Bioaerosol sampling and bioanalysis: Applicability of the next generation impactor for quantifying Legionella pneumophila in droplet aerosols by flow cytometry

Bioaerosol generation, sampling, and cultivation-independent quantification of pathogenic bacteria play a crucial role in studying dose-response effects of Legionella pneumophila. Here, the Next Generation Impactor (NGI), initially created for pharmaceutical inhaling studies, was assessed for its potential to sample airborne bioaerosols and to separate size-dependent wet droplets by incrementally increasing the airflow speed. This stainless-steel sampler was shown in this study to be suitable for sampling prior to cultivation-independent analysis of pathogen-containing bioaerosols using washable cups. The applicability was studied by quantifying the total and intact cell count of L. pneumophila by flow cytometry after being dispersed into a droplet aerosol. Our results demonstrate a high total sampling efficiency of 95.5% ± 11.8% despite a lower biological sampling efficiency of 59.7% ± 16.5% for dry aerosols. However, by elevating the relative humidity (RH) to 100% in a liquid aerosolization unit, the biological sampling efficiency increased to over 90% for L. pneumophila. More than 50% of the cells were found in stage 1 using the liquid aerosolization unit. In comparison, 80% of the cells were sampled in stages 4–6 at 30% RH. Specifically, while at 100% RH, the droplet size mattered, at 30% RH, the size distribution of dry particles, in this case L. pneumophila, was relevant due to evaporation processes, which explains the size differences. These findings indicate the potential of the NGI for further exploration and application in studying other aerosol-borne pathogens, especially concerning the size distribution of wet droplets, viability, or effect-based bioanalysis.