枝孢样枝孢霉微生物学特性的实验研究
发布时间：2018-07-23 12:03
【摘要】： 枝孢样枝孢霉是一种广泛存在于环境中的暗色真菌,在一定条件下可导致人和动物的暗色丝孢霉病。迄今为止,人类不但对枝孢样枝孢霉的基本微生物学特性及其致病情况尚缺乏了解,而且还不得不面临着对枝孢样枝孢霉所致暗色丝孢霉病诊断和治疗上的困难性与复杂性。因此,研究并评价枝孢样枝孢霉的致病性、温度耐受性、药物敏感性以及其基因多态性等诸多微生物学特性,将对于枝孢样枝孢霉所致暗色丝孢霉病的诊断、治疗及其预后具有重要的理论意义及实际价值。 目的 探讨枝孢样枝孢霉的致病性;观察并比较枝孢样枝孢霉与卡氏枝孢霉超微结构的差异,探讨其可能的致病性差异;检测枝孢样枝孢霉对8种常用抗真菌药物的敏感性,为治疗枝孢样枝孢霉感染提供抗真菌药物的参考依据;观察并比较枝孢样枝孢霉与几种常见致病性暗色真菌(裴氏着色真菌、紧密着色真菌、疣状瓶霉、卡氏支孢霉、皮炎外瓶霉、甄氏外瓶霉、链格孢)的耐热性,探讨温热治疗的必要性及可行性;研究枝孢样枝孢霉及常见致病性暗色真菌基因多态性,初步了解它们在同属内及同种内,属间及种间的基因遗传相似性及遗传变异性;枝孢样枝孢霉及常见致病性暗色真菌蛋白组学比较研究,以了解枝孢样枝孢霉与常见致病性暗色真菌蛋白质表达的差异。 方法与结果 (1)枝孢样枝孢霉对小鼠的致病性:将枝孢样枝孢霉的菌悬液分别接种于正常和免疫抑制小鼠的腹侧皮肤,于接种后的第15、30、60天处死动物,取接种部位的皮肤组织和各脏器组织进行组织病理学检查和逆培养。实验组小鼠在皮肤接种部位均出现不同程度损害。并且随着时间推移,损害加重。损害部位皮肤组织逆培养均阳性,实验小鼠均未见系统播散。 (2)将枝孢样枝孢霉与卡氏枝孢霉接种于马铃薯葡萄糖琼脂培养基上生长,在光学显微镜和透射电镜下对临床分离的枝孢样枝孢霉与卡氏枝孢霉的形态学及超微结构进行比较研究。受试的两种真菌在超微结构上有一定差异,枝孢样枝孢霉易于发生衰老,其真菌细胞壁可出现显著增厚等改变。 (3)枝孢样枝孢霉的抗真菌药物的敏感性试验:参照美国国家实验室标准委员会(NCCLS)推荐的产孢丝状真菌试验方案(M27-A)和(M38-P)。微量稀释法进行最小抑菌浓度(MIC)测定,所选菌悬液终浓度为(0.4～5)×106cfu/mL,孵育温度35℃,培养时间5～7d。测定枝孢样枝孢霉对氟康唑、伊曲康唑、特比萘芬、酮康唑、咪康唑、联苯苄唑、益康唑、制霉菌素的最小抑菌浓度(MIC)。结果显示,枝孢样枝孢霉对伊曲康唑、特比萘芬、酮康唑、咪康唑、联苯苄唑、益康唑、制霉菌素的敏感性均较高,而对氟康唑敏感性较差。 (4)真菌耐热试验:所选择的实验温度分别为37℃、45℃和55℃。随机取各种菌液3管及安置温度计的生理盐水试管同时固定于水浴锅内,待生理盐水试管的温度达到实验温度时,分别计时10min、30min、60min时,立即取出菌液并将其接种于装有沙氏培养基的平皿中央并观察生长情况。受试菌种的菌悬液经37℃、45℃不同时间处理后,再培养的生长情况未受影响,55℃处理后除皮炎外瓶霉、链格孢外其余菌种(包括枝孢样枝孢霉)在55℃不同时间处理后生长均受不同程度影响。而枝孢样枝孢霉、卡氏支孢霉、甄氏外瓶霉培养温度为37℃时即不生长,皮炎外瓶霉在42℃培养时即不生长,不同于其它菌株。 (5)随机扩增DNA多态性分析:应用GenTLETM DNA提取试剂盒提取菌丝体DNA,以随机扩增DNA多态法对18株暗色真菌的DNA指纹图谱进行分析。共选用10个随机引物进行扩增,筛选出3个具有稳定、清晰DNA扩增带型的引物,即引物1:5’-GAGCCCTCCA-3’,引物4:5’-GTCAGGGCAA-3’和引物5:5’-CAGCACCCAC-3’。18株菌DNA带型不完全相同,有一定的种间和种内的遗传变异性和遗传相似性腐生菌枝孢样枝孢霉与其他部分暗色真菌位于同一树组中。同种菌并不都具有遗传相似性。(6)双向电泳技术及蛋白质谱分析:,蛋白质首先根据其等电点在pH梯度胶内等电聚焦,然后按照它们的相对分子量大小进行SDS-PAGE第二次电泳分离,从而获得二维的蛋白质分离图谱。结果表明枝孢样枝孢霉的蛋白分离不易获得清晰分离图谱。 结论 (1)枝孢样枝孢霉对小鼠皮肤有较强的机会致病性。但对小鼠内脏器官的机会致病性较弱。 (2)枝孢样枝孢霉和卡氏枝孢霉的超微结构有一定差异,枝孢样枝孢霉易于发生衰老的现象在一定程度上提示其致病性要逊于卡氏枝孢霉。 (3)检测枝孢样枝孢霉对抗真菌药物的敏感性是必要的和有价值的,但体外试验仅可作为体内治疗选药的参考。 (4)并非所有暗色真菌经45℃或55℃处理后生长均被完全杀灭,部分暗色真菌仅表现为受到抑制。对局部热疗的适宜温度和作用形式因菌种不同而有差异。局部热疗与抗真菌药物联合应用对治疗暗色真菌病是有益的。 (5)随机扩增DNA多态性分析表明,包括枝孢样枝孢霉在内的暗色真菌在属内各种间不一定有遗传相似性;但腐生菌与致病菌的DNA带型未见明显特征性差异,似乎还有一定遗传相似性。 (6)枝孢样枝孢霉的蛋白分离困难,不易获得清晰的蛋白质分离图谱,这可能与超微结构所见枝孢样枝孢霉的真菌细胞壁明显增厚有关。仅培养条件的改变并不能改善枝孢样枝孢霉的蛋白质分离。
[Abstract]:Cladosporium Cladosporium is a dark colored fungus widely existed in the environment. Under certain conditions, it can cause dark mildew of human and animal. So far, human beings have not only understood the basic microbiological characteristics and pathogenicity of cladospora dendrite, but also do not have to face dark silk caused by cladosporsporum cladosporidium. Therefore, the study and evaluation of the pathogenicity, temperature tolerance, drug sensitivity and genetic polymorphisms of cladosporidium cladosporidium will be of great theoretical significance for the diagnosis, treatment and prognosis of cladosporsporum cladospora. Practical value.
objective
To investigate the pathogenicity of cladosporidium cladosporidium, observe and compare the differences in the ultrastructure of cladosporidium cladospora and Cladosporium Kagi, to explore the possible pathogenicity differences, and to detect the sensitivity of cladosporidium Cladosporium to 8 commonly used antifungal drugs, and to observe and compare branches of antifungal agents for the treatment of cladosporsporum cladosporidium infection. Cladosporidium spporaiceri and several common pathogenic fungi (Pei's coloring fungi, tight coloring fungi, verruciform vase, Cladosporium carinospora, dermatitis, vase, jensinella, cyclosporin), and the necessity and feasibility of thermotherapy, and a preliminary understanding of the genetic polymorphism of cladospora cladospora and common pathogenic dark fungi Their genetic similarity and genetic variability in Intergenera and intraspecific, interspecific and interspecific genes, and a comparative study of cladosporidia cladosporidia and common pathogenic dark fungus proteomics to understand the differences in protein expression between cladosporspora cladospora and common pathogenic dark fungi.
Methods and results
(1) the pathogenicity of cladosporidium cladosporidium to mice: inoculating the bacterial suspension of Cladosporium Cladosporium to the abdominal skin of normal and immunosuppressive mice respectively. The animals were killed on day 15,30,60 after inoculation. The skin tissues and organs of the inoculated sites were taken for histopathological examination and reverse culture. The experimental group was in the skin inoculation department. All lesions showed different degrees of damage, and as time went on, the damage was aggravated. The skin tissue of the lesion site was positive in reverse culture, and no systemic spread was found in the experimental mice.
(2) the morphology and ultrastructure of Cladosporium Cladosporium and cladospora carcasa were compared under optical and transmission electron microscopy. The ultrastructure of the two true bacteria was different in the ultrastructure. Spores are susceptible to senescence, and their fungal cell walls can be significantly thickened.
(3) sensitivity test of antifungal agents of Cladosporium cladospora: the minimum inhibitory concentration (MIC) was measured by microdilution method with reference to the National Laboratory Standards Committee (NCCLS) recommended by the National Laboratory for Laboratory Standards (NCCLS). The final concentration of the suspension was (0.4 to 5) x 106cfu/mL, the incubation temperature was 35, and the incubation time was 5 to 7d. The minimum inhibitory concentration (MIC) for fluconazole, itraconazole, terbinafine, ketoconazole, miconazole, bibenzazole, yicconazole, and nystatin showed that the sensitivity of cladosporspora dendrite to itraconazole, terbinafine, ketoconazole, miconazole, bibenzazole, yicconazole, nystatin and nystatin was high, but fluconazole was higher than that of fluconazole. The sensitivity is poor.
(4) the heat resistance test of fungi: the selected experimental temperatures were 37, 45 and 55, respectively. The normal saline test tubes of 3 tubes and the thermometer were randomly selected and fixed in the water bath at the same time. When the temperature of the saline test tubes reached the experimental temperature, 10min, 30min and 60min were timed, and the bacteria were immediately removed and inoculated to the sand. The growth conditions were not affected by the bacterial suspension of the tested strains at 37 and 45 degrees C. After treatment at 55 C, the other strains (including cladosporsporum Cladosporium) were treated with different degrees after treatment at 55 degrees centigrade. The growth of the other strains (including cladosporsporum Cladosporium) was affected by different degrees. Cladosporium Cladosporium, P. cariosum, don't grow at the temperature of 37 degrees centigrade. The dermatitis does not grow at 42 C and is different from other strains.
(5) random amplified DNA polymorphism analysis: GenTLETM DNA extraction kit was used to extract mycelial DNA, and the DNA fingerprint of 18 dark fungi was analyzed by random amplification of DNA polymorphism. A total of 10 random primers were selected to amplify and select 3 primers with stable and clear DNA enlargement type, namely, primer 1:5 '-GAGCCCTCCA-3', and primer 4 5 '-GTCAGGGCAA-3' and primers 5:5 '-CAGCACCCAC-3'.18 strains are not identical, there are certain interspecific and intraspecific genetic variability and genetic similarity in the same tree group with Cladosporium cladosporspora Cladosporium and other dark fungi. (6) two dimensional electrophoresis technology and protein Spectral analysis: the protein was first focused on the pH gradient gel according to its isoelectric point and then separated by SDS-PAGE second electrophoresis according to their relative molecular weight, thus obtaining a two-dimensional protein separation map. The results showed that the protein separation of cladosporidium cladospora was not easy to get a clear separation map.
conclusion
(1) Cladosporium Cladosporium has a strong opportunistic pathogenicity to the skin of mice, but the opportunistic pathogenicity of mice's visceral organs is weak.
(2) the ultrastructure of cladosporidium cladosporidium and Cladosporium kabiosporus had some difference, and the phenomenon of dendrospora Cladosporium could be easily senescent. To a certain extent, the pathogenicity of Cladosporium Cladosporium was inferior to Cladosporium cardulin.
(3) it is necessary and valuable to detect the sensitivity of Cladosporium spp. to antifungal agents, but in vitro tests can only be used as a reference for the selection of drugs in vivo.
(4) not all dark fungi were completely killed after treatment at 45 or 55 C, and some dark fungi were only inhibited. The appropriate temperature and form of action for local hyperthermia were different because of the strain. Local hyperthermia and antifungal drugs were beneficial to the treatment of dark coloured fungal disease.
(5) random amplified DNA polymorphism analysis showed that the dark fungi, including cladosporidia cladosporidia, did not have genetic similarity within the genus, but there was no obvious characteristic difference between saprophytic and pathogenic DNA bands, and there seemed to be a certain genetic similarity.
(6) the protein separation of Cladosporium cladospora is difficult, and it is not easy to obtain a clear protein separation map. This may be related to the obvious thickening of the cell wall of the fungal cell like cladosporidium cladospora in ultrastructure. Only the change of culture conditions can not improve the protein separation of cladosporidium cladosporidium.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R379
本文编号：2139352
[Abstract]:Cladosporium Cladosporium is a dark colored fungus widely existed in the environment. Under certain conditions, it can cause dark mildew of human and animal. So far, human beings have not only understood the basic microbiological characteristics and pathogenicity of cladospora dendrite, but also do not have to face dark silk caused by cladosporsporum cladosporidium. Therefore, the study and evaluation of the pathogenicity, temperature tolerance, drug sensitivity and genetic polymorphisms of cladosporidium cladosporidium will be of great theoretical significance for the diagnosis, treatment and prognosis of cladosporsporum cladospora. Practical value.
objective
To investigate the pathogenicity of cladosporidium cladosporidium, observe and compare the differences in the ultrastructure of cladosporidium cladospora and Cladosporium Kagi, to explore the possible pathogenicity differences, and to detect the sensitivity of cladosporidium Cladosporium to 8 commonly used antifungal drugs, and to observe and compare branches of antifungal agents for the treatment of cladosporsporum cladosporidium infection. Cladosporidium spporaiceri and several common pathogenic fungi (Pei's coloring fungi, tight coloring fungi, verruciform vase, Cladosporium carinospora, dermatitis, vase, jensinella, cyclosporin), and the necessity and feasibility of thermotherapy, and a preliminary understanding of the genetic polymorphism of cladospora cladospora and common pathogenic dark fungi Their genetic similarity and genetic variability in Intergenera and intraspecific, interspecific and interspecific genes, and a comparative study of cladosporidia cladosporidia and common pathogenic dark fungus proteomics to understand the differences in protein expression between cladosporspora cladospora and common pathogenic dark fungi.
Methods and results
(1) the pathogenicity of cladosporidium cladosporidium to mice: inoculating the bacterial suspension of Cladosporium Cladosporium to the abdominal skin of normal and immunosuppressive mice respectively. The animals were killed on day 15,30,60 after inoculation. The skin tissues and organs of the inoculated sites were taken for histopathological examination and reverse culture. The experimental group was in the skin inoculation department. All lesions showed different degrees of damage, and as time went on, the damage was aggravated. The skin tissue of the lesion site was positive in reverse culture, and no systemic spread was found in the experimental mice.
(2) the morphology and ultrastructure of Cladosporium Cladosporium and cladospora carcasa were compared under optical and transmission electron microscopy. The ultrastructure of the two true bacteria was different in the ultrastructure. Spores are susceptible to senescence, and their fungal cell walls can be significantly thickened.
(3) sensitivity test of antifungal agents of Cladosporium cladospora: the minimum inhibitory concentration (MIC) was measured by microdilution method with reference to the National Laboratory Standards Committee (NCCLS) recommended by the National Laboratory for Laboratory Standards (NCCLS). The final concentration of the suspension was (0.4 to 5) x 106cfu/mL, the incubation temperature was 35, and the incubation time was 5 to 7d. The minimum inhibitory concentration (MIC) for fluconazole, itraconazole, terbinafine, ketoconazole, miconazole, bibenzazole, yicconazole, and nystatin showed that the sensitivity of cladosporspora dendrite to itraconazole, terbinafine, ketoconazole, miconazole, bibenzazole, yicconazole, nystatin and nystatin was high, but fluconazole was higher than that of fluconazole. The sensitivity is poor.
(4) the heat resistance test of fungi: the selected experimental temperatures were 37, 45 and 55, respectively. The normal saline test tubes of 3 tubes and the thermometer were randomly selected and fixed in the water bath at the same time. When the temperature of the saline test tubes reached the experimental temperature, 10min, 30min and 60min were timed, and the bacteria were immediately removed and inoculated to the sand. The growth conditions were not affected by the bacterial suspension of the tested strains at 37 and 45 degrees C. After treatment at 55 C, the other strains (including cladosporsporum Cladosporium) were treated with different degrees after treatment at 55 degrees centigrade. The growth of the other strains (including cladosporsporum Cladosporium) was affected by different degrees. Cladosporium Cladosporium, P. cariosum, don't grow at the temperature of 37 degrees centigrade. The dermatitis does not grow at 42 C and is different from other strains.
(5) random amplified DNA polymorphism analysis: GenTLETM DNA extraction kit was used to extract mycelial DNA, and the DNA fingerprint of 18 dark fungi was analyzed by random amplification of DNA polymorphism. A total of 10 random primers were selected to amplify and select 3 primers with stable and clear DNA enlargement type, namely, primer 1:5 '-GAGCCCTCCA-3', and primer 4 5 '-GTCAGGGCAA-3' and primers 5:5 '-CAGCACCCAC-3'.18 strains are not identical, there are certain interspecific and intraspecific genetic variability and genetic similarity in the same tree group with Cladosporium cladosporspora Cladosporium and other dark fungi. (6) two dimensional electrophoresis technology and protein Spectral analysis: the protein was first focused on the pH gradient gel according to its isoelectric point and then separated by SDS-PAGE second electrophoresis according to their relative molecular weight, thus obtaining a two-dimensional protein separation map. The results showed that the protein separation of cladosporidium cladospora was not easy to get a clear separation map.
conclusion
(1) Cladosporium Cladosporium has a strong opportunistic pathogenicity to the skin of mice, but the opportunistic pathogenicity of mice's visceral organs is weak.
(2) the ultrastructure of cladosporidium cladosporidium and Cladosporium kabiosporus had some difference, and the phenomenon of dendrospora Cladosporium could be easily senescent. To a certain extent, the pathogenicity of Cladosporium Cladosporium was inferior to Cladosporium cardulin.
(3) it is necessary and valuable to detect the sensitivity of Cladosporium spp. to antifungal agents, but in vitro tests can only be used as a reference for the selection of drugs in vivo.
(4) not all dark fungi were completely killed after treatment at 45 or 55 C, and some dark fungi were only inhibited. The appropriate temperature and form of action for local hyperthermia were different because of the strain. Local hyperthermia and antifungal drugs were beneficial to the treatment of dark coloured fungal disease.
(5) random amplified DNA polymorphism analysis showed that the dark fungi, including cladosporidia cladosporidia, did not have genetic similarity within the genus, but there was no obvious characteristic difference between saprophytic and pathogenic DNA bands, and there seemed to be a certain genetic similarity.
(6) the protein separation of Cladosporium cladospora is difficult, and it is not easy to obtain a clear protein separation map. This may be related to the obvious thickening of the cell wall of the fungal cell like cladosporidium cladospora in ultrastructure. Only the change of culture conditions can not improve the protein separation of cladosporidium cladosporidium.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R379
【引证文献】
相关期刊论文 前1条
1 马晓平;古玉;刘玲;刘小敏;凌珊珊;侯加法;王承东;;大熊猫源枝孢样枝孢霉的生物学特性[J];中国兽医科学;2013年01期
，本文编号：2139352
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2139352.html
最近更新
教材专著
