IN-SITU IDENTIFICATION OF LEGIONELLACEAE USING 16S RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES AND CONFOCAL LASER-SCANNING MICROSCOPY

Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence a nalysis we constructed two oligonucleotide probes complementary to reg ions of 165 rRNA characteristic for Legionellaceae. Probe specificitie s were tested by whole-cell or dot-blot hybridization against 14 serog roups of Legionella pneumophila, 22 different Legionella spp. and 72 n on-legionellae reference strains. Using optimized conditions both prob es hybridized to all tested strains of L. pneumophila, Probes LEG226 a nd 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 mol ecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay wer e in goad 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. Eve n after incubation for 1 year a proportion of the released cells was s till detectable. In situ hybridization also facilitated visualization of Legionella spp. cells in model biofilms. A combination of in situ h ybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within ce lls of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probi ng with 165 rRNA-targeted oligonucleotides could. in the future, compl ement established techniques like immunofluorescence and PCR in ecolog ical and epidemiological studies of Legionellaceae.