A detection method for Legionella spp in (cooling) water: fluorescent in situ hybridisation (FISH) on whole bacteria

Abstract

Although traditional culture methods are appropriate for detection of Legionella species, such culture takes several days. Rapid detection (< 24 h) of individual Legionella is possible using fluorescent in situ hybridisation (FISH) on whole bacteria. Water samples were filtered and the concentrated bacteria were immediately detected (without culture) with a fluorescence microscope following appropriate labelling. The detection level was very high and quantification was possible. For the detection of all Legionella spp. the probe LEG705 was used, complementary to a 16S rRNA sequence conserved in all Legionella spp. For specific detection of L. pneumophila the probe LEGPNE1 was used. This probe is designed against a variable domain of the 16S rRNA sequence from L. pneumophila. CY3 and FLUOS labels were tested and CY3 showed clearly detectable bacteria with minimum background staining. This FISH technique is very sensitive, fast, reliable and individual bacteria are easily detected.

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

“…The NRCL anti-L. pneumophila sg 1 polyclonal antibody does not cross-react with other serogroups (data not shown). The Accurate Chemical & Scientific anti-L. pneumophila polyclonal antibody was found to be specific for sgs 1,6,7, and 12, as demonstrated by tests on L. pneumophila type strains (data not shown). The specificity of the MAbs from Dresden has already been reported for the different serogroups (10).…”

“…Additional problems with culture detection include low sensitivity, microbial contamination inhibiting Legionella growth, and the potential presence of viable but nonculturable bacteria (VBNC) (3,13,23). Methods based on direct detection, combining immunofluorescent labeling (IF) (19) or fluorescent in situ hybridization (FISH) (4,7,22) with detection by epifluorescence microscopy or flow cytometry (25), allow a more rapid detection of Legionella cells and avoid most of the problems encountered with culture. However, these techniques cannot be applied to the detection of rare events (15).…”

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

“…It is thus essential to use a more powerful technique for legionellae detection in environmental samples. Fluorescent in situ hybridization (FISH) using genus-specific fluorescently labelled oligonucleotide probe, complementary to a conserved target sequence of the 16S rRNA region, has been shown to be a useful method for the detection and identification of slow growing, fastidious or nonculturable micro-organisms without cultivation (Amann et al 1991;Kalmbach et al 1997;Declerck et al 2003). Two problems were encountered by the use of FISH for detecting bacteria in natural water: (i) the low level of targeted rRNA (low fluorescence) and (ii) the inability to distinguish viable from nonviable cells (Villarino et al 2000;Garcia-Armisen and Servais 2004).…”

Rapid quantification of bioaerosols containing L. pneumophila by Coriolis® μ air sampler and chemiluminescence antibody microarrays

“…For quantification of microorganisms with FISH a certain cultivation time on a filter is necessary before fluorescence labeled oligonucleotides are incubated with lysed cells. Otherwise the detection limit is very high (Declerck et al, 2003). The quantification is carried out with a fluorescence microscope.…”