枝孢样枝孢霉微生物学特性的实验研究
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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。
Relatively little information is available on the microbiol characteristics of cladosporium cladosporioides. Because of the increasing number of immunocompromised patients, the number of fungi causing subcutaneous phaeohyphomycosis has been increasing. Among them, members of the genera Alternaria, Bipolariz, Curvularia, Exophiala, Phialophora and Wangiella are the most common species. Despite the morphological differences among species, the illnesses they produce have many common features. Subcutaneous phaeohyphomycosis has pigmenta-occurred with equal frequency in tropical, subtropical and temperate areas partly because of the opportunistic nature of its etiologic agents.
Objective
To investigate the pathogenicity of Cladosporium cladosporioides. This study was also to investigate the difference on the ultrastructure and pathogenicity of the Cladosporium cladosporioides and Cladosporium carrionii. Investigate the antifungal susceptibility characteristic to know if it is sensitive to common antifungal drugs.To report the test of heat-tolerance of Cladosporium cladosporioides ,Cladosporium carrionii ,Fonsecaea pedrosoi, Fonsecaea compactum, Phialophora verrucosa, Cladosporium carrionii Exophiala dermatitidis,Exophiala jeanselmei and Alternaria alternate. To know the microbiological characteristics of medically Cladosporium cladosporioides and as well as to provide reference for the diagnosis and treatment of Cladosporium cladosporioides infection. Investigate the antifungal susceptibility characteristic to know if it is sensitive to common antifungal drugs. To investigate the DNA polymorphism of saprophytes and pathogenicity of Cladosporium species and to find out the relationship between them. To study the different expressions of proteins between Cladosporium cladosporioides and Cladosporium carrionii.
Methods and Results
1. The pathogenicity of Cladosporium cladosporioides: The cell suspension of Cladosporium cladosporioides was inoculated into gaster-skin of normal and immunocompromised ICR(Institute of Cancer Research) mice. Histopathologic examination and fungal culture were performed on sacrified mice on 15th d,30th d,60nd d respectively after inoculation. The abscesses were developed at the inoculated sites in all experimental mice. The necropsy revealed no disseminated infection of Cladosporium cladosporioides in all the test mice.
2. A comparative study was performed on the difference on the ultrastructure of the Cladosporium cladosporioides and Cladosporium carrionii. There are some differeces on the ultrastructure of the Cladosporium cladosporioides and Cladosporium carrionii. The cell wall of Cladosporium cladosporioides is prone to be more thicker and to be aging.
3. In vitro antifungal susceptibility tests of the isolate performed according to NCCLS guidelines. To detect the susceptibility of fluconazol, itraconazole, terbinafine, ketoconazole, miconazole, bifonazole,econazole and nystatin against Cladosporium cladosporioides.Using M38-P method the strains of Cladosporium cladosporioides were studied .The media containing the fungi in the concentrations of (0.4～5)×106cfu/mL were incubated at 35℃for5~7days. The tested Cladosporium cladosporioides were highly susceptible to itraconazole, terbinafine, ketoconazole, miconazole, bifonazole,econazole and nystatin,but its poorly susceptible to fluconazol.
4. Test the heat-tolerance of Cladosporium cladosporioides ,Cladosporium carrionii , Fonsecaea pedrosoi, Fonsecaea compactum, Phialophora verrucosa, Cladosporium carrionii ExopHiala dermatitidis,ExopHiala jeanselmei and Alternaria alternate. Our findings show that two of the eight strains(Exophiala dermatitidis and Alternaria alternate) has the peculiarity of heat-tolerance while the other six strains are not well tolerant to heat.
5. The total DNA was extracted with GenTLETM DNA extraction kit, Random amplification of polymorphic DNA(RAPD) assay was used to study the fingerprinting of 18 strains. Of ten random primers used, three(primer1,4,5) gave good reaction. The sequences of them were: primer1: 5’-GAGCCCTCCA-3’, primer4: 5’-GTCAGGGCAA-3’, primer1:5’-CAGCACCCAC-3’.There were some degrees of variation and genetic similarities in RAPD patterns of 18 isolates, with interspecies and intraspecies variation and genetic similarities . But the isolates of the same species didn’t show high genetic similarities. Saprobic Cladosporium cladosporioides located in the same group of dendrogram of some dematiaceous fungi with humanpathogenicity.
6. Proteins isolated from the Cladosporium cladosporioides were separated by two dimensional (2-D)gel electrophoresis.It is not easy to get sharp separation figure of protein from Cladosporium cladosporioides regardless of condition of culture.
Conclusion
Cladosporium cladosporioides is a kind of opportunistic fungus with relatively high virulence to skin but low virulence to viscera. According to our study, we conclude that it is feasible to compare the differences of Cladosporium cladosporioides from Cladosporium carrionii for diagnosis and treatment of phaeohyphomycosis.This also states that the pathogenicity of Cladosporium cladosporioides is a little poorer than that of the Cladosporium carrionii. In vitro susceptibility testing of each clinical isolate should be done.This finding is of some significance in guiding to determination of the species of these fungi and also for prevention and treatment of infections of dematiaceous fungi .It suggested that it is necessary to monitor the resistant strains in antifungal treatment. Local heat therapy may also be used for patients with subcutaneous phaeohyphomycosis, even though some of the etiologic phaeoid fungi possess considerable thermotolerance. These are some genetic similarities between some different species and strains, but no distinct characteristics are found between saprophytes and human-pathogens. Sharp separation figure of protein from Cladosporium cladosporioides should be studied.
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。
Relatively little information is available on the microbiol characteristics of cladosporium cladosporioides. Because of the increasing number of immunocompromised patients, the number of fungi causing subcutaneous phaeohyphomycosis has been increasing. Among them, members of the genera Alternaria, Bipolariz, Curvularia, Exophiala, Phialophora and Wangiella are the most common species. Despite the morphological differences among species, the illnesses they produce have many common features. Subcutaneous phaeohyphomycosis has pigmenta-occurred with equal frequency in tropical, subtropical and temperate areas partly because of the opportunistic nature of its etiologic agents.
Objective
To investigate the pathogenicity of Cladosporium cladosporioides. This study was also to investigate the difference on the ultrastructure and pathogenicity of the Cladosporium cladosporioides and Cladosporium carrionii. Investigate the antifungal susceptibility characteristic to know if it is sensitive to common antifungal drugs.To report the test of heat-tolerance of Cladosporium cladosporioides ,Cladosporium carrionii ,Fonsecaea pedrosoi, Fonsecaea compactum, Phialophora verrucosa, Cladosporium carrionii Exophiala dermatitidis,Exophiala jeanselmei and Alternaria alternate. To know the microbiological characteristics of medically Cladosporium cladosporioides and as well as to provide reference for the diagnosis and treatment of Cladosporium cladosporioides infection. Investigate the antifungal susceptibility characteristic to know if it is sensitive to common antifungal drugs. To investigate the DNA polymorphism of saprophytes and pathogenicity of Cladosporium species and to find out the relationship between them. To study the different expressions of proteins between Cladosporium cladosporioides and Cladosporium carrionii.
Methods and Results
1. The pathogenicity of Cladosporium cladosporioides: The cell suspension of Cladosporium cladosporioides was inoculated into gaster-skin of normal and immunocompromised ICR(Institute of Cancer Research) mice. Histopathologic examination and fungal culture were performed on sacrified mice on 15th d,30th d,60nd d respectively after inoculation. The abscesses were developed at the inoculated sites in all experimental mice. The necropsy revealed no disseminated infection of Cladosporium cladosporioides in all the test mice.
2. A comparative study was performed on the difference on the ultrastructure of the Cladosporium cladosporioides and Cladosporium carrionii. There are some differeces on the ultrastructure of the Cladosporium cladosporioides and Cladosporium carrionii. The cell wall of Cladosporium cladosporioides is prone to be more thicker and to be aging.
3. In vitro antifungal susceptibility tests of the isolate performed according to NCCLS guidelines. To detect the susceptibility of fluconazol, itraconazole, terbinafine, ketoconazole, miconazole, bifonazole,econazole and nystatin against Cladosporium cladosporioides.Using M38-P method the strains of Cladosporium cladosporioides were studied .The media containing the fungi in the concentrations of (0.4～5)×106cfu/mL were incubated at 35℃for5~7days. The tested Cladosporium cladosporioides were highly susceptible to itraconazole, terbinafine, ketoconazole, miconazole, bifonazole,econazole and nystatin,but its poorly susceptible to fluconazol.
4. Test the heat-tolerance of Cladosporium cladosporioides ,Cladosporium carrionii , Fonsecaea pedrosoi, Fonsecaea compactum, Phialophora verrucosa, Cladosporium carrionii ExopHiala dermatitidis,ExopHiala jeanselmei and Alternaria alternate. Our findings show that two of the eight strains(Exophiala dermatitidis and Alternaria alternate) has the peculiarity of heat-tolerance while the other six strains are not well tolerant to heat.
5. The total DNA was extracted with GenTLETM DNA extraction kit, Random amplification of polymorphic DNA(RAPD) assay was used to study the fingerprinting of 18 strains. Of ten random primers used, three(primer1,4,5) gave good reaction. The sequences of them were: primer1: 5’-GAGCCCTCCA-3’, primer4: 5’-GTCAGGGCAA-3’, primer1:5’-CAGCACCCAC-3’.There were some degrees of variation and genetic similarities in RAPD patterns of 18 isolates, with interspecies and intraspecies variation and genetic similarities . But the isolates of the same species didn’t show high genetic similarities. Saprobic Cladosporium cladosporioides located in the same group of dendrogram of some dematiaceous fungi with humanpathogenicity.
6. Proteins isolated from the Cladosporium cladosporioides were separated by two dimensional (2-D)gel electrophoresis.It is not easy to get sharp separation figure of protein from Cladosporium cladosporioides regardless of condition of culture.
Conclusion
Cladosporium cladosporioides is a kind of opportunistic fungus with relatively high virulence to skin but low virulence to viscera. According to our study, we conclude that it is feasible to compare the differences of Cladosporium cladosporioides from Cladosporium carrionii for diagnosis and treatment of phaeohyphomycosis.This also states that the pathogenicity of Cladosporium cladosporioides is a little poorer than that of the Cladosporium carrionii. In vitro susceptibility testing of each clinical isolate should be done.This finding is of some significance in guiding to determination of the species of these fungi and also for prevention and treatment of infections of dematiaceous fungi .It suggested that it is necessary to monitor the resistant strains in antifungal treatment. Local heat therapy may also be used for patients with subcutaneous phaeohyphomycosis, even though some of the etiologic phaeoid fungi possess considerable thermotolerance. These are some genetic similarities between some different species and strains, but no distinct characteristics are found between saprophytes and human-pathogens. Sharp separation figure of protein from Cladosporium cladosporioides should be studied.
引文
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