Abstract: 【Abstract】 Objective To evaluate the effect of microevolution on phenotypes and drug resistance of the Trichosporon asahii biofilm. Methods The standard strain of Trichosporon asahii was obtained from the Fungal Biodiversity Institute of the Royal Netherlands Academy of Arts and Sciences, the fluconazole-sensitive primary strain （TO） of Trichosporon asahii was isolated from a case of trichosporonosis diagnosed in the Department of Dermatology, the Seventh Medical Center of Chinese People′s Liberation Army General Hospital in 2000, and the fluconazole-resistant evolved strain （TEVO） of Trichosporon asahii was isolated from the above patient in 2014. Biofilms of the above-mentioned strains were formed in vitro, and tetrazolium salt XTT reduction assay was performed to evaluate growth kinetics of the Trichosporon asahii biofilm, and laser scanning confocal microscopy to determine the thickness of the biofilm; the sessile minimum inhibitory concentrations （SMICs） of fluconazole, itraconazole and voriconazole against the biofilms at different growth stages were determined in vitro for the evaluation of the resistance of the biofilms. One-way analysis of variance was used for comparisons among multiple groups, and Hartley test for testing homogeneity of variance. If the variance was homogeneous, least significant difference test was used for multiple comparisons; if the variance was heterogeneous, Tamhane′ T2 test was used for multiple comparisons. Results In the adhesion （0 h） and formation stages （4 - 24 hours） of the Trichosporon asahii biofilm, the metabolic activity of the evolved strain TEVO was the weakest （adhesion stage: F = 35.705, P < 0.001; formation stage: F = 15.042, P < 0.001）. At 48 hours after adhesion, the biofilms matured, and the TO strain showed the weakest metabolic activity （F = 10.985, P < 0.001）. In the maturation stage, the biofilm thickness of the TEVO strain （26.1 ± 1.18 μm） was significantly higher than that of the TO strain （22.8 ± 1.73 μm, P = 0.001）, but significantly lower than that of the standard strain （29.5 ± 1.28 μm, P = 0.001）. As drug susceptibility testing showed, the SMICs of azole antifungal agents against the TEVO strain were higher than those against the TO strain in the adhesion and formation stages of the Trichosporon asahii biofilm, and the SMICs of azole antifungal agents against the biofilms of the 3 strains of Trichosporon asahii were all over 1 024 mg/L in the maturation stage of the biofilm. Conclusion Under the dual pressure of host environment and antifungal drugs, adaptive changes took place in the phenotypes of the Trichosporon asahii biofilm with an increase in the resistance to azole antifungal drugs.

Key words: Trichosporon, Biofilms, Drug resistance, fungal, Biological evolution, Trichosporon asahii, Microevolution