Resistant mechanisms of Candida albicans to fluconazole

To detect the resistant mechanisms of Candida albicans to fluconazole (FCZ) at molecular biology level, since the resistance mechanisms of azole antifungal agents have been the focus of attention these years.

Thirty-two FCZ-resistant C. albicans were selected as our test strains (MICs > or = 64 micrograms/ml). With 14-alpha-demethylase gene (ERG16 gene, target enzyme encoding gene of azoles) as our object, we chose six sets of primers from the ERG16 gene to amplify the interested fragments, and conducted Southern blot hybridization, restriction fragment length polymorphism (RFLP), and single strand conformation polymorphism (SSCP) analysis for the fragments which were amplified by the six sets of primers, and pre-resistant sensitive strains were used as controls. Three representative fragments, A66, D66 and E78, were selected to be cloned and sequenced.

The polymerase chain reaction (PCR) amplification showed that several tested strains were negative for some primers. However, our Southern blot analysis reminded that their resistance did not result from the lack of target enzyme coding gene. SSCP analysis showed that differences were noted between the resistant and sensitive strains and inter-resistant strains. Statistical analysis showed that the most variable sequence lied in the amplifier of the sixth pair of primer, and all the tested 32 strains showed positive results. In the 11 mutation points we found, five resulted in amino acid alterations. It is likely that one or more mutational alterations (alone or in combination) might lead to the expression of an enzyme highly resistant to the inhibitory action of FCZ which in turn is responsible for the FCZ resistant trait in these strains.

One or more mutational alterations might lead to the azole resistant trait in this strains.