Mechanism of fluconazole resistance in Candida albicans biofilms:: Phase-specific role of efflux pumps and membrane sterols
Mechanism of fluconazole resistance in Candida albicans biofilms:: Phase-specific role of efflux pumps and membrane sterols
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DOI：
10.1128/iai.71.8.4333-4340.2003
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发表时间：
2003-08-01
影响因子：
3.1
通讯作者：
Ghannoum, MA
中科院分区：
文献类型：
--
作者：
Mukherjee, PK;Chandra, J;Ghannoum, MA
白色念珠菌生物膜的形成是通过三个不同的发展阶段，并与高氟康唑（FLU）的耐药性。在本研究中，我们使用了一组缺乏一个或多个药物外排泵Cdr 1 p，Cdr 2 p和Mdr 1 p的同基因念珠菌菌株，以确定它们在生物膜的FLU抗性中的作用。此外，研究了甾醇谱的变化作为耐药性的可能机制。我们的研究结果表明，亲本和突变株形成类似的生物膜。然而，由双突变体和三突变体形成的生物膜在6小时时比野生型菌株（MIC > 256 μ g/ml）对FLU更敏感（MIC分别为64和16 μ g/ml）。在稍后的时间点（12和48小时），所有菌株都对该唑类产生耐药性（MIC大于或等于256 μ g/ml），表明在生物膜形成的晚期阶段，外排泵不参与耐药性。北方印迹分析显示，念珠菌生物膜表达CDR和MDR 1基因在所有的发展阶段，而嗜酸性细胞表达这些基因仅在12和48小时的时间点。外排泵的功能进行了测定罗丹明（Rh 123）外排试验，揭示了显着差异Rh 123保留生物膜和反渗透细胞在早期阶段（P = 0.0006），但不是在后期阶段（12和48小时）。甾醇分析表明，麦角甾醇水平显着降低（P < 0.001），在中间和成熟阶段，与早期阶段的生物膜。这些研究表明，多组分，阶段特异性机制在真菌生物膜的抗真菌药物耐药性中起作用。
Candida albicans biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic Candida strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 mug/ml, respectively) than the wild-type strain (MIC > 256 mug/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC greater than or equal to 256 mug/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that Candida biofilms expressed CDR and MDR1 genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase (P = 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased (P < 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.