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Applied and Environmental Microbiology, August 2008, p. 4817-4824, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.02899-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability{triangledown}

Eric Dusserre,1,2,3,4 Christophe Ginevra,1,2,3,4 Sylvie Hallier-Soulier,5 François Vandenesch,1,2,3,4 Gabriel Festoc,5 Jerome Etienne,1,2,3,4 Sophie Jarraud,1,2,3,4 and Maëlle Molmeret1,2,3,4*

Université de Lyon,1 Université Lyon 1, Faculté Laennec, F-69008 Lyon,2 INSERM U851, Faculté de Médecine Laennec, Laboratoire de Bactériologie, 7 rue Guillaume Paradin, F-69372 Lyon cedex 08,3 Hospices Civils de Lyon, Centre de Biologie et Pathologie Est, Laboratoire de Bactériologie, Centre National de Référence des Légionelles, F-69677 Bron,4 Société GeneSystems, 1 rue du Courtil, Centre d'Affaires CICEA, Bâtiment 1, F-35170 Bruz, France5

Received 21 December 2007/ Accepted 23 May 2008

Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 106 genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 105 and 102 metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga. Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.

* Corresponding author. Mailing address: Centre National de Réference des Légionelles, INSERM U851, Faculté de Médecine Laennec, Laboratoire de Bactériologie, 7 rue Guillaume Paradin, F-69372 Lyon cedex 08, France. Phone: (33) 47 877 86 42. Fax: (33) 47 877 86 58. E-mail: maelle.molmeret{at}univ-lyon1.fr

{triangledown} Published ahead of print on 30 May 2008.

Applied and Environmental Microbiology, August 2008, p. 4817-4824, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.02899-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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