microbiology, Vol 141, 29-39, Copyright © 1995 by Society for General Microbiology

ARTICLES

In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy

W Manz, R Amann, R Szewzyk, U Szewzyk, TA Stenstrom, P Hutzler and KH Schleifer
Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, Germany.

Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.

This article has been cited by other articles:

				E. Hoque, S. Pflugmacher, J. Fritscher, and M. Wolf Induction of Glutathione S-Transferase in Biofilms and Germinating Spores of Mucor hiemalis Strain EH5 from Cold Sulfidic Spring Waters Appl. Envir. Microbiol., April 15, 2007; 73(8): 2697 - 2707. [Abstract] [Full Text] [PDF]

				S. A. Wilks and C. W. Keevil Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms Appl. Envir. Microbiol., August 1, 2006; 72(8): 5453 - 5462. [Abstract] [Full Text] [PDF]

				J. Langmark, M. V. Storey, N. J. Ashbolt, and T.-A. Stenstrom Accumulation and Fate of Microorganisms and Microspheres in Biofilms Formed in a Pilot-Scale Water Distribution System Appl. Envir. Microbiol., February 1, 2005; 71(2): 706 - 712. [Abstract] [Full Text] [PDF]

				C. Bouteleux, S. Saby, D. Tozza, J. Cavard, V. Lahoussine, P. Hartemann, and L. Mathieu Escherichia coli Behavior in the Presence of Organic Matter Released by Algae Exposed to Water Treatment Chemicals Appl. Envir. Microbiol., February 1, 2005; 71(2): 734 - 740. [Abstract] [Full Text] [PDF]

				M. W. Kuiper, B. A. Wullings, A. D. L. Akkermans, R. R. Beumer, and D. van der Kooij Intracellular Proliferation of Legionella pneumophila in Hartmannella vermiformis in Aquatic Biofilms Grown on Plasticized Polyvinyl Chloride Appl. Envir. Microbiol., November 1, 2004; 70(11): 6826 - 6833. [Abstract] [Full Text] [PDF]

				P. T. Sunde, I. Olsen, U. B. Gobel, D. Theegarten, S. Winter, G. J. Debelian, L. Tronstad, and A. Moter Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth Microbiology, May 1, 2003; 149(5): 1095 - 1102. [Abstract] [Full Text] [PDF]

				J. D. Rudney, R. Chen, and G. J. Sedgewick Intracellular Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in Buccal Epithelial Cells Collected from Human Subjects Infect. Immun., April 1, 2001; 69(4): 2700 - 2707. [Abstract] [Full Text] [PDF]

				D. Grimm, H. Merkert, W. Ludwig, K.-H. Schleifer, J. Hacker, and B. C. Brand Specific Detection of Legionella pneumophila: Construction of a New 16S rRNA-Targeted Oligonucleotide Probe Appl. Envir. Microbiol., July 1, 1998; 64(7): 2686 - 2690. [Abstract] [Full Text]

				C. M. Buswell, Y. M. Herlihy, L. M. Lawrence, J. T. M. McGuiggan, P. D. Marsh, C. W. Keevil, and S. A. Leach Extended Survival and Persistence of Campylobacter spp. in Water and Aquatic Biofilms and Their Detection by Immunofluorescent-Antibody and -rRNA Staining Appl. Envir. Microbiol., February 1, 1998; 64(2): 733 - 741. [Abstract] [Full Text]