1887

Abstract

Biofilm formation is a major virulence attribute of and is directly associated with therapeutic failure. One method by which acquires antifungal resistance is the expression of drug-resistance genes. This study aimed to evaluate the transcriptional regulation of several genes associated with antifungal resistance of under planktonic, recently adhered and biofilm growth modes and in biofilms in response to antifungal agents. Initially, the antifungal susceptibility of cultures in different growth modes was evaluated by standard antifungal susceptibility testing. Next, to assess , , , , and expression, RNA was harvested from cells in each growth mode, and from biofilms after drug treatment, and subjected to quantitative real-time RT-PCR (qRT-PCR). Biofilm was more resistant to antifungals than recently adhered cells and stationary-phase planktonic cultures. Transcriptional expression of , , , and was lower in recently adhered than in the stationary-phase planktonic cultures. In contrast, levels were significantly increased in recently adhered and biofilm modes of growth. The expression of in biofilms greatly increased on challenge with amphotericin B but not with the other drugs tested (<0.01). was significantly upregulated by ketoconazole (<0.01). Caspofungin and amphotericin B significantly upregulated expression, whereas they significantly downregulated expression (<0.01). These results indicate that the expression of drug-resistance genes is associated with higher drug resistance of biofilms, and lay a foundation for future large-scale genome-wide expression analysis.

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2011-09-01
2021-10-17
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References

  1. Baillie G. S., Douglas L. J. 1999; Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol 310:644–656 [View Article][PubMed]
    [Google Scholar]
  2. Beck-Sagué C. M., Jarvis W. R. National Nosocomial Infections Surveillance System 1993; Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980–1990. J Infect Dis 167:1247–1251 [View Article][PubMed]
    [Google Scholar]
  3. Beggs W. H. 1989; Development of phenotypic resistance to direct lethal miconazole action by Candida albicans entering stationary phase. Mycopathologia 108:201–206 [View Article][PubMed]
    [Google Scholar]
  4. Brajtburg J., Powderly W. G., Kobayashi G. S., Medoff G. 1990; Amphotericin B: current understanding of mechanisms of action. Antimicrob Agents Chemother 34:183–188[PubMed] [CrossRef]
    [Google Scholar]
  5. Chandra J., Mukherjee P. K., Leidich S. D., Faddoul F. F., Hoyer L. L., Douglas L. J., Ghannoum M. A. 2001; Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res 80:903–908 [View Article][PubMed]
    [Google Scholar]
  6. Edlind T. D., Katiyar S. K. 2004; The echinocandin “target” identified by cross-linking is a homolog of Pil1 and Lsp1, sphingolipid-dependent regulators of cell wall integrity signaling. Antimicrob Agents Chemother 48:4491 [View Article][PubMed]
    [Google Scholar]
  7. Ellepola A. N., Samaranayake L. P. 2001; Adjunctive use of chlorhexidine in oral candidoses: a review. Oral Dis 7:11–17 [View Article][PubMed]
    [Google Scholar]
  8. Ellis D. 2002; Amphotericin B: spectrum and resistance. J Antimicrob Chemother 49:Suppl. 17–10[PubMed] [CrossRef]
    [Google Scholar]
  9. Franz R., Kelly S. L., Lamb D. C., Kelly D. E., Ruhnke M., Morschhäuser J. 1998; Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains. Antimicrob Agents Chemother 42:3065–3072[PubMed]
    [Google Scholar]
  10. Franz R., Ruhnke M., Morschhäuser J. 1999; Molecular aspects of fluconazole resistance development in Candida albicans . Mycoses 42:453–458 [View Article][PubMed]
    [Google Scholar]
  11. Gavin A.-C., Bösche M., Krause R., Grandi P., Marzioch M., Bauer A., Schultz J., Rick J. M., Michon A. M. et al. 2002; Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–147 [View Article][PubMed]
    [Google Scholar]
  12. Holmes A. R., Lin Y.-H., Niimi K., Lamping E., Keniya M., Niimi M., Tanabe K., Monk B. C., Cannon R. D. 2008; ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates. Antimicrob Agents Chemother 52:3851–3862 [View Article][PubMed]
    [Google Scholar]
  13. Jayatilake J. A., Samaranayake L. P., Lu Q., Jin L. J. 2007; IL-1α, IL-1ra and IL-8 are differentially induced by Candida in experimental oral candidiasis. Oral Dis 13:426–433 [View Article][PubMed]
    [Google Scholar]
  14. Jin Y., Samaranayake L. P., Samaranayake Y., Yip H. K. 2004; Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol 49:789–798 [View Article][PubMed]
    [Google Scholar]
  15. Kusch H., Biswas K., Schwanfelder S., Engelmann S., Rogers P. D., Hecker M., Morschhäuser J. 2004; A proteomic approach to understanding the development of multidrug-resistant Candida albicans strains. Mol Genet Genomics 271:554–565 [View Article][PubMed]
    [Google Scholar]
  16. Lopez-Ribot J. L., McAtee R. K., Lee L. N., Kirkpatrick W. R., White T. C., Sanglard D., Patterson T. F. 1998; Distinct patterns of gene expression associated with development of fluconazole resistance in serial Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob Agents Chemother 42:2932–2937[PubMed]
    [Google Scholar]
  17. Lu Q., Jayatilake J. A., Samaranayake L. P., Jin L. 2006; Hyphal invasion of Candida albicans inhibits the expression of human β-defensins in experimental oral candidiasis. J Invest Dermatol 126:2049–2056 [View Article][PubMed]
    [Google Scholar]
  18. Lupetti A., Danesi R., Campa M., Del Tacca M., Kelly S. 2002; Molecular basis of resistance to azole antifungals. Trends Mol Med 8:76–81 [View Article][PubMed]
    [Google Scholar]
  19. Mateus C., Crow S. A. Jr, Ahearn D. G. 2004; Adherence of Candida albicans to silicone induces immediate enhanced tolerance to fluconazole. Antimicrob Agents Chemother 48:3358–3366 [View Article][PubMed]
    [Google Scholar]
  20. Morschhäuser J. 2002; The genetic basis of fluconazole resistance development in Candida albicans . Biochim Biophys Acta 1587:240–248 [CrossRef]
    [Google Scholar]
  21. Mukherjee P. K., Chandra J., Kuhn D. M., Ghannoum M. A. 2003; Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols. Infect Immun 71:4333–4340 [View Article][PubMed]
    [Google Scholar]
  22. Nakamura K., Niimi M., Niimi K., Holmes A. R., Yates J. E., Decottignies A., Monk B. C., Goffeau A., Cannon R. D. 2001; Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters. Antimicrob Agents Chemother 45:3366–3374 [View Article][PubMed]
    [Google Scholar]
  23. Navarro-García F., Sánchez M., Nombela C., Pla J. 2001; Virulence genes in the pathogenic yeast Candida albicans . FEMS Microbiol Rev 25:245–268 [View Article][PubMed]
    [Google Scholar]
  24. Nett J. E., Crawford K., Marchillo K., Andes D. R. 2010; Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene. Antimicrob Agents Chemother 54:3505–3508 [View Article][PubMed]
    [Google Scholar]
  25. Perea S., López-Ribot J. L., Kirkpatrick W. R., McAtee R. K., Santillán R. A., Martínez M., Calabrese D., Sanglard D., Patterson T. F. 2001; Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 45:2676–2684 [View Article][PubMed]
    [Google Scholar]
  26. Pfaffl M. W. 2001; A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45 [View Article][PubMed]
    [Google Scholar]
  27. Pfaller M. A., Diekema D. J. 2007; Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20:133–163 [View Article][PubMed]
    [Google Scholar]
  28. Prasad R., Kapoor K. 2004; Multidrug resistance in yeast Candida . Int Rev Cytol 242:215–248 [View Article][PubMed]
    [Google Scholar]
  29. Ramage G., Bachmann S., Patterson T. F., Wickes B. L., López-Ribot J. L. 2002; Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother 49:973–980 [View Article][PubMed]
    [Google Scholar]
  30. Rognon B., Kozovska Z., Coste A. T., Pardini G., Sanglard D. 2006; Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance. Microbiology 152:3701–3722 [View Article][PubMed]
    [Google Scholar]
  31. Ruhnke M., Hartwig K., Kofla G. 2008; New options for treatment of candidaemia in critically ill patients. Clin Microbiol Infect 14:Suppl. 446–54 [View Article][PubMed]
    [Google Scholar]
  32. Samaranayake L. P., MacFarlane T. W. 1990 Oral Candidosis London: Wright;
    [Google Scholar]
  33. Samaranayake Y. H., Dassanayake R. S., Jayatilake J. A., Cheung B. P., Yau J. Y., Yeung K. W., Samaranayake L. P. 2005; Phospholipase B enzyme expression is not associated with other virulence attributes in Candida albicans isolates from patients with human immunodeficiency virus infection. J Med Microbiol 54:583–593 [View Article][PubMed]
    [Google Scholar]
  34. Sanglard D., Kuchler K., Ischer F., Pagani J. L., Monod M., Bille J. 1995; Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39:2378–2386[PubMed] [CrossRef]
    [Google Scholar]
  35. Seneviratne C. J., Jin L., Samaranayake L. P. 2008a; Biofilm lifestyle of Candida: a mini review. Oral Dis 14:582–590 [View Article][PubMed]
    [Google Scholar]
  36. Seneviratne C. J., Jin L. J., Samaranayake Y. H., Samaranayake L. P. 2008b; Cell density and cell aging as factors modulating antifungal resistance of Candida albicans biofilms. Antimicrob Agents Chemother 52:3259–3266 [View Article][PubMed]
    [Google Scholar]
  37. Seneviratne C. J., Wang Y., Jin L., Abiko Y., Samaranayake L. P. 2008c; Candida albicans biofilm formation is associated with increased anti-oxidative capacities. Proteomics 8:2936–2947 [View Article][PubMed]
    [Google Scholar]
  38. Tumbarello M., Posteraro B., Trecarichi E. M., Fiori B., Rossi M., Porta R., de Gaetano Donati K., La Sorda M., Spanu T. et al. 2007; Biofilm production by Candida species and inadequate antifungal therapy as predictors of mortality for patients with candidemia. J Clin Microbiol 45:1843–1850 [View Article][PubMed]
    [Google Scholar]
  39. Vanden Bossche H., Marichal P., Odds F. C. 1994; Molecular mechanisms of drug resistance in fungi. Trends Microbiol 2:393–400 [View Article][PubMed]
    [Google Scholar]
  40. Walker L. A., Gow N. A., Munro C. A. 2010; Fungal echinocandin resistance. Fungal Genet Biol 47:117–126 [View Article][PubMed]
    [Google Scholar]
  41. White T. C. 1997; Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41:1482–1487[PubMed]
    [Google Scholar]
  42. Wisplinghoff H., Bischoff T., Tallent S. M., Seifert H., Wenzel R. P., Edmond M. B. 2004; Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 39:309–317 [View Article][PubMed]
    [Google Scholar]
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