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 × 103 intact cells m-3 for IMS-FCM and 7.8 × 102 intact cells m-3 for qPCR indicating a comparable sensitivity as in culture (LOD = 1.5 × 103 culturable cells m-3). Over a working range of 103 - 106 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.

Microbiological examination of water and aerosols from four industrial evaporative cooling systems in regard to risk of Legionella emissions and methodological suggestions for surveillance.

The hygienic risk associated with evaporative cooling systems in Germany is currently only assessed by determining concentrations of Legionella spp. in the corresponding cooling waters. Relevant for the health risk is however the load of Legionella in emitted aerosols. In this work aerosol emissions from four industrial cooling systems (A - D) were analyzed. A microbiological air bioburden factor (MABF) is suggested to be useful to assess the overall microbiological load of emitted air and to judge the efficiency of droplet separation and overall microbiological retention. Whereas the MABF by itself only serves as a technical quality assurance (QA) parameter, the hygienic relevance has to be seen in combination with the assessment of Legionella either contained in the aerosol or in the cooling water. Plate counting of colonies was an appropriate method to quantify Legionella spp. in aerosols given the short time of flight at the chosen sampling locations and resulting low risk of desiccation. qPCR data on the other hand proved more reproducible than the culture approach to quantify Legionella spp. concentrations in cooling water-. The application of qPCR also allowed to assess the relative proportion of Legionella pneumophila within the total pool of Legionella which adds epidemiological relevance to risk assessment. A traffic light system was proposed to guide interpretation of qPCR data. The four industrial systems greatly differed in all measured parameters leading to different associated risks.