| C. albicans resistant to azole agents has been a big problem along with the abuse of broad-spectrum antibiotics and an increasing number of immunocompromised patient populations. It is a more important investigation to know about Candidiasis and its drug resistant condition and study its resistant mechanisms. Therefore, we conduct a primary study of clinical C. albicans and induced resistant strains on the resistance mechamisms. This provided reliable theory for prevention and treatment of Candidiasis. The C. albicans isolates used in this study represent a collection of 56 selected isolates from 3 hospitals in Beijing and Changchun. The isolates were identified as C. albicans and for in vitro antifungal drug susceptibility testing; Two susceptible of them and one standard C. albicans ATCC10231 were selected to have inducible azole resistance associated with a heterogeneous phenotype. Moreover, the point mutations in target enzyme of azole drugs encoding gene ERG11 were compared with the two strains before induction. Random amplified polymorphic DNA was used to investigate the relationship between genotype and drug resistance phenotype among 56 isolates and induced strains. In our study processing, 56 isolates were identified by morphology, blood serum germ tube formation, pachy-membrance sporulation, assimilation carbon source essay, CHROMagar Candida chromogenic media and API 20C Kits, etc. We had obtained 56 clinical C.albicans strains from 74 clinical specimens. The result suggested Candidiasis caused by Candida albicans is the most common mycosis. Minimal inhibitory concentrations (MICs) of frequently used five antifungal drugs against 56 C. albicans isolates were obtained with the document M27-A published by the National Committee for Clinical Laboratory Standards. We have found 8.9% fluconazole resistant strains, 7.1% itraconazole resistant strains, and four of five fluconazole resistant strains were resistance to itraconazole, moreover, their MICs of ketoconazole were big. This suggested azole agents have cross-resistance, but the resistance degree is different partly because of different dosages and usage frequency. In view of characteristic of fungi, fluconazole plus abendazole resistance induction was performed with time-increased MICs. In serum permission concentration, abendazole made overexpression of efflux pumps CDR1or (and) CDR2, which resulted in its drug resistance. And it reduced the induced generations. The clinical fluconazole susceptible strains had been resistant to fluconazole when they were transferred of culture in gradually increasing fluconazole concentration or pulsing abendazole (2μg/ml) in it. When fluconazole concentration increased to 16μg/ml (64MIC), after 40 generations, fluconazole MICs of the two strains were 64μg/ml and 128μg/ml, respectively. Transferred of culture in fluconazole (16μg/ml) pulsing abendazole (2μg/ml), after 35 generations, fluconazole MICs of the two strains were 128μg/ml and >128μg/ml, respectively. Standard C.albicans had been resistant to fluconazole when it was transferred of culture in gradually increasing fluconazole concentration or pulsing abendazole (2μg/ml) in it. When fluconazole concentration increased to 16μg/ml (16MIC), after 45 generations, fluconazole MICs was 64μg/ml. Transferred of culture in fluconazole (8μg/ml) pulsing abendazole (2μg/ml), after 35 generations, fluconazole MICs was 64μg/ml. This resultsuggested clinical C. albicans strains are susceptible to azole resistance, probably because of their reinforcemented suitability. In this study, RAPD was used to investigate the relationship between drug resistance phenotype and DNA polymorphism of 56 C.albicans isolates and the induced stains. All three primers generated polymorphic bands among the 56 strains. These results suggested the matrix of genetic distance between five fluconazole resistant C. albicans was very little. Our results showed these strains had high similarity and good agreements among three primers. The relationship between drug resistance phenotype and DNA polymorphism of 56 C.albicans isolates was conformed. Alteration of DNA fingerprinting profiles by comparing strains before and after induction was probably of point mutations, fragment insertion or deletion, which affected definited conjugated sites of primers. Those result in more amplification bands, less amplification bands or alteration of bands length. Probably, It was resulted from the increased plasmalemma permeability, overexpression of some gene or these factors affected on it together. ERG11 gene fragment of the standard C. albicans induced by fluconazole and one fluconazole resistant isolate were cloned into pMD18-T vectors. Comparing the sequence clone plasmid with published sequence of Genbank (AB071956), the clinical resistant isolate had eleven point mutations, including T348A(D116E), A383C(K128T), C549T, C658T, C996T, A1020G, G1083A, T1110C, C1203T, C1284T and A1440N. Two point mutations which resulted in alteration of amino acid are D116E and K128T. Comparing the sequence of the standard C. albicans after induction with published sequence of Genbank (AB071956), it had three point... |
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