暗色丝孢霉病致病菌奔马赭霉的实验研究

Experimental Study on the Phaeohyphomycosis Caused by Ochroconis gallopava

目的对我国临床首次分离的引起肺部暗色丝孢霉病的奔马赭霉进行形态学、生理、生化以及分子生物学特点的研究,并与1株标准株比较,为临床诊断和治疗提供帮助.方法选取沙氏葡萄糖蛋白胨琼脂、马铃薯葡萄糖琼脂、玉米琼脂、察氏琼脂、脑心琼脂、燕麦琼脂培养基接种2株菌,观察菌落形态、生长速度及光镜和电镜下形态.还进行了温度实验、环己亚胺抑制实验和碳源同化实验,体外药敏实验和分子生物学特性研究.结果奔马赭霉菌落呈棕色,镜下可见暗色分隔菌丝,分生孢子为棒状,最适宜的生长温度为35℃,环己亚胺可以抑制奔马赭霉的生长.碳源同化实验结果显示:2株菌同化的碳源中有14种是相同的,有1种是不同的.4种抗真菌药物体外药敏实验的MIC值分别为氟康唑32～64μg/mL、伊曲康唑1μg/mL、两性霉素B 2μg/mL、特比萘芬0.25～0.5μg/mL.2株菌的DNA扩增测序结果为:2株菌ITS区片段长度均为743 bp,仅有3个碱基的差别;18S区片段长度为506 bp,2株菌序列相似度为100%.结论通过形态、生理、生化特点及分子生物学特点进一步支持奔马赭霉的鉴定.

Objective To study the morphology, physiology, biochemistry and molecular biologic characteristics of Ochroconis gallopava which was firstly isolated from a pulmonary phaeohyphomycosis in China and compare it with a standard strain to assist in the clinical diagnosis and treatment. Methods The clinical isolate and standard strain were inoculated on SDA, PDA, CMA, CDA, BHIA and OA, respectively to observe the colony morphology and growth rate, and slide cultures were prepared and were examined under light microscope and electron microscope. Thermo-tolerance study, cycloheximide resistance test, API 20C, antifungal susceptibility test and PCR for the internal transcribed spacer of rDNA were also performed. Results Ochroconis gallopava grew rapidly on most media. The colonies appeared to be brown in color with smooth surface. There were aerial mycelia on SDA and PDA. Microscopic observations revealed pale brown and septate hyphae with club-shaped conidia. The growth of O. gallopava was optimal at 35 ℃ and was inhibited by cycloheximide. The two strains assimilated the same 14 glycogens, but 1 different, in API 20C AUX. MICs of fluconazole, itraconazole, amphotericin B and terbinafine were 32 ~ 64 μg/mL, 1 μg/mL, 2 μg/mL and 0.25 ~ 0.5 μg/mL, respectively. PCR results showed that the ITS segment was 743 bp with 3 bp difference in both strains. The length of 18S region was 506 bp, their sequences were the same entirely. Conclusions The clinical isolate is proved as a domestic strain of Ochroconis gallopava at the level of morphology, physiology, biochemistry and molecular biologic characteristics.