| Objective:To analyze the fluconazole resistance in hyphal-form Candida albicans, the suitable culture method to induce C. albicans to form pseudo-hyphae in vitro was selected, the endonuclease maps of the hyphal-form and the maps between the hyphal-form and yeast-form were respectively compared, and randomly amplified polymorphic DNA (RAPD) fingerprints were analyzed among susceptible, susceptible dose dependent and resistant C. albicans strains with fluconazole.Method:A series of 16 strains C. albicans from the same body were induced to form pseudo-hyphae in RPMI 1640 medium and DMEM medium at 37 癈 for twenty-four hours respectively. The pseudo-hyphae and yeast were counted and the ratio between pseudo-hypha and total cells was calculated. C. albicans strains were induced to form pseudo-hyphae in RPMI 1640 medium for seven days by transferring twelve times. The genome DNAs of the hyphal-form of C. albicans was cleaved by two restriction enzymes EcoR I and Bgl II, the products were electrophored and photoed. DNAs isolated from hyphal-form C. albicans were amplified with a RAPD primer, 5'... TCACCACGGT...3', and the PCR-amplified DNAs were separated by denaturing polyacrylamine gel electrophoresis (PAGE) and photoed.Results:1. The ratio of hyphal-form of the substrain CA-l-CA-12 was higher than that of3the substrain CA-13-CA-17 after the strains were cultured in DMEM medium at 37℃ and significant difference was observed (jf=0.000, t test).2. Greater than 80% hyphal-form of C. albicans were observed after the stains were cultured in RPMI 1640 medium at 37 ℃ for eight or twelve hours, but the only 20.10% hyphal-form in substrain CA-17 after being cultured for twelve hours.3. The ratio of pseudo-hyphae of the strains above in RPMI 1640 medium was higher than that of in DMEM medium at the same time except a few stains occasionally.4. The ratio of hyphal-form of all the strains was more than 99% after being cultured for seven days by transferring twelve times in RPMI 1640 medium at 37℃.5. There was no difference among the EcoR I endonuclease map of genome DNAs of the hyphal-form C. albicans, which was the same as that of the yeast-form. The Bglll endonuclease maps had some difference between genome DNAs of the hyphal-form C. albicans and there was one more band of about 2kb in substrain CA-4-CA-17 than in substrain CA-l-CA-3, but the Bglll endonuclease map of the yeast-form had the similar difference only between substrain CA-1 and other substrains.6. On amplification with the same arbitrary sequence decamer primer, the results demonstrated a band of about 450bp was specific to substain CA-14-CA-17.Conclusion:1. The ratio of hyphal-form C. albicans of the susceptible strains C. albicans was obviously higher than that of susceptible dose dependent and resistant stains in DMEM medium at the same time, which implied that DMEM medium had a potential to distinguish the resistant strains with fluconazole in clinical lab quickly.2. RPMI 1640 medium to induce hyphae at 37℃ for seven days was thought to be the favorable condition for C. albicans to form pseudo-hyphae.3. The EcoR I endonuclease maps between hyphal-form and yeast-form was identical, but the Bgl II endonuclease maps between them was not identical. Theendonuclease map of hyphal-form truly reflected genome DNAs of C. albicans in vivo.4. On amplification with the primer 51... TCACCACGGT ...3', there was obvious difference in RAPD maps between more resistant strains (MIC^32.0|ag/ml) and more susceptible strains C. albicans (MIC^16.0|ag/ml).
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