Development of a new CARD-FISH protocol for quantification of Legionella pneumophila and its application in two hospital cooling towers

Aims: Open cooling towers are frequent sources of infections with Legionella pneumophila. The gold standard for the detection of Leg.similar to pneumophila is based on cultivation lasting up to 10 days and detecting only culturable cells. Alternative fluorescence in situ hybridization (FISH) protocols have been proposed, but they result in faint fluorescence signals and lack specificity because of cross-hybridization with other Legionella species. Our aim was thus to develop a new FISH protocol for rapid and specific detection of Leg.similar to pneumophila in water samples. Methods and Results: A novel catalysed reporter deposition FISH (CARD-FISH) protocol for the detection of Leg.similar to pneumophila was developed, which significantly enhanced signal intensity as well as specificity of the probe through the use of a novel competitor probe. The developed protocol was compared with the culture method for monitoring the seasonal development of culturable and nonculturable Leg.similar to pneumophila in two hospital cooling tower systems. Seasonal fluctuations of Leg.similar to pneumophila concentrations detected via CARD-FISH were related to the development of the total bacterial community in both cooling towers, with temperature and biocide as the main factors controlling this development. Conclusions: Our results clearly showed that the majority of the Leg.similar to pneumophila cells were in a nonculturable state. Thus, detection of Leg.similar to pneumophila with culture methods may underestimate the total numbers of Leg.similar to pneumophila present. Significance and Impact of the Study: Rapid, sensitive and specific detection and quantification of Leg.similar to pneumophila in water systems is prerequisite for reliable risk estimation. The new protocol significantly improves current methodology and can be used to monitor and screen for Leg.similar to pneumophila concentrations in cooling towers or other water systems.