摘要目的:研究体内微进化对阿萨希毛孢子菌生物膜相关表型及耐药性的影响。方法:阿萨希毛孢子菌标准株源自荷兰真菌多态性保藏中心，阿萨希毛孢子菌原代株TO（氟康唑敏感）为解放军总医院第七医学中心皮肤科1例毛孢子菌病的临床分离株，进化株TEVO（氟康唑耐药）在该患者2014年复诊时分离。体外构建上述菌株的生物膜，使用四甲基氮盐（XTT）还原法及激光共聚焦扫描显微镜评估生物膜生长动力学并测定生物膜厚度；体外测定氟康唑、伊曲康唑、伏立康唑对不同生长阶段生物膜的最低抑菌浓度（SMIC），评估生物膜的耐药性。多组间比较采用单因素方差分析，Hartley检验对数据进行方差同质性检验，若方差齐，选用LSD检验进行组间多重比较，若方差不齐，选用Tamhane′ T2检验进行组间多重比较。 结果:在阿萨希毛孢子菌生物膜黏附（0 h）及形成阶段（4 ~ 24 h），进化株TEVO代谢活性最弱（ F黏附 = 35.705， P < 0.001； F形成 = 15.042， P < 0.001）。而黏附后第48小时生物膜成熟，此时生物膜代谢活性最弱的菌株为TO株（ F = 10.985， P < 0.001）。生物膜厚度测量结果显示，成熟阶段TEVO株生物膜厚度[（26.1 ± 1.18）μm]大于TO株[（22.8 ± 1.73）μm， P = 0.001]，但小于标准株[（29.5 ± 1.28）μm， P = 0.001]。药物敏感性实验结果显示，在阿萨希毛孢子菌生物膜黏附及形成阶段，唑类抗真菌药物对TEVO株的SMIC值大于TO株；生物膜成熟阶段，3株菌生物膜的SMIC值均大于1 024 mg/L。 结论:在宿主内环境和抗真菌药物的双重压力下，阿萨希毛孢子菌生物膜相关表型发生了适应性改变，并增强了对唑类抗真菌药物的耐药性。

abstractsObjective: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.