Quantification of Viable but Non-Culturable Cells of Legionella pneumophila

Legionella pneumophila, among other bacteria, may enter a viable but non-culturable state as a means for survival in stressful conditions. Bacterial cells in the viable but non-culturable state cannot grow on standard medium; however, they continue to exhibit characteristics that are associated with live cells, such as respiration, transcription, and cell wall integrity. The present paper outlines a detailed protocol for the detection of viable but non-culturable L. pneumophila cells via Syto® 9 and propidium iodide staining coupled with flow cytometry

Viable but Not Culturable Forms of Legionella pneumophila Generated After Heat Shock Treatment Are Infectious for Macrophage-Like and Alveolar Epithelial Cells After Resuscitation on Acanthamoeba polyphaga

An erratum to this article can be found at http://dx.doi.org/10.1007/s00248-015-0580-0.International audienceLegionella pneumophila, the causative agent of legionellosis is transmitted to human through aerosols from environmental sources and invades lung’s macrophages. It also can invade and replicate within various protozoan species in environmental reservoirs. Following exposures to various stresses, L. pneumophila enters a non-replicative viable but non-culturable (VBNC) state. Here, we evaluated whether VBNC forms of three L. pneumophila serogroup 1 strains (Philadelphia GFP 008, clinical 044 and environmental RNN) infect differentiated macrophage-like cell lines (U937 and HL-60), A549 alveolar cells and Acanthamoeba polyphaga. VBNC forms obtained following shocks at temperatures ranging from 50 to 70 °C for 5 to 60 min were quantified using a flow cytometric assay (FCA). Their loss of culturability was checked on BCYE agar medium. VBNC forms were systematically detected upon a 70 °C heat shock for 30 min. When testing their potential to resuscitate upon amoebal infection, VBNC forms obtained after 30 min at 70 °C were re-cultivated except for the clinical strain. No resuscitation or cell lysis was evidenced when using U937, HL-60, or A549 cells despite the use of various contact times and culture media. None of the strains tested could infect A. polyphaga, macrophage-like or alveolar epithelial cells after a 60-min treatment at 70 °C. However, heat-treated VBNC forms were able to infect macrophage-like or alveolar epithelial cells following their resuscitation on A. polyphaga. These results suggest that heat-generated VBNC forms of L. pneumophila (i) are not infectious for macrophage-like or alveolar epithelial cells in vitro although resuscitation is still possible using amoeba, and (ii) may become infectious for human cell lines following a previous interaction with A. polyphaga

Involvement of Minerals in Adherence of Legionella pneumophila to Surfaces

International audienceLegionella pneumophila is the causative agent of 90 % of Legionnaires' disease cases. This bacterium lives naturally in fresh water and can colonize biofilms, which play an important role in the protection of Legionella against environmental stress factors. Relationship between the presence of minerals in water and Legionella adherence to surfaces is not well-known. In this study, we studied influence of minerals on bacterial adherence. For the first time, to our knowledge, this report shows that calcium and magnesium in a less extent, enhances the adherence of Legionella to surfaces compared to the bacteria behavior in distilled water. Treatment with proteinase K of live cells showed that surface proteins do not seem to play a crucial role in bacteria adherence to surfaces. Our results represent a first step in understanding effect of ions on Legionella adherence to surfaces. Such field of research could be helpful to better understand biofilm colonization by this bacterium to improve Legionella risk management in water networks