This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wellinghausen, N.
Right arrow Articles by Marre, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wellinghausen, N.
Right arrow Articles by Marre, R.
Agricola
Right arrow Articles by Wellinghausen, N.
Right arrow Articles by Marre, R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2001, p. 3985-3993, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.3985-3993.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Detection of Legionellae in Hospital Water Samples by Quantitative Real-Time LightCycler PCR

Nele Wellinghausen,* Cathrin Frost, and Reinhard Marre

Department of Medical Microbiology and Hygiene, University of Ulm, D-89081 Ulm, Germany

Received 11 April 2001/Accepted 12 June 2001

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.

* Corresponding author. Mailing address: Department of Medical Microbiology and Hygiene, University of Ulm, Robert-Koch-Str. 8, D-89081 Ulm, Germany. Phone: 49-731-500 24603. Fax: 49-731-500 23473. E-mail: nele.wellinghausen{at}medizin.uni-ulm.de.

Applied and Environmental Microbiology, September 2001, p. 3985-3993, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.3985-3993.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Feazel, L. M., Baumgartner, L. K., Peterson, K. L., Frank, D. N., Harris, J. K., Pace, N. R. (2009). Opportunistic pathogens enriched in showerhead biofilms. Proc. Natl. Acad. Sci. USA 106: 16393-16399 [Abstract] [Full Text]  
  • Delgado-Viscogliosi, P., Solignac, L., Delattre, J.-M. (2009). Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples. Appl. Environ. Microbiol. 75: 3502-3512 [Abstract] [Full Text]  
  • Ginevra, C., Lopez, M., Forey, F., Reyrolle, M., Meugnier, H., Vandenesch, F., Etienne, J., Jarraud, S., Molmeret, M. (2009). Evaluation of a Nested-PCR-Derived Sequence-Based Typing Method Applied Directly to Respiratory Samples from Patients with Legionnaires' Disease. J. Clin. Microbiol. 47: 981-987 [Abstract] [Full Text]  
  • Dusserre, E., Ginevra, C., Hallier-Soulier, S., Vandenesch, F., Festoc, G., Etienne, J., Jarraud, S., Molmeret, M. (2008). A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability. Appl. Environ. Microbiol. 74: 4817-4824 [Abstract] [Full Text]  
  • Diederen, B. M. W., Kluytmans, J. A. J. W., Vandenbroucke-Grauls, C. M., Peeters, M. F. (2008). Utility of Real-Time PCR for Diagnosis of Legionnaires' Disease in Routine Clinical Practice. J. Clin. Microbiol. 46: 671-677 [Abstract] [Full Text]  
  • Bencini, M. A., van den Brule, A. J. C., Claas, E. C. J., Hermans, M. H. A., Melchers, W. J. G., Noordhoek, G. T., Salimans, M. M. M., Schirm, J., Vink, C., van der Zee, A., Jansen, R. (2007). Multicenter Comparison of Molecular Methods for Detection of Legionella spp. in Sputum Samples. J. Clin. Microbiol. 45: 3390-3392 [Abstract] [Full Text]  
  • Yaradou, D. F., 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 [Abstract] [Full Text]  
  • Shih, H.-Y., Lin, Y. E. (2006). Caution on interpretation of legionella results obtained using real-time PCR for environmental water samples.. Appl. Environ. Microbiol. 72: 6859-6859 [Full Text]  
  • Stolhaug, A., Bergh, K. (2006). Identification and Differentiation of Legionella pneumophila and Legionella spp. with Real-Time PCR Targeting the 16S rRNA Gene and Species Identification by mip Sequencing. Appl. Environ. Microbiol. 72: 6394-6398 [Abstract] [Full Text]  
  • Temmerman, R., Vervaeren, H., Noseda, B., Boon, N., Verstraete, W. (2006). Necrotrophic Growth of Legionella pneumophila.. Appl. Environ. Microbiol. 72: 4323-4328 [Abstract] [Full Text]  
  • Joly, P., Falconnet, P.-A., 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. Microbiol. 72: 2801-2808 [Abstract] [Full Text]  
  • Giglio, S., Monis, P. T., Saint, C. P. (2005). Legionella Confirmation Using Real-Time PCR and SYTO9 Is an Alternative to Current Methodology. Appl. Environ. Microbiol. 71: 8944-8948 [Abstract] [Full Text]  
  • Kelly, D. M., Greene, C. M., Meachery, G., O'Mahony, M., Gallagher, P. M., Taggart, C. C., O'Neill, S. J., McElvaney, N. G. (2005). Endotoxin Up-regulates Interleukin-18: Potential Role for Gram-Negative Colonization in Sarcoidosis. Am. J. Respir. Crit. Care Med. 172: 1299-1307 [Abstract] [Full Text]  
  • Borella, P., Montagna, M. T., Stampi, S., Stancanelli, G., Romano-Spica, V., Triassi, M., Marchesi, I., Bargellini, A., Tato, D., Napoli, C., Zanetti, F., Leoni, E., Moro, M., Scaltriti, S., Ribera D'Alcala, G., Santarpia, R., Boccia, S. (2005). Legionella Contamination in Hot Water of Italian Hotels. Appl. Environ. Microbiol. 71: 5805-5813 [Abstract] [Full Text]  
  • Yanez, M. A., Carrasco-Serrano, C., Barbera, V. M., Catalan, V. (2005). Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene. Appl. Environ. Microbiol. 71: 3433-3441 [Abstract] [Full Text]  
  • Delgado-Viscogliosi, P., Simonart, T., Parent, V., Marchand, G., Dobbelaere, M., Pierlot, E., Pierzo, V., Menard-Szczebara, F., Gaudard-Ferveur, E., Delabre, K., Delattre, J. M. (2005). Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water. Appl. Environ. Microbiol. 71: 4086-4096 [Abstract] [Full Text]  
  • Roch, N., Maurin, M. (2005). Antibiotic susceptibilities of Legionella pneumophila strain Paris in THP-1 cells as determined by real-time PCR assay. J Antimicrob Chemother 55: 866-871 [Abstract] [Full Text]  
  • Raggam, R. B., Leitner, E., Berg, J., Muhlbauer, G., Marth, E., Kessler, H. H. (2005). Single-Run, Parallel Detection of DNA from Three Pneumonia-Producing Bacteria by Real-Time Polymerase Chain Reaction. J. Mol. Diagn. 7: 133-138 [Abstract] [Full Text]  
  • Stocher, M., Berg, J. (2004). Internal Control DNA for PCR Assays Introduced into Lambda Phage Particles Exhibits Nuclease Resistance. Clin. Chem. 50: 2163-2166 [Full Text]  
  • Labrenz, M., Brettar, I., Christen, R., Flavier, S., Botel, J., Hofle, M. G. (2004). Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea. Appl. Environ. Microbiol. 70: 4971-4979 [Abstract] [Full Text]  
  • Skovhus, T. L., Ramsing, N. B., Holmstrom, C., Kjelleberg, S., Dahllof, I. (2004). Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples. Appl. Environ. Microbiol. 70: 2373-2382 [Abstract] [Full Text]  
  • Aurell, H., Catala, P., Farge, P., Wallet, F., Le Brun, M., Helbig, J. H., Jarraud, S., Lebaron, P. (2004). Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry. Appl. Environ. Microbiol. 70: 1651-1657 [Abstract] [Full Text]  
  • Herpers, B. L., de Jongh, B. M., van der Zwaluw, K., van Hannen, E. J. (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 [Abstract] [Full Text]  
  • Templeton, K. E., Scheltinga, S. A., Sillekens, P., Crielaard, J. W., van Dam, A. P., Goossens, H., Claas, E. C. J. (2003). 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. 41: 4016-4021 [Abstract] [Full Text]  
  • Newby, D. T., Hadfield, T. L., Roberto, F. F. (2003). Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5'-Exonuclease, and Hybridization Probe Assays. Appl. Environ. Microbiol. 69: 4753-4759 [Abstract] [Full Text]  
  • Wilson, D. A., Yen-Lieberman, B., Reischl, U., Gordon, S. M., Procop, G. W. (2003). Detection of Legionella pneumophila by Real-Time PCR for the mip Gene. J. Clin. Microbiol. 41: 3327-3330 [Abstract] [Full Text]  
  • Calvo-Bado, L. A., Morgan, J. A. W., Sergeant, M., Pettitt, T. R., Whipps, J. M. (2003). Molecular Characterization of Legionella Populations Present within Slow Sand Filters Used for Fungal Plant Pathogen Suppression in Horticultural Crops. Appl. Environ. Microbiol. 69: 533-541 [Abstract] [Full Text]  
  • Reischl, U., Linde, H.-J., Lehn, N., Landt, O., Barratt, K., Wellinghausen, N. (2002). Direct Detection and Differentiation of Legionella spp. and Legionella pneumophila in Clinical Specimens by Dual-Color Real-Time PCR and Melting Curve Analysis. J. Clin. Microbiol. 40: 3814-3817 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»