Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry

Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge

doi:10.1016/j.resmic.2015.01.004

Cited by 23 publications

(14 citation statements)

References 22 publications

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“…Among them, the presence in water samples of viable but not culturable (VBNC) forms contributes to underestimation of the number of potentially pathogenic Legionella detected by standard methods. Our preliminary studies on anthropic reservoirs have demonstrated the presence of VBNC forms of Legionella after water disinfection treatments (heat 20 and chlorine 21 ). Finally, the role of Legionella intracellular forms (intra-amoebic) has been little investigated in environmental samples 22 even though these forms are considered more infectious 19 .…”

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confidence: 91%

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Characterization of aerosols containing Legionella generated upon nebulization

Allegra

Leclerc

Massard

et al. 2016

Sci Rep

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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.

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confidence: 91%

“…VBNC cells can be detected and quantified by a flow cytometric assay (FCA) 23 . Assessment of their presence is important because they are able to infect amoebas as well as macrophage-like and epithelial cells from human respiratory mucosa after resuscitation on amoebas 21 24 .…”

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confidence: 99%

Characterization of aerosols containing Legionella generated upon nebulization

Allegra

Leclerc

Massard

et al. 2016

Sci Rep

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“…TCC measurements, based on a single stain protocol, are suitable to detect changes in bacterial concentrations, which the inflow of wastewater, for example, would cause ( Prest et al, 2013 ). To assess the effect of oxidative disinfection on bacteria, i.e., primarily cell membrane damage, a viability staining protocol can be applied to determine for example the intact cell concentration (ICC) ( Phe et al, 2005 ; Berney et al, 2007 ; Ramseier et al, 2011 ; Mustapha et al, 2015 ; Lee et al, 2016 ). Ideally, both measurements (TCC and ICC) are performed in parallel to maximize information gain ( Van Nevel et al, 2017b ).…”

Section: Introductionmentioning

confidence: 99%

Laboratory-Scale Simulation and Real-Time Tracking of a Microbial Contamination Event and Subsequent Shock-Chlorination in Drinking Water

et al. 2017

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Rapid contamination of drinking water in distribution and storage systems can occur due to pressure drop, backflow, cross-connections, accidents, and bio-terrorism. Small volumes of a concentrated contaminant (e.g., wastewater) can contaminate large volumes of water in a very short time with potentially severe negative health impacts. The technical limitations of conventional, cultivation-based microbial detection methods neither allow for timely detection of such contaminations, nor for the real-time monitoring of subsequent emergency remediation measures (e.g., shock-chlorination). Here we applied a newly developed continuous, ultra high-frequency flow cytometry approach to track a rapid pollution event and subsequent disinfection of drinking water in an 80-min laboratory scale simulation. We quantified total (TCC) and intact (ICC) cell concentrations as well as flow cytometric fingerprints in parallel in real-time with two different staining methods. The ingress of wastewater was detectable almost immediately (i.e., after 0.6% volume change), significantly changing TCC, ICC, and the flow cytometric fingerprint. Shock chlorination was rapid and detected in real time, causing membrane damage in the vast majority of bacteria (i.e., drop of ICC from more than 380 cells μl-1 to less than 30 cells μl-1 within 4 min). Both of these effects as well as the final wash-in of fresh tap water followed calculated predictions well. Detailed and highly quantitative tracking of microbial dynamics at very short time scales and for different characteristics (e.g., concentration, membrane integrity) is feasible. This opens up multiple possibilities for targeted investigation of a myriad of bacterial short-term dynamics (e.g., disinfection, growth, detachment, operational changes) both in laboratory-scale research and full-scale system investigations in practice.

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“…In the context of this study, we demonstrated a new data that the Legionella physiological states were not modified by the nebulization process or by its transportation through the 12-stage DLPI or the anatomical model. Thermally or chlorine-induced VBNC Legionella pneumophila may still infect amoebae and pulmonary cells after resuscitation on amoebae (Cervero-Aragó et al, 2019;Epalle et al, 2015;Mustapha et al, 2015). VBNC cell concentrations in the water network must be included in the QMRA models.…”

Section: Discussionmentioning

confidence: 99%

A valuable experimental setup to model exposure to Legionella’s aerosols generated by shower-like systems

et al. 2020

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The mechanism underlying Legionella aerosolization and entry into the respiratory tract remains poorly documented. In previous studies, we characterized the aerodynamic behaviour of Legionella aerosols and assessed their regional deposition within the respiratory tract using a human-like anatomical model. The aim of this study was to assess whether this experimental setup could mimic the exposure to bioaerosols generated by showers. To achieve this objective we performed experiments to measure the mass median aerodynamic diameter (MMAD) as well as the emitted dose and the physiological state of the airborne bacteria generated by a shower and two nebulizers (vibrating-mesh and jet nebulizers). The MMADs of the dispersed bioaerosols were characterized using a 12-stage cascade low-pressure impactor. The amount of dispersed airborne bacteria from a shower was quantified using a Coriolis® Delta air sampler and compared to the airborne bacteria reaching the thoracic region in the experimental setup. The physiological state and concentration of airborne Legionella were assessed by qPCR for total cells, culture for viable and cultivable Legionella (VC), and flow cytometry for viable but noncultivable Legionella (VBNC). In summary, the experimental setup developed appears to mimic the bioaerosol emission of a shower in terms of aerodynamic size distribution. Compared to the specific case of a shower used as a reference in this study, the experimental setup developed underestimates by 2 times (when the jet nebulizer is used) or overestimates by 43 times (when the vibrating-mesh nebulizer is used) the total emitted dose of airborne bacteria. To our knowledge, this report is the first showing that an experimental model mimics so closely an exposure to Legionella aerosols produced by showers to assess human lung deposition and infection in well-controlled and safe conditions.

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Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry

Cited by 23 publications

References 22 publications

Characterization of aerosols containing Legionella generated upon nebulization

Characterization of aerosols containing Legionella generated upon nebulization

Laboratory-Scale Simulation and Real-Time Tracking of a Microbial Contamination Event and Subsequent Shock-Chlorination in Drinking Water

A valuable experimental setup to model exposure to Legionella’s aerosols generated by shower-like systems

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