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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

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An Erratum to this article was published on 27 February 2015

Abstract

Legionella 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.

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Acknowledgments

We would like to thank Dr. F. Grattard from Centre Hospitalier Universitaire de Saint Etienne, France, for the clinical and environmental strains used during this study. We would like to thank Dr. C. Ginevra and Dr. S. Jarraud from the Centre National de Référence des Légionelles for providing us with the GFP-modified Philadelphia strain. We would like to thank Dr A. Guignandon from Laboratoire de Biologie intégrative de Tissu Osseux, St Etienne, France, for his help in videomicroscopy. We thank Dr O. Delezay for his help in cells culture. T. Epalle, as a PhD student, is supported by a grant from Région Rhône-Alpes, France through the Environment Academic Research Committee. We would like to sincerely thank Région Rhône-Alpes for its support.

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  1. Groupe Immunité des Muqueuses et Agents Pathogènes (GIMAP), EA 3064, SFR 143, University of Lyon, 42023, Saint-Etienne, France

    Thibaut Epalle, Françoise Girardot, Séverine Allegra, Cécile Maurice-Blanc & Olivier Garraud

  2. Groupe Immunité des Muqueuses et Agents Pathogènes (GIMAP), EA 3064, SFR 143, Faculté de Médecine J. Lisfranc, Université Jean Monnet, 15 rue Ambroise Paré, 42023, Saint-Etienne, France

    Serge Riffard

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  1. Thibaut Epalle
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Correspondence to Serge Riffard.

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Epalle, T., Girardot, F., Allegra, S. et al. 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 . Microb Ecol 69, 215–224 (2015). https://doi.org/10.1007/s00248-014-0470-x

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