Skip to main content
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Feb;61(2):407–412. doi: 10.1128/aem.61.2.407-412.1995

Detection of Legionella species in reclaimed water and air with the EnviroAmp Legionella PCR kit and direct fluorescent antibody staining.

C J Palmer 1, G F Bonilla 1, B Roll 1, C Paszko-Kolva 1, L R Sangermano 1, R S Fujioka 1
PMCID: PMC167300  PMID: 7574578

Abstract

Reclaimed water is an important resource for areas with inadequate water supplies. However, there have been few studies on the variety of microorganisms found in this type of water, since typically reclaimed water is examined only for the presence of coliform bacteria. Many microorganisms, including the legionellae, are known to be more resistant to chlorine than are coliform bacteria. Previously, we detected > 10(3) Legionella cells per ml in primary and secondary sewage effluents and observed no significant reduction in population numbers throughout the treatment process. In this study, we detected Legionella spp. in chlorinated effluent by using an EnviroAmp Legionella PCR kit and direct fluorescent antibody (DFA) staining. However, we were not able to isolate Legionella spp. from either natural or seeded reclaimed water samples. This suggests that the Legionella spp. detected by the PCR and DFA methods may be injured or viable but nonculturable after exposure to the high residual chlorine levels typically found in this type of water source. The numbers of coliform bacteria were low (< 2 cells per 100 ml) in most reclaimed water samples and were not correlated with the presence or absence of Legionella spp. We also collected air samples from above a secondary aeration basin and analyzed them by using the PCR, DFA, and plate culture methods. Legionella spp. were detected in the air obtained from above the secondary basin with all three methods. We concluded that the PCR was superior to the culture and DFA methods for detecting Legionella spp. in environmental water samples.

Full Text

The Full Text of this article is available as a PDF (193.8 KB).

Selected References

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

  1. Bopp C. A., Sumner J. W., Morris G. K., Wells J. G. Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium. J Clin Microbiol. 1981 Apr;13(4):714–719. doi: 10.1128/jcm.13.4.714-719.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dondero T. J., Jr, Rendtorff R. C., Mallison G. F., Weeks R. M., Levy J. S., Wong E. W., Schaffner W. An outbreak of Legionnaires' disease associated with a contaminated air-conditioning cooling tower. N Engl J Med. 1980 Feb 14;302(7):365–370. doi: 10.1056/NEJM198002143020703. [DOI] [PubMed] [Google Scholar]
  3. Fang G. D., Yu V. L., Vickers R. M. Disease due to the Legionellaceae (other than Legionella pneumophila). Historical, microbiological, clinical, and epidemiological review. Medicine (Baltimore) 1989 Mar;68(2):116–132. doi: 10.1097/00005792-198903000-00005. [DOI] [PubMed] [Google Scholar]
  4. Fenstersheib M. D., Miller M., Diggins C., Liska S., Detwiler L., Werner S. B., Lindquist D., Thacker W. L., Benson R. F. Outbreak of Pontiac fever due to Legionella anisa. Lancet. 1990 Jul 7;336(8706):35–37. doi: 10.1016/0140-6736(90)91532-f. [DOI] [PubMed] [Google Scholar]
  5. Fliermans C. B., Cherry W. B., Orrison L. H., Smith S. J., Tison D. L., Pope D. H. Ecological distribution of Legionella pneumophila. Appl Environ Microbiol. 1981 Jan;41(1):9–16. doi: 10.1128/aem.41.1.9-16.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. King C. H., Shotts E. B., Jr, Wooley R. E., Porter K. G. Survival of coliforms and bacterial pathogens within protozoa during chlorination. Appl Environ Microbiol. 1988 Dec;54(12):3023–3033. doi: 10.1128/aem.54.12.3023-3033.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Kuchta J. M., States S. J., McGlaughlin J. E., Overmeyer J. H., Wadowsky R. M., McNamara A. M., Wolford R. S., Yee R. B. Enhanced chlorine resistance of tap water-adapted Legionella pneumophila as compared with agar medium-passaged strains. Appl Environ Microbiol. 1985 Jul;50(1):21–26. doi: 10.1128/aem.50.1.21-26.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mangione E. J., Remis R. S., Tait K. A., McGee H. B., Gorman G. W., Wentworth B. B., Baron P. A., Hightower A. W., Barbaree J. M., Broome C. V. An outbreak of Pontiac fever related to whirlpool use, Michigan 1982. JAMA. 1985 Jan 25;253(4):535–539. [PubMed] [Google Scholar]
  10. Miller L. A., Beebe J. L., Butler J. C., Martin W., Benson R., Hoffman R. E., Fields B. S. Use of polymerase chain reaction in an epidemiologic investigation of Pontiac fever. J Infect Dis. 1993 Sep;168(3):769–772. doi: 10.1093/infdis/168.3.769. [DOI] [PubMed] [Google Scholar]
  11. Palmer C. J., Tsai Y. L., Paszko-Kolva C., Mayer C., Sangermano L. R. Detection of Legionella species in sewage and ocean water by polymerase chain reaction, direct fluorescent-antibody, and plate culture methods. Appl Environ Microbiol. 1993 Nov;59(11):3618–3624. doi: 10.1128/aem.59.11.3618-3624.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Paszko-Kolva C., Shahamat M., Colwell R. R. Effect of temperature on survival of Legionella pneumophila in the aquatic environment. Microb Releases. 1993 Sep;2(2):73–79. [PubMed] [Google Scholar]
  13. Vives-Corrons J. L., Jou J. M., Pastor C., Reverter J. C., Jou C. Characteristics of the external quality assessment (EQA) scheme for haematology in Spain. Qual Assur Health Care. 1991;3(2):75–83. doi: 10.1093/intqhc/3.2.75. [DOI] [PubMed] [Google Scholar]
  14. 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]

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

RESOURCES