阿萨希毛孢子菌生物膜形态学、耐药机制和影响因素的研究

发布时间：2018-03-22 01:05

本文选题：阿萨希毛孢子菌　切入点：生物膜　出处：《山西医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 研究背景毛孢子菌属于半知菌亚门,芽生菌纲,隐球酵母目,隐球酵母科。在自然界中广泛存在,是口腔、皮肤、指甲的正常寄生菌。它可引起浅部感染,如毛发、指甲、皮肤的白毛结节病和免疫缺陷患者的系统性或播散性感染,主要见于恶性肿瘤、血液病、呼吸功能不全、慢性肾功能不全、糖尿病、肝硬化或艾滋病患者。其主要风险因素有：抗生素、肾上腺皮质激素、免疫抑制剂、细胞毒性药物的使用,和各种医疗设备的应用如机械通气、导尿术、静脉插管、移植、手术操作或持续性腹膜透析等。目前尚有关于健康者感染此病原菌导致系统性感染的报告。阿萨希毛孢子菌是其重要临床致病菌。阿萨希毛孢子菌(asahii trichosporon,T. asahii)所致播散性毛孢子菌病的临床研究中发现,尽管使用抗真菌药物,但感染仍持续存在。2006年Giovanni等发现与浮游状态的菌株相比,阿萨希毛孢子菌生物膜细胞对两性霉素B、卡泊芬净、伏立康唑和氟康唑的最低抑菌浓度(MICs)明显增加,国内李继红等也发现固着相即生物膜细胞的MIC比浮游相成倍提高。导致耐药性增加的原因是什么? 研究目的 1本课题首先模拟体内环境在常用医学材料表面构建阿萨希毛孢子菌(T. asahii)生物膜(biofilm,BF)模型,证明其存在并在倒置显微镜及扫描电镜下观察其超微结构形态特征,从形态结构方面探索生物膜耐药的机理。 2采用FDA/PI双荧光染色和激光共聚焦显微镜相结合的技术以2μm为步距观察阿萨希毛孢子菌生物膜在不同时间菌的分布情况和成熟期不同层面生物膜菌分布和活性,分析其耐药机制。 3初步观察不同材料对阿萨希毛孢子菌(T. asahii)生物膜(biofilm,BF)形成能力的影响。研究方法 1以来源于本院皮肤科临床分离株阿萨希毛孢子菌株(BZP07002)1株为研究对象,在聚芳脂材料上构建T. asahii生物膜模型,不同时间观察其在倒置显微镜和扫描电镜下的形态特征,并同时用XTT法和细胞计数法测定生物膜活性进行定量分析。 2激光共聚焦显微镜下观察经FDA/PI染色生物膜不同时间菌分布和成熟期不同层面菌分布、活性及生物膜厚度。 3将聚芳脂(PAT)、聚苯乙烯(PS)、聚氯乙烯(PVC)材料剪成1×1cm2大小,放入24孔板中构建T. asahii生物膜模型,采用XTT法测定生物膜活性进行定量分析,不同时间观察T. asahii生物膜在倒置显微镜下的形态特征,将聚芳脂、聚苯乙烯上培养72h的生物膜在扫描电镜下观察。结果 1.T. asahii可在聚芳脂表面形成生物膜,其形成过程包括了真菌表面粘附、微菌落形成和生物膜成熟；镜下T. asahii生物膜的孢子、菌丝、假菌丝等多种形态相互堆积缠绕,与基质共同形成一个复杂的三维立体结构。随时间延长,生物膜活性不断升高,XTT法和活菌计数结果呈正相关(r=0.936,P0.001)； 2.随时间延长,阿萨希毛孢子菌逐渐由散在粘附到聚集成团块,活菌和死菌的量都增加；T. asahii生物膜不同层面的活性曲线坡度缓和,无明显规律,生物膜的厚度为14.3μm到31μm不等。 3.聚芳脂、聚苯乙烯、聚氯乙烯3种材料上均能形成生物膜,且形成的生物膜广泛,几乎覆盖整个材料表面,不同材料上形成生物膜的活性有差别(F=14.743,P0.01),48h时活性由高到低为聚芳脂=聚氯乙烯聚苯乙烯。倒置显微镜和扫描电镜下观察发现聚芳脂、聚氯乙烯形成的生物膜可见孢子、菌丝、假菌丝结构,聚苯乙烯上形成以孢子为主要结构的微生物群落。结论 1.体外可形成T. asahii生物膜；T. asahii生物膜比其浮游状态下具有更加复杂的结构； 2.随时间延长T. asahii生物膜活性不断增高,不同层面T. asahii生物膜的活性无明显差异,分析细胞低生长率并非T. asahii生物膜的耐药机制。3.T. asahii可在不同的材料上形成生物膜,但不同材料上形成生物膜的能力不同。聚芳脂、聚氯乙烯比聚苯乙烯更易于真菌的粘附；且以菌丝、假菌丝为主要结构的微生物群落活力比单纯孢子的活力强。
[Abstract]:Research background
Spore bacteria belong to deuteromycotina, blastomycete Gang, Cryptococcus yeast, yeast Cryptococcus. Exist widely in nature is the mouth, skin, normal parasite nails. It can cause superficial infections, such as hair, nails, skin white haired patients with sarcoidosis and immunodeficiency of systemic or disseminated infection, mainly in malignant tumor, blood disease, respiratory insufficiency, chronic renal insufficiency, diabetes, cirrhosis or AIDS patients. The main risk factors are: antibiotics, glucocorticoids, immunosuppressants, use of cytotoxic drugs, and other medical equipment applications such as mechanical ventilation, venous catheterization. Intubation, transplantation, surgical operation or continuous peritoneal dialysis. There are about to lead to systemic infection infection Report of the pathogen and health. ASA Trichosporon is one of the most important clinical pathogens.
A Csathy Mao (asahii Trichosporon, T. spores asahii) found in clinical research due to disseminated trichosporosis in, despite the use of antifungal drugs, but the infection persists in.2006 Giovanni found with planktonic strains and compared to ASA spore bacteria biofilm cells of amphotericin B, caspofungin, MIC voriconazole and fluconazole (MICs) increased significantly, domestic Li Jihong also found that phase sessile biofilm cells MIC times higher than planktonic phase. What is the cause of the increase of drug resistance?
research objective
1 this paper simulated in vivo environment in the construction of ASA and Trichosporon surface (T. asahii) commonly used in medical materials biofilm (biofilm, BF) model, and prove the existence and to observe the ultrastructure morphological features in the inverted microscope and scanning electron microscope, the mechanism of biofilm resistance from the cable structure.
2 by FDA/PI double fluorescent staining and laser confocal microscope technology combined with 2 m to step away from the observation that ASA spore bacteria biofilm distribution in different time and different levels of bacteria mature biofilm bacteria distribution and activity analysis of its drug resistance mechanism.
3 the effects of different materials on the formation ability of T. asahii (T. asahii) biofilm (biofilm, BF) were preliminarily observed.
research method
Since 1 from the Department of dermatology in our hospital clinical isolates of ASA and Trichosporon strains 1 strains (BZP07002) as the research object, construct the T. asahii biofilm model in aromatic lipid materials, different time were observed under inverted microscope and scanning electron microscope morphological characteristics, and at the same time by XTT method and cell counting method for the determination of biological membrane the activity of quantitative analysis.
Under 2 laser confocal microscopy, the distribution of bacteria in different layers of the biofilm and the distribution of bacteria, activity and the thickness of the biofilm were observed at different time of FDA/PI staining.
3 aromatic lipid (PAT), polystyrene (PS), polyvinyl chloride (PVC) material is cut into 1 x 1cm2 size, into 24 well plates to construct T. asahii biofilm model, determination of biological activity were quantitatively analyzed by XTT method, different time to observe T. asahii biofilm morphology under inverted microscope, the poly aryl fat, polystyrene biofilm 72h under scanning electron microscope.
Result
1.T. asahii can form biofilms in aromatic lipid surface, its formation process consists of fungal adherence, microcolony formation and mature biofilm; microscopic T. asahii biofilm spores, mycelium, various forms of pseudohypha and stacking winding, together form a complex three-dimensional structure and matrix. With the extension of time biological activity, increasing XTT method and viable count were positively correlated (r=0.936, P0.001);
2., with the extension of time, the amount of viable and dead bacteria increased gradually from sporadic adherence to aggregated agglomerates, and the slope of T. asahii biofilms varied at different levels without obvious regularity. The thickness of biofilms ranged from 14.3 m to 31 m.
3. aromatic resin, polystyrene, polyvinyl chloride 3 materials can form biofilm, biofilm formation and wide, covering almost the entire surface of the material, the formation of biofilm on different materials have different activity (F=14.743, P0.01), 48h activity from high to low fat = poly aryl polyvinyl chloride polystyrene. Found polyaryl inverted microscope and scanning electron microscope observation of lipid, biofilm formation of visible spores, hyphae and pseudohyphae of PVC, structure, formation of the microbial community with spores for the main structure of polystyrene.
conclusion
1. the T. asahii biofilm can be formed in vitro, and the T. asahii biofilm has a more complex structure than its floating state.
2. with the prolonging of T. asahii biofilm activity increased, no significant difference between the different levels of T. asahii biofilm activity, analysis of cell growth rate is not low T. asahii biofilm resistance mechanism of.3.T. asahii biofilm formation in different materials, but the ability of biofilm formation in different materials. Different aromatic grease the adhesion of PVC more easily than polystyrene and fungi; mycelium, pseudohypha activity of microbial community as the main structure than single spore viability.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R379

【参考文献】