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

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.

This is a preview of subscription content, log in to check access.

Figure 1
Figure 2

References

  1. 1.

    Albrich JM, Hurst JK (1982) Oxidative inactivation of Escherichia coli by hypochlorous acid—rates and differentiation of respiratory from other reaction sites. FEBS Lett 144:157–161

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    American Public Health Association (1998) Standard methods for the examination of water and wastewater, 20thth edn. American Public Health Association, American Water Works Association, Water Environmental Federation, Washington DC, pp 4.63–64.64

    Google Scholar 

  3. 3.

    Barrette WC, Albrich JM, Hurst JK (1987) Hypochlorous acid promoted loss of metabolic energy in Escherichia coli. Infect Immun 55:2518–2525

    PubMed  CAS  Google Scholar 

  4. 4.

    Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. Appl Environ Microbiol 73:3283–3290

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Boulos L, Prevost M, Barbeau B, Coallier J, Desjardins R (1999) LIVE/DEAD® BacLight(TM): application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Meth 37:77–86

    Article  CAS  Google Scholar 

  6. 6.

    Costa J, Tiago I, da Costa MS, Verissimo A (2005) Presence and persistence of Legionella spp. in groundwater. Appl Environ Microbiol 71:663–671

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Garcia MT, Jones S, Pelaz C, Millar RD, Abu Kwaik Y (2007) Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection. Environ Microbiol 9:1267–1277

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Howard K, Inglis TJJ (2003) The effect of free chlorine on Burkholderia pseudomallei in potable water. Water Res 37:4425–4432

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Hsu SC, Martin R, Wentworth BB (1984) Isolation of Legionella species from drinking water. Appl Environ Microbiol 48:830–832

    PubMed  CAS  Google Scholar 

  10. 10.

    Hussong D, Colwell RR, O’Brien M, Weiss E, Pearson AD, Weiner RM, Burge WD (1987) Viable Legionella pneumophila not detectable by culture on agar media. Nat Biotechnol 5:947–950

    Article  Google Scholar 

  11. 11.

    James BW, Mauchline WS, Dennis PJ, Keevil CW, Wait R (1999) Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low nutrient environments. Appl Environ Microbiol 65:822–827

    PubMed  CAS  Google Scholar 

  12. 12.

    Keevil CW (2002) Pathogens in environmental biofilms. In: Bitton G (ed) The encyclopedia of environmental microbiology. Wiley, New York, pp 2339–2356

    Google Scholar 

  13. 13.

    Keevil CW (2003) Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy. Water Sci Technol 47:105–116

    PubMed  CAS  Google Scholar 

  14. 14.

    Kim BR, Anderson JE, Mueller SA, Gaines WA, Kendall AM (2002) Literature review—efficacy of various disinfectants against Legionella in water systems. Water Res 36:4433–4444

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Kuchta JM, States SJ, McGlaughlin JE, Overmeyer JH, Wadowsky RM, McNamara AM, Wolford RS, Yee RB (1985) Enhanced chlorine resistance of tap water adapted Legionella pneumophila as compared with agar medium passaged strains. Appl Environ Microbiol 50:21–26

    PubMed  CAS  Google Scholar 

  16. 16.

    Kuchta JM, States SJ, McNamara AM, Wadowsky RM, Yee RB (1983) Susceptibility of Legionella pneumophila to chlorine in tap water. Appl Environ Microbiol 46:1134–1139

    PubMed  CAS  Google Scholar 

  17. 17.

    Lebaron P, Parthuisot N, Catala P (1998) Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl Environ Microbiol 64:1725–1730

    PubMed  CAS  Google Scholar 

  18. 18.

    Lisle JT, Pyle BH, McFeters GA (1999) The use of multiple indices of physiological activity to access viability in chlorine disinfected Escherichia coli O157: H7. Lett Appl Microbiol 29:42–47

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR (1977) Legionnaires disease—isolation of a bacterium and demonstration of its role in other respiratory disease. N Engl J Med 297:1197–1203

    PubMed  CAS  Google Scholar 

  20. 20.

    McDougald D, Rice SA, Weichart D, Kjelleberg S (1998) Nonculturability: adaptation or debilitation? FEMS Microbiol Ecol 25:1–9

    Article  CAS  Google Scholar 

  21. 21.

    Moberg L, Karlberg B (2000) An improved N,N′-diethyl-p-phenylenediamine (DPD) method for the determination of free chlorine based on multiple wavelength detection. Anal Chim Acta 407:127–133

    Article  CAS  Google Scholar 

  22. 22.

    Ohno A, Kato N, Yamada K, Yamaguchi K (2003) Factors influencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water. Appl Environ Microbiol 69:2540–2547

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Oliver JD (2005) The viable but nonculturable state in bacteria. J Microbiol 43:93–100

    PubMed  Google Scholar 

  24. 24.

    Oliver JD, Dagher M, Linden K (2005) Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater. J Water Health 3:249–257

    PubMed  CAS  Google Scholar 

  25. 25.

    Pasculle W (2000) Update on Legionella. Clin Microbiol Newslett 22:97–101

    Article  Google Scholar 

  26. 26.

    Phe MH, Dossot M, Block JC (2004) Chlorination effect on the fluorescence of nucleic acid staining dyes. Water Res 38:3729–3737

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Phe MH, Dossot M, Guilloteau H, Block JC (2005) Nucleic acid fluorochromes and flow cytometry prove useful in assessing the effect of chlorination on drinking water bacteria. Water Res 39:3618–3628

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Schoenen D (2002) Role of disinfection in suppressing the spread of pathogens with drinking water: possibilities and limitations. Water Res 36:3874–3888

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Skaliy P, Thompson TA, Gorman GW, Morris GK, McEachern HV, Mackel DC (1980) Laboratory studies of disinfectants against Legionella pneumophila. Appl Environ Microbiol 40:697–700

    PubMed  CAS  Google Scholar 

  30. 30.

    Steinert M, Emody L, Amann R, Hacker J (1997) Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl Environ Microbiol 63:2047–2053

    PubMed  CAS  Google Scholar 

  31. 31.

    Stocks SM (2004) Mechanism and use of the commercially available viability stain, BacLight. Cytom Part A 61A:189–195

    Article  CAS  Google Scholar 

  32. 32.

    Szewzyk U, Szewzyk R, Manz W, Schleifer KH (2000) Microbiological safety of drinking water. Annu Rev Microbiol 54:81–127

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Tobin JO, Swann RA, Bartlett CLR (1981) Isolation of Legionella pneumophila from water systems: methods and preliminary results. Br Med J 282:515–517

    Article  CAS  Google Scholar 

  34. 34.

    Tobin JOH, Bartlett CLR, Waitkins SA, Barrow GI, Macrae AD, Taylor AG, Fallon RJ, Lynch FRN (1981) Legionnaires’ disease: further evidence to implicate water storage and distribution systems as sources. Br Med J 282:573–573

    Article  CAS  Google Scholar 

  35. 35.

    Wadowsky RM, Yee RB, Mezmar L, Wing EJ, Dowling JN (1982) Hot water systems as sources of Legionella pneumophila in hospital and non-hospital plumbing fixtures. Appl Environ Microbiol 43:1104–1110

    PubMed  CAS  Google Scholar 

  36. 36.

    West AA, Rogers J, Lee JV, Keevil CW (1992) Lack of dormancy in Legionella pneumophila? In: Barbaree JM, Breiman RF, Dufour AP (eds) Legionella current state and emerging perspectives. American Society for Microbiology Press, Washington DC, pp 201–203

    Google Scholar 

  37. 37.

    Yamamoto H, Hashimoto Y, Ezaki T (1996) Study of nonculturable Legionella pneumophila cells during multiple nutrient starvation. FEMS Microbiol Ecol 20:149–154

    Article  CAS  Google Scholar 

  38. 38.

    Yee RB, Wadowsky RM (1982) Multiplication of Legionella pneumophila in unsterilized tap water. Appl Environ Microbiol 43:1330–1334

    PubMed  CAS  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

Corresponding author

Correspondence to M. S. Gião.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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 (2009). https://doi.org/10.1007/s00248-008-9472-x

Download citation

Keywords

  • Chlorine
  • Free Chlorine
  • Chlorine Concentration
  • Residual Chlorine
  • Drinking Water Distribution System