Immunomagnetic separation coupled with flow cytometry for the analysis of Legionella pneumophila in aerosols

Legionella pneumophila are pathogenic bacteria that can be found in high concentrations in artificial water systems like evaporative cooling towers, which have been the source of frequent outbreaks in recent years. Since inhaled L. pneumophila can lead to Legionnaires’ disease, the development of suitable sampling and rapid analysis strategies for these bacteria in aerosols is therefore of great relevance. In this work, different concentrations of viable L. pneumophila Sg 1 were nebulized and sampled by the cyclone sampler Coriolis® µ under defined conditions in a bioaerosol chamber. To quantify intact Legionella cells, the collected bioaerosols were subsequently analyzed by immunomagnetic separation coupled with flow cytometry (IMS-FCM) on the platform rqmicro.COUNT. For analytical comparison, measurements with qPCR and cultivation were performed. Limits of detection (LOD) of 2.9 × 10 3 intact cells m −3 for IMS-FCM and 7.8 × 10 2 intact cells m −3 for qPCR indicating a comparable sensitivity as in culture (LOD = 1.5 × 10 3 culturable cells m −3 ). Over a working range of 10 3 − 10 6 cells mL −1 , the analysis of nebulized and collected aerosol samples with IMS-FCM and qPCR provides higher recovery rates and more consistent results than by cultivation. Overall, IMS-FCM is a suitable culture-independent method for quantification of L. pneumophila in bioaerosols and is promising for field application due to its simplicity in sample preparation.