Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 May;63(5):2047–2053. doi: 10.1128/aem.63.5.2047-2053.1997

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.

M Steinert 1, L Emödy 1, R Amann 1, J Hacker 1
PMCID: PMC168494  PMID: 9143134

Abstract

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.

Full Text

The Full Text of this article is available as a PDF (606.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amann R. I., Krumholz L., Stahl D. A. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 1990 Feb;172(2):762–770. doi: 10.1128/jb.172.2.762-770.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barker J., Lambert P. A., Brown M. R. Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila. Infect Immun. 1993 Aug;61(8):3503–3510. doi: 10.1128/iai.61.8.3503-3510.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berger K. H., Isberg R. R. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol. 1993 Jan;7(1):7–19. doi: 10.1111/j.1365-2958.1993.tb01092.x. [DOI] [PubMed] [Google Scholar]
  4. Berger K. H., Merriam J. J., Isberg R. R. Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene. Mol Microbiol. 1994 Nov;14(4):809–822. doi: 10.1111/j.1365-2958.1994.tb01317.x. [DOI] [PubMed] [Google Scholar]
  5. Bozue J. A., Johnson W. Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion. Infect Immun. 1996 Feb;64(2):668–673. doi: 10.1128/iai.64.2.668-673.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brand B. C., Sadosky A. B., Shuman H. A. The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages. Mol Microbiol. 1994 Nov;14(4):797–808. doi: 10.1111/j.1365-2958.1994.tb01316.x. [DOI] [PubMed] [Google Scholar]
  7. Cianciotto N. P., Eisenstein B. I., Mody C. H., Engleberg N. C. A mutation in the mip gene results in an attenuation of Legionella pneumophila virulence. J Infect Dis. 1990 Jul;162(1):121–126. doi: 10.1093/infdis/162.1.121. [DOI] [PubMed] [Google Scholar]
  8. Cianciotto N. P., Eisenstein B. I., Mody C. H., Toews G. B., Engleberg N. C. A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection. Infect Immun. 1989 Apr;57(4):1255–1262. doi: 10.1128/iai.57.4.1255-1262.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cianciotto N. P., Fields B. S. Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5188–5191. doi: 10.1073/pnas.89.11.5188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. England A. C., 3rd, Fraser D. W., Mallison G. F., Mackel D. C., Skaliy P., Gorman G. W. Failure of Legionella pneumophila sensitivities to predict culture results from disinfectant-treated air-conditioning cooling towers. Appl Environ Microbiol. 1982 Jan;43(1):240–244. doi: 10.1128/aem.43.1.240-244.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fields B. S. The molecular ecology of legionellae. Trends Microbiol. 1996 Jul;4(7):286–290. doi: 10.1016/0966-842x(96)10041-x. [DOI] [PubMed] [Google Scholar]
  12. Fraser D. W. Legionellosis: evidence of airborne transmission. Ann N Y Acad Sci. 1980;353:61–66. doi: 10.1111/j.1749-6632.1980.tb18906.x. [DOI] [PubMed] [Google Scholar]
  13. Hobbie J. E., Daley R. J., Jasper S. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol. 1977 May;33(5):1225–1228. doi: 10.1128/aem.33.5.1225-1228.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoge C. W., Breiman R. F. Advances in the epidemiology and control of Legionella infections. Epidemiol Rev. 1991;13:329–340. doi: 10.1093/oxfordjournals.epirev.a036076. [DOI] [PubMed] [Google Scholar]
  15. Horwitz M. A. Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes. J Exp Med. 1987 Nov 1;166(5):1310–1328. doi: 10.1084/jem.166.5.1310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Horwitz M. A. The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med. 1983 Dec 1;158(6):2108–2126. doi: 10.1084/jem.158.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kilvington S., Price J. Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. J Appl Bacteriol. 1990 May;68(5):519–525. doi: 10.1111/j.1365-2672.1990.tb02904.x. [DOI] [PubMed] [Google Scholar]
  18. Kuchta J. M., Navratil J. S., Shepherd M. E., Wadowsky R. M., Dowling J. N., States S. J., Yee R. B. Impact of Chlorine and Heat on the Survival of Hartmannella vermiformis and Subsequent Growth of Legionella pneumophila. Appl Environ Microbiol. 1993 Dec;59(12):4096–4100. doi: 10.1128/aem.59.12.4096-4100.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Manz W., Amann R., Szewzyk R., Szewzyk U., Stenström T. A., Hutzler P., Schleifer K. H. In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Microbiology. 1995 Jan;141(Pt 1):29–39. doi: 10.1099/00221287-141-1-29. [DOI] [PubMed] [Google Scholar]
  20. Marra A., Shuman H. A. Isolation of a Legionella pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings. J Bacteriol. 1989 Apr;171(4):2238–2240. doi: 10.1128/jb.171.4.2238-2240.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moffat J. F., Tompkins L. S. A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infect Immun. 1992 Jan;60(1):296–301. doi: 10.1128/iai.60.1.296-301.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morgan J. A., Rhodes G., Pickup R. W. Survival of nonculturable Aeromonas salmonicida in lake water. Appl Environ Microbiol. 1993 Mar;59(3):874–880. doi: 10.1128/aem.59.3.874-880.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Newsome A. L., Baker R. L., Miller R. D., Arnold R. R. Interactions between Naegleria fowleri and Legionella pneumophila. Infect Immun. 1985 Nov;50(2):449–452. doi: 10.1128/iai.50.2.449-452.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nilsson L., Oliver J. D., Kjelleberg S. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. J Bacteriol. 1991 Aug;173(16):5054–5059. doi: 10.1128/jb.173.16.5054-5059.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oliver J. D., Bockian R. In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus. Appl Environ Microbiol. 1995 Jul;61(7):2620–2623. doi: 10.1128/aem.61.7.2620-2623.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Oliver J. D., Hite F., McDougald D., Andon N. L., Simpson L. M. Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl Environ Microbiol. 1995 Jul;61(7):2624–2630. doi: 10.1128/aem.61.7.2624-2630.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pope C. D., O'Connell W., Cianciotto N. P. Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection. Infect Immun. 1996 Feb;64(2):629–636. doi: 10.1128/iai.64.2.629-636.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Preyer J. M., Oliver J. D. Starvation-induced thermal tolerance as a survival mechanism in a psychrophilic marine bacterium. Appl Environ Microbiol. 1993 Aug;59(8):2653–2656. doi: 10.1128/aem.59.8.2653-2656.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rahman I., Shahamat M., Chowdhury M. A., Colwell R. R. Potential virulence of viable but nonculturable Shigella dysenteriae type 1. Appl Environ Microbiol. 1996 Jan;62(1):115–120. doi: 10.1128/aem.62.1.115-120.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rahman I., Shahamat M., Kirchman P. A., Russek-Cohen E., Colwell R. R. Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae type 1. Appl Environ Microbiol. 1994 Oct;60(10):3573–3578. doi: 10.1128/aem.60.10.3573-3578.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rogers J., Dowsett A. B., Dennis P. J., Lee J. V., Keevil C. W. Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems. Appl Environ Microbiol. 1994 Jun;60(6):1842–1851. doi: 10.1128/aem.60.6.1842-1851.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rollins D. M., Colwell R. R. Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl Environ Microbiol. 1986 Sep;52(3):531–538. doi: 10.1128/aem.52.3.531-538.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rowbotham T. J. Isolation of Legionella pneumophila from clinical specimens via amoebae, and the interaction of those and other isolates with amoebae. J Clin Pathol. 1983 Sep;36(9):978–986. doi: 10.1136/jcp.36.9.978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rowbotham T. J. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol. 1980 Dec;33(12):1179–1183. doi: 10.1136/jcp.33.12.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sanden G. N., Morrill W. E., Fields B. S., Breiman R. F., Barbaree J. M. Incubation of water samples containing amoebae improves detection of legionellae by the culture method. Appl Environ Microbiol. 1992 Jun;58(6):2001–2004. doi: 10.1128/aem.58.6.2001-2004.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wadowsky R. M., Butler L. J., Cook M. K., Verma S. M., Paul M. A., Fields B. S., Keleti G., Sykora J. L., Yee R. B. Growth-supporting activity for Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors. Appl Environ Microbiol. 1988 Nov;54(11):2677–2682. doi: 10.1128/aem.54.11.2677-2682.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Winn W. C., Jr Legionnaires disease: historical perspective. Clin Microbiol Rev. 1988 Jan;1(1):60–81. doi: 10.1128/cmr.1.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wintermeyer E., Flügel M., Ott M., Steinert M., Rdest U., Mann K. H., Hacker J. Sequence determination and mutational analysis of the lly locus of Legionella pneumophila. Infect Immun. 1994 Mar;62(3):1109–1117. doi: 10.1128/iai.62.3.1109-1117.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wintermeyer E., Ludwig B., Steinert M., Schmidt B., Fischer G., Hacker J. Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells. Infect Immun. 1995 Dec;63(12):4576–4583. doi: 10.1128/iai.63.12.4576-4583.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES