1887

Abstract

biofilms are intrinsically resistant to antimicrobial agents. Previous work demonstrated that the antifungal activity of fluconazole against biofilms is notably enhanced by doxycycline. In order to explore the synergistic mechanism of fluconazole and doxycycline, we investigated the changes of efflux pump gene expression, intracellular calcium concentration and cell cycle distribution after drug intervention in this study. The expression levels of , and were determined by real-time PCR, and the results showed that fluconazole alone could stimulate the high expression of , and , and the combination of doxycycline and fluconazole downregulated the gene overexpression induced by fluconazole. Intracellular calcium concentration was determined using Fluo-3/AM by observing the fluorescence with flow cytometry. A calcium fluctuation, which started 4 h and peaked 8 h after the treatment with fluconazole, was observed. The combined drugs also initiated a calcium fluctuation after 4 h treatment and showed a peak at 16 h, and the peak was higher than that stimulated by fluconazole alone. The cell cycle was measured using flow cytometry. Fluconazole alone and the combined drugs both induced a reduction in the percentages of S-phase cells and an elevation in the percentages of cells in the G/M phase. The results of this research showed that the synergism of fluconazole and doxycycline against biofilms is associated with blockade of the efflux pump genes , and , and stimulation of high intracellular calcium concentration. The findings of this study are of great significance in the search for new antifungal mechanisms.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.072421-0
2014-07-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/jmm/63/7/956.html?itemId=/content/journal/jmm/10.1099/jmm.0.072421-0&mimeType=html&fmt=ahah

References

  1. Albertson G. D., Niimi M., Cannon R. D., Jenkinson H. F.. ( 1996;). Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. . Antimicrob Agents Chemother 40:, 2835–2841.[PubMed]
    [Google Scholar]
  2. Bader T., Schröppel K., Bentink S., Agabian N., Köhler G., Morschhäuser J.. ( 2006;). Role of calcineurin in stress resistance, morphogenesis, and virulence of a Candida albicans wild-type strain. . Infect Immun 74:, 4366–4369. [CrossRef][PubMed]
    [Google Scholar]
  3. Bink A., Pellens K., Cammue B. P. A., Thevissen K.. ( 2011;). Anti-biofilm strategies: how to eradicate Candida biofilms. ? The Open Mycology Journal 5:, 29–38. [CrossRef]
    [Google Scholar]
  4. Cruz M. C., Goldstein A. L., Blankenship J. R., Del Poeta M., Davis D., Cardenas M. E., Perfect J. R., McCusker J. H., Heitman J.. ( 2002;). Calcineurin is essential for survival during membrane stress in Candida albicans. . EMBO J 21:, 546–559. [CrossRef][PubMed]
    [Google Scholar]
  5. Fiori A., Van Dijck P.. ( 2012;). Potent synergistic effect of doxycycline with fluconazole against Candida albicans is mediated by interference with iron homeostasis. . Antimicrob Agents Chemother 56:, 3785–3796. [CrossRef][PubMed]
    [Google Scholar]
  6. Foland T. B., Dentler W. L., Suprenant K. A., Gupta M. L. Jr, Himes R. H.. ( 2005;). Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae. . Yeast 22:, 971–978. [CrossRef][PubMed]
    [Google Scholar]
  7. Ford J. M., Hait W. N.. ( 1990;). Pharmacology of drugs that alter multidrug resistance in cancer. . Pharmacol Rev 42:, 155–199.[PubMed]
    [Google Scholar]
  8. Gao Y., Zhang C., Lu C., Liu P., Li Y., Li H., Sun S.. ( 2013;). Synergistic effect of doxycycline and fluconazole against Candida albicans biofilms and the impact of calcium channel blockers. . FEMS Yeast Res 13:, 453–462. [CrossRef][PubMed]
    [Google Scholar]
  9. Gupta S. S., Ton V. K., Beaudry V., Rulli S., Cunningham K., Rao R.. ( 2003;). Antifungal activity of amiodarone is mediated by disruption of calcium homeostasis. . J Biol Chem 278:, 28831–28839. [CrossRef][PubMed]
    [Google Scholar]
  10. Hawser S. P., Douglas L. J.. ( 1995;). Resistance of Candida albicans biofilms to antifungal agents in vitro. . Antimicrob Agents Chemother 39:, 2128–2131. [CrossRef][PubMed]
    [Google Scholar]
  11. Jacks T., Weinberg R. A.. ( 1996;). Cell-cycle control and its watchman. . Nature 381:, 643–644. [CrossRef][PubMed]
    [Google Scholar]
  12. Jung H. J., Park K., Lee I. S., Kim H. S., Yeo S. H., Woo E. R., Lee D. G.. ( 2007;). S-phase accumulation of Candida albicans by anticandidal effect of amentoflavone isolated from Selaginella tamariscina. . Biol Pharm Bull 30:, 1969–1971. [CrossRef][PubMed]
    [Google Scholar]
  13. Kauh E. A., Bjornsti M. A.. ( 1995;). SCT1 mutants suppress the camptothecin sensitivity of yeast cells expressing wild-type DNA topoisomerase I. . Proc Natl Acad Sci U S A 92:, 6299–6303. [CrossRef][PubMed]
    [Google Scholar]
  14. Lu H., Zhu Z., Dong L., Jia X., Sun X., Yan L., Chai Y., Jiang Y., Cao Y.. ( 2011;). Lack of trehalose accelerates H2O2-induced Candida albicans apoptosis through regulating Ca2+ signaling pathway and caspase activity. . PLoS ONE 6:, e15808. [CrossRef][PubMed]
    [Google Scholar]
  15. McConkey D. J., Orrenius S.. ( 1997;). The role of calcium in the regulation of apoptosis. . Biochem Biophys Res Commun 239:, 357–366. [CrossRef][PubMed]
    [Google Scholar]
  16. Miceli M. H., Bernardo S. M., Lee S. A.. ( 2009;). In vitro analyses of the combination of high-dose doxycycline and antifungal agents against Candida albicans biofilms. . Int J Antimicrob Agents 34:, 326–332. [CrossRef][PubMed]
    [Google Scholar]
  17. Oliver B. G., Silver P. M., Marie C., Hoot S. J., Leyde S. E., White T. C.. ( 2008;). Tetracycline alters drug susceptibility in Candida albicans and other pathogenic fungi. . Microbiology 154:, 960–970. [CrossRef][PubMed]
    [Google Scholar]
  18. Pfaffl M. W.. ( 2001;). A new mathematical model for relative quantification in real-time RT-PCR. . Nucleic Acids Res 29:, e45. [CrossRef][PubMed]
    [Google Scholar]
  19. Phillips A. J., Sudbery I., Ramsdale M.. ( 2003;). Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. . Proc Natl Acad Sci U S A 100:, 14327–14332. [CrossRef][PubMed]
    [Google Scholar]
  20. Pina-Vaz C., Rodrigues A. G., Costa-de-Oliveira S., Ricardo E., Mårdh P. A.. ( 2005;). Potent synergic effect between ibuprofen and azoles on Candida resulting from blockade of efflux pumps as determined by FUN-1 staining and flow cytometry. . J Antimicrob Chemother 56:, 678–685. [CrossRef][PubMed]
    [Google Scholar]
  21. Pozniakovsky A. I., Knorre D. A., Markova O. V., Hyman A. A., Skulachev V. P., Severin F. F.. ( 2005;). Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast. . J Cell Biol 168:, 257–269. [CrossRef][PubMed]
    [Google Scholar]
  22. 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. [CrossRef][PubMed]
    [Google Scholar]
  23. Reedy J. L., Filler S. G., Heitman J.. ( 2010;). Elucidating the Candida albicans calcineurin signaling cascade controlling stress response and virulence. . Fungal Genet Biol 47:, 107–116. [CrossRef][PubMed]
    [Google Scholar]
  24. Sabourin M., Nitiss J. L., Nitiss K. C., Tatebayashi K., Ikeda H., Osheroff N.. ( 2003;). Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks. . Nucleic Acids Res 31:, 4373–4384. [CrossRef][PubMed]
    [Google Scholar]
  25. Sanglard D., Ischer F., Monod M., Bille J.. ( 1997;). Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. . Microbiology 143:, 405–416. [CrossRef][PubMed]
    [Google Scholar]
  26. Schmitt M. E., Brown T. A., Trumpower B. L.. ( 1990;). A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. . Nucleic Acids Res 18:, 3091–3092. [CrossRef][PubMed]
    [Google Scholar]
  27. Shi W., Chen Z., Chen X., Cao L., Liu P., Sun S.. ( 2010;). The combination of minocycline and fluconazole causes synergistic growth inhibition against Candida albicans: an in vitro interaction of antifungal and antibacterial agents. . FEMS Yeast Res 10:, 885–893. [CrossRef][PubMed]
    [Google Scholar]
  28. Wang H., Liang Y., Zhang B., Zheng W., Xing L., Li M.. ( 2011;). Alkaline stress triggers an immediate calcium fluctuation in Candida albicans mediated by Rim101p and Crz1p transcription factors. . FEMS Yeast Res 11:, 430–439. [CrossRef][PubMed]
    [Google Scholar]
  29. Watamoto T., Samaranayake L. P., Egusa H., Yatani H., Seneviratne C. J.. ( 2011;). Transcriptional regulation of drug-resistance genes in Candida albicans biofilms in response to antifungals. . J Med Microbiol 60:, 1241–1247. [CrossRef][PubMed]
    [Google Scholar]
  30. Wu X. Z., Chang W. Q., Cheng A. X., Sun L. M., Lou H. X.. ( 2010;). Plagiochin E, an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway. . Biochim Biophys Acta 1800:, 439–447. [CrossRef][PubMed]
    [Google Scholar]
  31. Zhang Y. Q., Rao R.. ( 2007;). Global disruption of cell cycle progression and nutrient response by the antifungal agent amiodarone. . J Biol Chem 282:, 37844–37853. [CrossRef][PubMed]
    [Google Scholar]
  32. Zore G. B., Thakre A. D., Jadhav S., Karuppayil S. M.. ( 2011;). Terpenoids inhibit Candida albicans growth by affecting membrane integrity and arrest of cell cycle. . Phytomedicine 18:, 1181–1190. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.072421-0
Loading
/content/journal/jmm/10.1099/jmm.0.072421-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error