Characterization of aerosols containing Legionella generated upon nebulization

Abstract

Legionella pneumophila is, by far, the species most frequently associated with Legionnaires' disease (LD). Human infection occurs almost exclusively by aerosol inhalation which places the bacteria in juxtaposition with alveolar macrophages. LD risk management is based on controlling water quality by applying standardized procedures. However, to gain a better understanding of the real risk of exposure, there is a need (i) to investigate under which conditions Legionella may be aerosolized and (ii) to quantify bacterial deposition into the respiratory tract upon nebulization. In this study, we used an original experimental set-up that enables the generation of aerosol particles containing L. pneumophila under various conditions. Using flow cytometry in combination with qPCR and culture, we determined (i) the size of the aerosols and (ii) the concentration of viable Legionella forms that may reach the thoracic region. We determined that the 0.26-2.5 μm aerosol size range represents 7% of initial bacterial suspension. Among the viable forms, 0.7% of initial viable bacterial suspension may reach the pulmonary alveoli. In conclusion, these deposition profiles can be used to standardize the size of inoculum injected in any type of respiratory tract model to obtain new insights into the dose response for LD.

Figure 1. Experimental set-up for the generation and characterization of aerosols.

Figure 2. Representative experimental results obtained with FCA and culture for different aerodynamic size ranges of airborne particles (n = 3, repeated twice).

Left: FCA results in percentages. Cells are distributed according to propidium iodide fluorescence intensity versus GFP fluorescence intensity. Green: VC, blue: VBNC, red: DC. Right: culture in colony-forming units (CFU). Aerosolized suspension of Lp1 008 EPF cells at 2.10⁷ CFU/mL.

Figure 3. Microscopic epifluorescent image (Gx400) of aerosolized Legionella suspension.

(A) Lp1 008 EPF cells. (B) Lp1 008 SPF cells.

Figure 4. Typical cumulative distribution of airborne droplets characterized by a low-pressure impactor (DLPI).

NaF quantification by an electrochemical method (expressed in % of mass reaching each stage of the DLPI, i.e., in % of total mass of NaF aerosolized). Legionella quantification by qPCR (expressed in % of genetic units reaching each stage of the DLPI, i.e., in % of total airborne Legionella). Particles larger than 10 μm were not plotted.