Department of Pharmacy, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, PR China.
School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong Province, PR China.
J Med Microbiol. 2014 Jul;63(Pt 7):956-961. doi: 10.1099/jmm.0.072421-0. Epub 2014 May 8.
Candida albicans biofilms are intrinsically resistant to antimicrobial agents. Previous work demonstrated that the antifungal activity of fluconazole against C. albicans 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 CDR1, CDR2 and MDR1 were determined by real-time PCR, and the results showed that fluconazole alone could stimulate the high expression of CDR1, CDR2 and MDR1, 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 G2/M phase. The results of this research showed that the synergism of fluconazole and doxycycline against C. albicans biofilms is associated with blockade of the efflux pump genes CDR1, CDR2 and MDR1, and stimulation of high intracellular calcium concentration. The findings of this study are of great significance in the search for new antifungal mechanisms.
白色念珠菌生物膜具有内在的抗微生物药物耐药性。以前的工作表明，多西环素显著增强了氟康唑对白色念珠菌生物膜的抗真菌活性。为了探索氟康唑和多西环素的协同作用机制，本研究在药物干预后，研究了外排泵基因表达、细胞内钙浓度和细胞周期分布的变化。通过实时 PCR 测定 CDR1、CDR2 和 MDR1 的表达水平，结果表明氟康唑单独作用可刺激 CDR1、CDR2 和 MDR1 的高表达，而多西环素与氟康唑联合作用可下调氟康唑诱导的基因过表达。通过观察荧光用流式细胞术测定细胞内钙浓度。用 Fluo-3/AM 观察到，在氟康唑处理后 4 小时开始并在 8 小时达到峰值的钙波动。联合药物在 4 小时处理后也引发了钙波动，并在 16 小时达到峰值，峰值高于氟康唑单独作用。用流式细胞术测定细胞周期。氟康唑单独和联合药物均诱导 S 期细胞百分比降低，G2/M 期细胞百分比升高。该研究结果表明，氟康唑和多西环素对白色念珠菌生物膜的协同作用与外排泵基因 CDR1、CDR2 和 MDR1 的阻断以及细胞内钙浓度的升高有关。本研究的发现对寻找新的抗真菌机制具有重要意义。
