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Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable Legionella pneumophila

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Abstract

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires’ disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l−1), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD® BacLight™). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l−1 of free chlorine and in 10 min when the concentration is increased to 1.2 mg l−1. However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.

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Acknowledgments

This work was supported by the Portuguese Institute Fundação para a Ciência e Tecnologia (PhD grant SFRH/BD/17088/2004) and has been undertaken as part of a research project which is supported by the European Commission within the Fifth Framework Programme, “Energy, Environment and sustainable development programme”, no. EVK1-CT-2002-00108. Disclaimer states that the author is solely responsible for the work; it does not represent the opinion of the Community, and the Community is not responsible for any use that might be made of data appearing therein.

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Authors and Affiliations

  1. Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal

    M. S. Gião, N. F. Azevedo & M. J. Vieira

  2. School of Biological Sciences, Biomedical Sciences Building, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, UK

    M. S. Gião, S. A. Wilks, N. F. Azevedo & C. W. Keevil

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  1. M. S. Gião
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  2. S. A. Wilks
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  3. N. F. Azevedo
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  4. M. J. Vieira
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  5. C. W. Keevil
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Correspondence to M. S. Gião.

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Gião, M.S., Wilks, S.A., Azevedo, N.F. et al. Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable Legionella pneumophila . Microb Ecol 58, 56–62 (2009). https://doi.org/10.1007/s00248-008-9472-x

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  • DOI: https://doi.org/10.1007/s00248-008-9472-x

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