Comparison Of Purpurin And Fluconazole Effecting On The Formation Of Candida Albivans Biofilms

Objectives:This study was to construct Candida albicans biofilms model in vitro which can be repeated. Objecting and analyzing the effect on biofilms structure and mitochondria by purpurin and fluconazole, and detecting the expression level of adhesion genes ALS1, ALS4, EFG1, HWP1 to the preliminary exploration of mechanism about the adhesion process of Candida albicans under purpurin.Methods:1. Constrution and identification of Candida albicans biofilms:Slecting the standard strains of Candida albicans ATCC90028 to forming the biofilms in vitro, and observing the morphological structure using the inverted phase contrast microscope, scanning electron microscope and confocal microscopy. Using the XTT reduction technique to measure the growth of Candida albicans bioflms, especially to determine the logarithmic growth phase and declining phase.2. The impact of purpurin and fluconazole against formation of Candida albicans biofilms:To make sure the optimal concentrion of purpurin against Candida albicnas biofilms, we used the XTT reduction assays and cytotoxicity experiments. And also using the inverted phase contrast microscope, scanning electron microscope and confocal microscopy to observe the morphological structure of treated biofilms with purpurin and fluconazole.3. The impact of purpurin and fluconazole against Candida albicans biofilms adhesion process:Observing the effect of adhesion process with purpurin and fluconazole via the inverted phase contrast microscope, the activity of mitochondria via confocal microscopy in a qualitative method. By way of weighing the dry weight and detecting the expression level of gene ALS1, ALS4, EFG1, HWP1 in a quantitive method to exploring the mechanism about the adhesion.Results:1. The model of biofilms was stable and reproducible, microscopic biofilm was a tipical network structure, the hyphae structure and extracellular matrix were more apparent after staining with 0.01% crystal violet. The growth curve was nearly a "S" shape, and the logarithmic phase was about 12-48h, stable period was about 48-60h, after the 60h entering into the decling phase.2. The final concentration of purpurin was 20μg/mL by XTT reduction assays and cytotoxicity experiments, the concentration had an effective consequent and no toxicity on human fibrolast cells. Under the inverted phase contrast microscope, scanning electron microscope and confocal microscopy, the biofilms teated with different concentrations of purpurin and fluconazole were observed. And the biofilms were more loose and the hyphae structure were less than any other groups.3. Under the inverted phase contrast microscope, with 20μg/mL purpurin biofilm cells were less than any other groups. Incubated for 48h, after digestion and centrifugation, the weight of biofilms were lowest under 20μg/mL purpurin, comparing with other two groups the P values were less than 0.05. However, the genes expression levels were higher than the fluconazole groups, lower than normal groups. And the P values were less than 0.05. Finally, the activity of mitochondrial were significantly reduced with 20μg/mL purpunn.Conclusions:1. Standard strains of Candida albicans ATCC90028 could form the stable and repeatable biofilm model in vitro.2. Compared with fluconazole group and normal group, purpurin groups had a significantly inhibition on biofilms.3. Compared with fluconazole group and normal group, purpurin groups had a significantly inhibition on adhension process about biofilms.