Study On Trichostatin A Enhance In Vitro Activities Of Azoles Against Candida Albicans And Mechanism Research

Part1In vitro study of enhance effect of trichostatin A on azoles againgst Candida albicansChapter1In vitro study of the effect of trichostatin A on the activities of azoles against Candida albicans and other common pathogenic fungiObjective To choose Candida albicans strains of different susceptibilities, histone deacetylases mutant strains and other common pathogenic fungi, investigate the effect of trichostatin A on the in vitro activity of azoles against these pathogenic fungi. Methods According to microdilution method, test the in vitro susceptibility of0.25μg/ml trichostatin A combination with fluconazole, itraconazole, and voriconazole against Candida albicans susceptible strains, resistant strains and other common pathogenic fungi. Then test the minimum fungicide concentration of Candida albicans. Investigate the in vitro activities of azoles against histone deacetylases mutant strains HDA1△and RPD3△. Results Combination of0.25μg/ml trichostatin A with fluconazole, itraconazole, and voriconazole decreased the trailing growth of Candida albicans in different levels, reduced the growth quantity of strains, lower the24h MIC of few strains. However, there was no effect on minimum fungicide concentration. Compared with the homologous strain, HDA1△and RPD3△strains showed increase of in vitro susceptibility. Trichostatin A had no effect on in vitro susceptibilities of other common pathogenic fungi. Conclusion Trichostatin A had the in vitro effect to enhance the activity of azoles against Candida albicans, no effect on minimum fungicide concentration. Trichostatin A was fall to enhance the in vitro activity of azoles against other common pathogenic fungi. Maybe the histone deacetylases encode gene HDAIand RPD3related to the in vitro susceptibility of Candida albicans.Capter2Preliminary study on mechanism of enhance effect by trichostatin A to azoles against Candida albicansObjective Evaluate the expression of histone deacetylase encoding gene of Candida albicans and changes of the expression of histone deacetylase encoding gene and efflux pumps encoding gene during the formation of Candida albicans acquired drug-resistance. Application Rhodamine123to investigate the influence of trichostatin A on the efflux pumps function of Candida albicans. Methods Induce Candida albicans resistance to fluconazole in vitro and collect the in vitro and in vivo acquired resistant strains. Then, extraction of Candida albicans RNA with Trizol, ACT1as internal control gene, evaluate the expression level of histone deacetylase encoding genes HDA1、RPD3、 HOS1、HOS2.HOS3by fluorescent qualitative RT-PCR. Meanwhile the expression level of histone deacetylase encoding gene and efflux pumps encoding gene CDR1、CDR2、 MDR1、FLU1of in vitro and in vivo acquired drug-resistance Candida albicans were evaluated by fluorescent qualitative RT-PCR. Treat clinical strains and the induction resistant strains with Rhodamine123, to evaluate the efflux pumps function by test fluorescence of Candida albicans cells. Results Different expression levels of histone deacetylase encoding genes were found in Candida albicans strains before fluconazole choose, which is unrelated with in vitro sensitivity and the enchance effect by trichostatin A. The expression of HDA1、RPD3、HOS1、HOS2and HOS3rising to different levels during the progress of acquired drug-resistance. The expression of efflux pump gene CDR2、MDR1increased during the progress of in vitro acquired drug-resistance. After a short time contact with0.25μg/ml trichostatin A, the efflux pumps function of Candida albicans clinical strains had no significant changes. The fluorescence of fluconazole/trichostatin A group was higher than fluconazole group. Conclusion Candida albicans histone deacetylase modification is probably one of the upstream mechanisms of the efflux pumps encoding gene expression level changing.Part2In vitro antifungal susceptibilities of common antifungals agents against Candida speciesCapter1Comparison of two methods for determining MICs of Candida species by microdilution methodsObjective To evaluate the in vitro antifungal susceptibility of Candida species by microdilution methods and compare visual method and stereo microscope method to determining the MIC(minimal inhibitory concentration) results. Methods According to CLSI M27-A3microdilution method, in vitro susceptibility of203Candida isolates to4azoles antifungals (miconazole, ketoconazole, econazole and bifonazole) and4non-azoles antifungals (nystatin, liranaftate, naftifine and terbinafine) were measured, and reading MIC results by visual method and stereo microscope method at the same time. Results1. Azoles:the agreements between two reading MIC methods range from87.68%to96.06%.2. Non-azoles:the agreements between two reading MIC methods range from91.63%to99.51%.3. Compared with visual method, the advantages of stereo microscope include:could get a stereoscopic image; easy to observe the micro fungi growth; easy to distinguish the tested strains and contamination fungi.4. Except for liranaftate, naftifine and bifonazole, other tested antifungals has perfect in vitro fungistasis to Candida species. Conclusion Application of stereo microscope could assist to read MIC results of in vitro susceptibility test by microdilution method, it also has good agreements with visual reading results during Candida species testing. Combination of two methods is helpful for the determination of MICs of in vitro susceptibility research.Capter2In vitro activity of luliconazole and other6imidazoles antifungals to common Candida speciesObjective Evaluate the in vitro activity of luliconazole and other6imidazoles antifungals to common Candida species clinical isolates. Methods According to CLSI microdilution method M27-A3, test in vitro susceptibility of5kinds of183isolates of Candida species against luliconazole, ketoconazole, miconazole, econazole, clotrimazole, sertaconazole and bifonazole. Results The results showed that the minimal inhibitory concentration (geometric mean) of ketoconazole, miconazole, econazole, clotrimazole, sertaconazole and bifonazole were0.03～8(0.067) μg/ml、0.03～16(0.071)μg/ml、0.03～8(0.207)μg/ml、0.03～8(0.061)μg/ml、0.03～16(0.187)μg/ml and0.03～>16(1.050)μg/ml respectively. Luliconazole had superior in vitro activity to5kinds of Candida species, the range of MIC (minimal inhibitory concentration) was0.03～8μg/ml, geometric mean was0.087μg/ml, MIC50and MIC90were0.06μg/ml and0.5μg/ml, respectively. Including luliconazole, there were some relatively insensitive strains of each antifungals. Conclusion Except for bifonazole, including luliconazole, other6imidazoles have excellent in vitro activity to Candida species, but there also exist some relatively insensitive strains among these antfungals.