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Characterization of aerosols containing Legionella generated upon nebulization

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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.
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f2: 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.

Mentions: Bacterial detection and viability before and after aerosolization were assessed using culture, qPCR and FCA on Lp1 008 suspensions of different concentrations and stages: exponential phase forms (EPFs) and stationary phase forms (SPFs) according to a procedure described by Faulkner and Garduno27. As determined by qPCR, only 15.8 ± 2.4% of the initial total inoculum (in genomic units (GU)) was recovered from the 13-DLPI stages after aerosolization. The remaining part of the inoculum was either kept in the nebulizer reservoir or disseminated in the artificial throat. The percentage recovered was the same for both suspensions at both concentrations, indicating good reproducibility of the nebulization process. As shown by FCA analysis and cultivability on BCYE media, the percentages of VC (35.7 ± 1.3%), VBNC (55.7 ± 1.1%) and dead cells (DC; 8.7 ± 0.9%) were the same for both aerosolized suspensions and those recovered at each DLPI stage (Fig. 2).


Characterization of aerosols containing Legionella generated upon nebulization
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037422&req=5

f2: 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.
Mentions: Bacterial detection and viability before and after aerosolization were assessed using culture, qPCR and FCA on Lp1 008 suspensions of different concentrations and stages: exponential phase forms (EPFs) and stationary phase forms (SPFs) according to a procedure described by Faulkner and Garduno27. As determined by qPCR, only 15.8 ± 2.4% of the initial total inoculum (in genomic units (GU)) was recovered from the 13-DLPI stages after aerosolization. The remaining part of the inoculum was either kept in the nebulizer reservoir or disseminated in the artificial throat. The percentage recovered was the same for both suspensions at both concentrations, indicating good reproducibility of the nebulization process. As shown by FCA analysis and cultivability on BCYE media, the percentages of VC (35.7 ± 1.3%), VBNC (55.7 ± 1.1%) and dead cells (DC; 8.7 ± 0.9%) were the same for both aerosolized suspensions and those recovered at each DLPI stage (Fig. 2).

View Article: PubMed Central - PubMed

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.

No MeSH data available.