Study on the Pharmacological Action and Induced Resistance Mechanism of Terbinafine to Candida Albicans
|Keywords||Candida Terbinafine Susceptibility testing Minimum inhibitory concentration (MIC) Candida albicans Induced Resistance Gene Chip Pharmacological effects Mechanisms of resistance|
PartⅠIn vitro susceptibility of Candida isolates to Terbinafine using a colorimetric system and the microbroth methodObjective: To evaluate the antifungal susceptibility of the Candida that isolated from clinic in ourlocality to terbinafine.Methods: The antifungal susceptibilities to terbinafine of the 2 standard strains of Candida albicans（ATCC90028 and ATCC10213）and the 129 clinical isolates of the Candida that isolated from clinic in ourlocality to terbinafine were assessed by the NCCLS stanard method（M-27A）, and the minimum inhibitory concentrations（MICs）of terbinafine were determined by oxidoreduction indicator（Alamar blue）.Results: The MICs of the 2 standard isolates of candida Albicans to terbinafine were as follows: candida Albicans ATCC90028 was 8μg/ml, and candida Albicans ATCC10213 was 2μg/ml; the total MICs of the 129 Candida clinical isolates to terbinafine were 0.0313～16μg/ml,total MIC50 were 4μg/ml, total MIC90 were 8μg/ml;among of them, the MICs of the Candida albicans were 0.0313～16μg/ml, MIC50 were 4μg/ml, MIC90 were 8μg/ml; the MICs of the Candida tropicalis were 2～16μg/ml, MIC50 were 8μg/mL, MIC90 were 16μg/mL; the MICs of the Monilia guilliermondii were 1～4μg/ml, MIC50 were 8μg/ml, MIC90 were 16μg/ml; the MICs of the Candida parapsilosis were 1～4μg/ml, MIC50 were 2μg/ml, MIC90 were 4μg/ml. the MICs of the Candida krusei were 1～4μg/ml; the MICs of the Candida lusitaniae were 14～≥16μg/ml.Conclusion: The colorimetric system and the reference microbroth method, performed according to NCCLs guidelines, are reproducible and stable, and terbinafine is active against the Candida that isolated from clinic in our locality. PartⅡStudy on stepwise induction of terbinafine resistance and reversion in Candida albicans in vitroObjective: To establish a model of terbinafine-resistance Candida albicans induced by terbinafine in vitro, investigate the stability of resistance and the biolonical phenotype variation between the terbinafine-resistant Candida albicans and its matched susceptible strain.Methods: Terbinafine-sensitive Candida albicans strain ATCC90028 was experimentally serially subcultured in YPD medium containing increasing concentrations of terbinafine to generate Candida albicans strain ATCC90028-R that is resistant to terbinafine. The susceptibilities of this parental strain and its resistant filial generation to terbinafine were measured respectively. The resistant filial generation that induced by 1024μg/ml of terbinafine was subcultured on drug-free medium for 10 times consecutively and then their susceptibilities to terbinafine were measured respectively to investigate the stability of resistance. and the biolonical phenotype variation between the terbinafine-resistant Candida albicans and its matched susceptible strain were compared by naked eye, light microscope and electron microscope respectively.Results: Terbinafine-susceptible Candida albicans strain ATCC90028（MIC=8μg/ml）was successfully induced by terbinafine to generate terbinafine-resistant Candida albicans strain ATCC90028-R（MIC≥512μg/ml）, and the induced terbinafine-resistant strain maintained drug resistance when it was subcultured on drug-free medium for 10 passages consecutively. But its growth and multiply is torpid, and its cell morphous is irregular.Conclusion: The stable terbinafine-resistant Candida albicans strain ATCC90028-R wassuccessfully induced and proved by in vitro tests. PartⅢGenome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysisObjectives: The aim of this study was to identify changes in the gene expression profile of Candida albicans upon exposure to the allylamine antifungal agent terbinafine, provide new insight into the molecular mechanism of terbinafine against Candida albicans.Methods: Candida albicans ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 MIC（4mg/L）for 90 min. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarrays analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-PCR（RT-PCR）.Results: A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ABC or MFS（CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1）, genes involved in stress response and detoxification（CDR1, AGP2, HOL3）, and gene involved in the ergosterol biosynthesis pathway（ERG12）. The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.Conclusions: We presume that the up-regulation of the genes encoding the multidrug resistance efflux pumps CDR1 and HOL3 may contribute to the terbinafine resistance in Candida albicans. However, the roles of other affected genes in terbinafine resistance remains unclear, further study of these genes and their respective gene products play in the context of antifungal resistance are warranted. PartⅣGenome-wide expression profiling reveals differential gene expression in terbinafine susceptible and experimentally induced resistant strains of Candida albicansObjectives: The aim of this study was to identify changes in the gene expression profile of Candida albicans associated with the acquisition of experimentally induced resistance to terbinafine by genomic expression profiling.Methods: Candida albicans strain ATCC 90028 was experimentally serially passed in increasing concentrations of terbinafine to generate Candida albicans strain ATCC90028-R that is resistant to terbinafine. Genome-wide expression profiling in the resulting terbinafine-resistant Candida albicans strain ATCC90028-R and its terbinafine-susceptible parent Candida albicans strain ATCC90028 was compared for over 6000 genes by a genome-wide DNA microarray analysis. To validate results, eight differentially expressed genes measured by DNA microarray were further confirmed by semi-quantitative RT-PCR.Results: Of the over 6000 genes represented on the microarray, a total of 109 genes were found to be differentially expressed between the terbinafine-resistant Candida albicans strain ATCC90028-R and its terbinafine-susceptible parent Candida albicans strain ATCC90028, specifically, 46 genes were up-regulated while 63 genes were down-regulated at a level at least 2.0-fold in Candida albicans strain ATCC90028-R, in addition to the the membrane transport genes IPF5324 and SIT1 were up-regulated. 11 protein synthesis genes, 7 energy generation genes, the membrane transport genes HXT5 and STL1, the stress response and detoxification genes CRD2 and SOD22, and the ergosterol biosynthesis gene ERG27 were down-regulated, respectively, The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis.Conclusion: The findings suggest these differential gene expression may lend insight into the elucidation of mechanisms of resistance to terbinafine in Candida albicans strain, furthermore, other genes are implicated as having a potential role in the terbinafine antifungal resistant phenotype, further evaluation of the role of these genes and their respective gene products play in terbinafine resistance is warranted.