| The incidence of Candida infections had dramatically increased in recent years, due partly to the onset of AIDS and improved medical techniques including the use of wide-spectrum antibiotics, immunosuppressive therapy, and organ transplantation. Systemic infections caused by Candida have been now a major impact on the severe patients lives. Being one of the limited antifungal agents, fluconazole acted efficiently by inhibiting the biosynthesis of ergosterole-the major membrane sterol of fungi. However, with increased use of these agents treatment failures occurred for the emergence of azole-resistant strains of C. albicans.Several mechanisms of resistance to fluconazole have been described in C. albicans. Point mutations in the gene encoding the target of the azoles-lanosterol demethylase (ERG11), increased expression of ERG11, have been identified associated with fluconazole resistance; Two classes of efflux pumps have been implicated in azole resistance, of which encoding gene are CDR1,CDR2, and MDR1; Other mechanisms include alteration in the ergoterol biosynthesis pathway, chromosomal nondisjunction, et al. The overexpression of CDR1, CDR2, and MDR1 is a common theme in azole antifungal resistance in C. albicans, and some researchers found that the overexpression of CDR1,CDR2, MDR1 were correlated with transcriptional regulators. However, little is known about the transcriptional regulation networks. With the progressed sequencing work on Candida albicans in Stanford University in America, the genome database becomes abundant, which attracts researchers and leads them in searching for potential regulatory mechanisms and other resistant genes.Due to the clonal nature of C. albicans, it is critical that matched sets of sensitive and resistant isolates of a single strain be used when characterizing molecular mechanisms of resistance. In this research, 22 C. albicans isolates from vagina were induced fluconazole resistance in media with fluconazole. Eric-PCR (a kind of RAPD method) and DifferentialDisplay PCR (DD-PCR) were then adopt in comparing DNA and RNA fingerprints between these matched resistant isolates and their susceptible ancestor, handling together with other studys on phenotype such as growth activity (fitness), biochemical character, resistance evolution and maintenance. We hope our study will lend insight into the azole resistance mechanisms study, and be helpful in clinical diagnosis, therapy, drug evolution, and resistance epidemiological monitoration.Methods and Results1. Susceptibility testing of C. albicans The MICs of fluconazole was determined by using broth microdulution techniques as described by the National Committee for Clinical Laboratory Standards. All 22 C. albicans isolates were susceptible to fluconazole (MIC<8ug/ml).2. Experimental induction of fluconazole resistance 22 C. albicans isolates were incubated in YEPD media with increasing fluconazole concentration. 4 fluconazole resistant strains appeared after being inducted for 45-80 days, with MIC values as 435-r (64-128M-g/ml ), A06-r(64 g/ml), B07-r (64 g/ml), C01-r (64 g/ml).3. Phenotypic analysis All resistant isolates had a similar growth activity with their susceptible ancestors in drug-free media (p>0.05); Each resistant isolates showed no difference either growing in drug-in media or in drug-free media (p>0.05); Strain 435, A06, and C01 had a same metabolic profile (YBC card) with their resistant filial isolates, while strain B07 had one reaction different-susceptible parent shows GLY (+) and the matched resistant isolate shows negative reaction; All 4 resistant filial isolates maintained fluconazole resistance after growing in drug-free media for 24 days.4. ERIC-PCR genotype comparison All 22 C. albicans isolates had some common bands in ERIC-PCR profiles, remarkably different to C. parapsilosis standard strain ATCC 22019; Distinctions still exist in the same C. albican strain, however, 4 different ERIC-PCR genotypes were showed in these 22 C.
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