1887

Abstract

hybridization of whole cells with rRNA-targeted, fluorescently labelled oligonucleotide probes is a powerful method to specifically detect micro-organisms in their natural habitat without cultivation and subsequent identification by phenotypic characterization. To examine the use of this method for the specific detection of pathogenic species, we have designed an oligonucleotide probe which binds to the 18S rRNA of and the two most important pathogenic species, and differentiates them from other clinically relevant species. After establishing suitable hybridization conditions, we confirmed the specificity of our probe O20 in RNA dot blot hybridizations with a series of reference strains and clinical isolates of medically important species. All and strains hybridized with the probe, whereas all strains of and did not. When we used the fluorescently labelled probe O20 to specifically detect single cells of the two target species by hybridization, both and reacted strongly with the probe and could be clearly differentiated from and although the latter organism contains only two nucleotide mismatches in the probe target region. This discrimination capacity was also seen when mixed suspensions of and were hybridized with the probe. After infection of a human endothelial cell line with and cells adhering to the endothelial cells were easily distinguishable from the cells by fluorescent hybridization with probe O20. In addition, germ tubes and hyphae of were also efficiently labelled. The application of fluorescently labelled rRNA-targeted oligonucleotide probes therefore appears to be a valuable tool for the specific detection and identification of different members of the genus which does not require any cultivation.

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1996-10-01
2021-10-24
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References

  1. Amann R. I., Binder B. J., Olson R. J., Chisholm S. W., Devereux R., Stahl D. A. 1990a; Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925
    [Google Scholar]
  2. Amann R. I., Krumholz L., Stahl D. A. 1990b; Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770
    [Google Scholar]
  3. Amann R. I., Springer N., Ludwig W., Görtz H. D., Schleifer K. -H. 1991; Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature 351:161–164
    [Google Scholar]
  4. Amann R. I., Ludwig W., Schleifer K. -H. 1995; Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169
    [Google Scholar]
  5. Anaissie E. 1992; Opportunistic mycoses in the immunocompromised host: experience at a cancer center and review. Clin Infect Dis 14: (Suppl.1) 43–53
    [Google Scholar]
  6. Calderone R., Braun P. 1991; Adherence and receptor relationships of Candida albicans . Microbiol Rev 55:1–20
    [Google Scholar]
  7. DeLong E. F., Wickham G. S., Pace N. R. 1989; Phylogenetic stains: ribosomal RNA-based probes for the identification of single microbial cells. Science 243:1360–1363
    [Google Scholar]
  8. van Deventer A. J. M., Goessens W. H. F., van Belkum A., van Vliet H. J. A., van Etten E. W. M., Verbrugh H. A. 1995; Improved detection of Candida albicans by PCR in blood of neutropenic mice with systemic candidiasis. J Clin Microbiol 33:625–628
    [Google Scholar]
  9. Edgell C.-J. S., McDonald G. C., Graham J. B. 1983; Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sei USA 803734–3737
    [Google Scholar]
  10. Fraser V. J., Jones M., Dunkel J., Storfer S., Medoff G., Dunagan W. C. 1992; Candidemia in a tertiary care hospital: epidemiology, risk factors and predictors of mortality. Clin Infect Dis 15:415–421
    [Google Scholar]
  11. Golemis E. A., Gyuris J., Brent R. 1993; Saccharomyces cerevisiae. In Current Protocols in Molecular Biology 2 Chapter 13 Ausubel F. M., Brent R., Kingston R.E.., Moore D. D., Seidman J. G., Smith J. A., Struhl K. Edited by New York: John Wiley & Sons;
    [Google Scholar]
  12. Hostetter M. 1994; Adhesins and ligands involved in the interaction of Candida spp. with epithelial and endothelial surfaces. Clin Microbiol Rev 7:29–42
    [Google Scholar]
  13. Jones J. M. 1990; Laboratory diagnosis of invasive candidiasis. Clin Microbiol Rev 3:32–45
    [Google Scholar]
  14. Jordan J. A. 1994; PCR identification of four medically important Candida species by using a single primer pair. J Clin Microbiol 32:2962–2967
    [Google Scholar]
  15. Kwon-Chung K. J., Bennett J. E. 1992; Candidiasis. In Medical Mycology pp. 280–336 Philadelphia: Lea & Febiger;
    [Google Scholar]
  16. McConaughy B. L., Laird C. D., McCarthy B. J. 1969; Nucleic acid reassociation in formamide. Biochemistry 8:3289–3295
    [Google Scholar]
  17. Mankin A. S., Skryabin K. G., Rubtsov P. M. 1986; Identification of ten additional nucleotides in the primary structure of yeast 18S rRNA. Gene 44:143–145
    [Google Scholar]
  18. Manz W., Amann R. I., Ludwig W., Wagner M., Schleifer K. -H. 1992; Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst Appl Microbiol 15:593–600
    [Google Scholar]
  19. Montone K. T., Litzky L. A. 1995; Rapid method for detection of Aspergillus 5S ribosomal RNA using a genus-specific oligonucleotide probe. Clin Microbiol Infect Dis 103:48–51
    [Google Scholar]
  20. Neefs J. -M., van de Peer Y., de Rijk P., Chapelle S., de Wachter R. 1993; Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res 21:3025–3049
    [Google Scholar]
  21. Odds F. C. 1988 Candida and Candidosis. London: W. B. Saunders;
    [Google Scholar]
  22. Ollert M. W., Söhnchen R., Korting H. G., Ollert U., Bräutigam S., Bräutigam W. 1993; Mechanisms of adherence of Candida albicans to cultured human epidermal keratinocytes. Infect Immun 61:4560–4568
    [Google Scholar]
  23. Pfaller M. A., Messer S. A., Hollis R. J. 1994; Strain delineation and antifungal susceptibilities of epidemiologically related and unrelated isolates of Candida lusitaniae . Diagn Microbiol Infect Dis 20:127–133
    [Google Scholar]
  24. Poulsen L. K., Lan F., Kristensen C. S., Hobolth P., Molin S., Krogfelt K. A. 1994; Spacial distribution of Escherichia coli in the mouse large intestine inferred from rRNA in situ hybridization. Infect Immun 62:5191–5194
    [Google Scholar]
  25. Reiss E., Morrison C. J. 1993; Nonculture methods for diagnosis of disseminated Candidiasis. Clin Microbiol Rev 6:311–323
    [Google Scholar]
  26. Rex J. H., Rinaldi M. G., Pfaller M. A. 1995; Resistance of Candida species to fluconazole. Antimicrob Agents Chemother 39:1–8
    [Google Scholar]
  27. Rüchel R., Borg-von Zepelin M., Eiffert H., Muche R. 1991; Bacteria accompanying clinical Candida isolates from respiratory secretions and the genitourinary tract. Mycoses 34:235–238
    [Google Scholar]
  28. Soli D. R. 1986; The regulation of cellular differentiation in the dimorphic yeast Candida albicans . BioEssays 5:5–11
    [Google Scholar]
  29. Vanden Bossche H., Marichal P., Odds F. C. 1994; Molecular mechanisms of drug resistance in fungi. Trends Microbiol 2:393–400
    [Google Scholar]
  30. Wagner M., Erhart R., Manz W., Amann R. I., Lemmer H., Wedi D., Schleifer K. -H. 1994; Development of an rRNA- targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appi Environ Microbiol 60:792–800
    [Google Scholar]
  31. Wilcox J. N. 1993; Fundamental principles of in situhybridization. J Histochem Cytochem 41:1725–1733
    [Google Scholar]
  32. Wingard J. R., Merz W. G., Rinaldi M. G., Johnson T. R., Karp J. E., Sarai R. 1991; Increase in Candida krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with fluconazole. N Engl J Med 325:1274–1277
    [Google Scholar]
  33. Wingard J. R., Merz W. G., Rinaldi M. G., Miller C. B., Karp J. E., Sarai R. 1993; Association of Torulopsis glabrata infections with fluconazole prophylaxis in neutropenic bone marrow transplant patients. Antimicrob Agents Chemother 37:1847–1849
    [Google Scholar]
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