Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS-FCM method). The method requires 120?min and can discriminate viable and membrane-damaged cells. The recovery is over 85% of spiked Lp SG 1 cells in 1?l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also viable but not culturable (VNBC) cells are detected by our method, this result was expected. The IMS-FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 112. This and the fact that no Lp-containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS-FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems.