooling) water. Water Sci Technol. 2003;47:103-7. https://doi.org/10.2166/wst.2003.0173

Delgado-Viscogliosi P, Solignac L, Delattre JM. Viability PCR, a culture-independent method for rapid and selective quantifi cation of viable Legionella pneumophila cells in environmental water samples. Appl Environ Microbiol. 2009;75:3502-12. https://doi.org/10.1128/AEM.02878-08