枝孢樣枝孢霉微生物學特性的實驗研究
發(fā)布時間：2018-07-23 12:03
【摘要】： 枝孢樣枝孢霉是一種廣泛存在于環(huán)境中的暗色真菌,在一定條件下可導致人和動物的暗色絲孢霉病。迄今為止,人類不但對枝孢樣枝孢霉的基本微生物學特性及其致病情況尚缺乏了解,而且還不得不面臨著對枝孢樣枝孢霉所致暗色絲孢霉病診斷和治療上的困難性與復雜性。因此,研究并評價枝孢樣枝孢霉的致病性、溫度耐受性、藥物敏感性以及其基因多態(tài)性等諸多微生物學特性,將對于枝孢樣枝孢霉所致暗色絲孢霉病的診斷、治療及其預后具有重要的理論意義及實際價值。 目的 探討枝孢樣枝孢霉的致病性;觀察并比較枝孢樣枝孢霉與卡氏枝孢霉超微結(jié)構(gòu)的差異,探討其可能的致病性差異;檢測枝孢樣枝孢霉對8種常用抗真菌藥物的敏感性,為治療枝孢樣枝孢霉感染提供抗真菌藥物的參考依據(jù);觀察并比較枝孢樣枝孢霉與幾種常見致病性暗色真菌(裴氏著色真菌、緊密著色真菌、疣狀瓶霉、卡氏支孢霉、皮炎外瓶霉、甄氏外瓶霉、鏈格孢)的耐熱性,探討溫熱治療的必要性及可行性;研究枝孢樣枝孢霉及常見致病性暗色真菌基因多態(tài)性,初步了解它們在同屬內(nèi)及同種內(nèi),屬間及種間的基因遺傳相似性及遺傳變異性;枝孢樣枝孢霉及常見致病性暗色真菌蛋白組學比較研究,以了解枝孢樣枝孢霉與常見致病性暗色真菌蛋白質(zhì)表達的差異。 方法與結(jié)果 (1)枝孢樣枝孢霉對小鼠的致病性:將枝孢樣枝孢霉的菌懸液分別接種于正常和免疫抑制小鼠的腹側(cè)皮膚,于接種后的第15、30、60天處死動物,取接種部位的皮膚組織和各臟器組織進行組織病理學檢查和逆培養(yǎng)。實驗組小鼠在皮膚接種部位均出現(xiàn)不同程度損害。并且隨著時間推移,損害加重。損害部位皮膚組織逆培養(yǎng)均陽性,實驗小鼠均未見系統(tǒng)播散。 (2)將枝孢樣枝孢霉與卡氏枝孢霉接種于馬鈴薯葡萄糖瓊脂培養(yǎng)基上生長,在光學顯微鏡和透射電鏡下對臨床分離的枝孢樣枝孢霉與卡氏枝孢霉的形態(tài)學及超微結(jié)構(gòu)進行比較研究。受試的兩種真菌在超微結(jié)構(gòu)上有一定差異,枝孢樣枝孢霉易于發(fā)生衰老,其真菌細胞壁可出現(xiàn)顯著增厚等改變。 (3)枝孢樣枝孢霉的抗真菌藥物的敏感性試驗:參照美國國家實驗室標準委員會(NCCLS)推薦的產(chǎn)孢絲狀真菌試驗方案(M27-A)和(M38-P)。微量稀釋法進行最小抑菌濃度(MIC)測定,所選菌懸液終濃度為(0.4～5)×106cfu/mL,孵育溫度35℃,培養(yǎng)時間5～7d。測定枝孢樣枝孢霉對氟康唑、伊曲康唑、特比萘芬、酮康唑、咪康唑、聯(lián)苯芐唑、益康唑、制霉菌素的最小抑菌濃度(MIC)。結(jié)果顯示,枝孢樣枝孢霉對伊曲康唑、特比萘芬、酮康唑、咪康唑、聯(lián)苯芐唑、益康唑、制霉菌素的敏感性均較高,而對氟康唑敏感性較差。 (4)真菌耐熱試驗:所選擇的實驗溫度分別為37℃、45℃和55℃。隨機取各種菌液3管及安置溫度計的生理鹽水試管同時固定于水浴鍋內(nèi),待生理鹽水試管的溫度達到實驗溫度時,分別計時10min、30min、60min時,立即取出菌液并將其接種于裝有沙氏培養(yǎng)基的平皿中央并觀察生長情況。受試菌種的菌懸液經(jīng)37℃、45℃不同時間處理后,再培養(yǎng)的生長情況未受影響,55℃處理后除皮炎外瓶霉、鏈格孢外其余菌種(包括枝孢樣枝孢霉)在55℃不同時間處理后生長均受不同程度影響。而枝孢樣枝孢霉、卡氏支孢霉、甄氏外瓶霉培養(yǎng)溫度為37℃時即不生長,皮炎外瓶霉在42℃培養(yǎng)時即不生長,不同于其它菌株。 (5)隨機擴增DNA多態(tài)性分析:應用GenTLETM DNA提取試劑盒提取菌絲體DNA,以隨機擴增DNA多態(tài)法對18株暗色真菌的DNA指紋圖譜進行分析。共選用10個隨機引物進行擴增,篩選出3個具有穩(wěn)定、清晰DNA擴增帶型的引物,即引物1:5’-GAGCCCTCCA-3’,引物4:5’-GTCAGGGCAA-3’和引物5:5’-CAGCACCCAC-3’。18株菌DNA帶型不完全相同,有一定的種間和種內(nèi)的遺傳變異性和遺傳相似性腐生菌枝孢樣枝孢霉與其他部分暗色真菌位于同一樹組中。同種菌并不都具有遺傳相似性。(6)雙向電泳技術(shù)及蛋白質(zhì)譜分析:,蛋白質(zhì)首先根據(jù)其等電點在pH梯度膠內(nèi)等電聚焦,然后按照它們的相對分子量大小進行SDS-PAGE第二次電泳分離,從而獲得二維的蛋白質(zhì)分離圖譜。結(jié)果表明枝孢樣枝孢霉的蛋白分離不易獲得清晰分離圖譜。 結(jié)論 (1)枝孢樣枝孢霉對小鼠皮膚有較強的機會致病性。但對小鼠內(nèi)臟器官的機會致病性較弱。 (2)枝孢樣枝孢霉和卡氏枝孢霉的超微結(jié)構(gòu)有一定差異,枝孢樣枝孢霉易于發(fā)生衰老的現(xiàn)象在一定程度上提示其致病性要遜于卡氏枝孢霉。 (3)檢測枝孢樣枝孢霉對抗真菌藥物的敏感性是必要的和有價值的,但體外試驗僅可作為體內(nèi)治療選藥的參考。 (4)并非所有暗色真菌經(jīng)45℃或55℃處理后生長均被完全殺滅,部分暗色真菌僅表現(xiàn)為受到抑制。對局部熱療的適宜溫度和作用形式因菌種不同而有差異。局部熱療與抗真菌藥物聯(lián)合應用對治療暗色真菌病是有益的。 (5)隨機擴增DNA多態(tài)性分析表明,包括枝孢樣枝孢霉在內(nèi)的暗色真菌在屬內(nèi)各種間不一定有遺傳相似性;但腐生菌與致病菌的DNA帶型未見明顯特征性差異,似乎還有一定遺傳相似性。 (6)枝孢樣枝孢霉的蛋白分離困難,不易獲得清晰的蛋白質(zhì)分離圖譜,這可能與超微結(jié)構(gòu)所見枝孢樣枝孢霉的真菌細胞壁明顯增厚有關(guān)。僅培養(yǎng)條件的改變并不能改善枝孢樣枝孢霉的蛋白質(zhì)分離。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R379
本文編號：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R379
【引證文獻】
相關(guān)期刊論文 前1條
1 馬曉平;古玉;劉玲;劉小敏;凌珊珊;侯加法;王承東;;大熊貓源枝孢樣枝孢霉的生物學特性[J];中國獸醫(yī)科學;2013年01期
，本文編號：2139352
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