短帚霉的形态学观察及其体内药物敏感性实验研究
发布时间：2019-05-28 20:49
【摘要】： 目的：短帚霉(Scopulariopsis brevicaulis)是一种腐生性条件致病真菌,通常引起甲真菌病,较少引起其他部位的感染,但目前国外已经有关于深部组织感染的报道。此菌分离于我科门诊病人,经中国医学科学院微生物研究所鉴定为Scopulariopsis brevicaulis。 本研究一方面通过对短帚霉在四种不同培养基上的培养情况进行观察,以便寻找出适合其生长的培养基及温度；另一方面,应用氟康唑、伊曲康唑和特比萘芬三种常用抗真菌药物治疗小鼠的短帚霉系统感染,通过对试验小鼠生存率以及心、肝、脾、肺组织的真菌逆培养阳性率和肾脏组织载菌量的检测来评价它们在小鼠体内抗短帚霉的活性,为临床上治疗短帚霉系统感染提供实验室依据；并通过组织病理学检查,为进一步了解此菌引起小鼠的病理形态学改变提供实验室依据。 方法： 1形态学观察试验 1.1大体形态 取我科保存菌种常温下复苏,3天后转种于沙堡氏琼脂培养基(SDA)、马铃薯葡萄糖琼脂(PDA)、米饭培养基和玉米吐温80琼脂上,分别置37℃和27℃温箱培养2周,每日测量并记录菌落直径,观察菌落形态。 1.2平皿培养的光镜下观察每日分别挑取37℃和27℃的四种培养基的菌落标本置于载玻片上,经乳酸酚棉蓝染色,光镜下观察。 2生理学实验 2.1尿素酶试验将菌落接种于尿素琼脂培养基上,室温培养1周,观察培养基变色情况。 3体内药敏试验 3.1短帚霉系统感染动物模型的制作： 取我科保存菌种复苏转种,用无菌生理盐水制备成菌悬液,通过腹腔注射途径接种至应用环磷酰胺免疫抑制的小鼠体内,每只小鼠腹腔注射菌悬液为0.5ml。 3.2生存观察 接种后2h开始给药,氟康唑、伊曲康唑、特比萘芬均为10mg/(kg.d),腹腔注射,每天1次,连续5天。连续观察各组小鼠接种后的一般状况及小鼠接种后21天内的自然死亡情况,记录死亡日期和每天的死亡只数。 3.3组织逆培养 接种真菌悬液后的第6天各组分别处死组织载菌量部分小鼠,取出心、肝、脾、肺分别置于组织研磨器中,研碎后取少许组织分3点接种于改良沙堡氏固体培养基,置于27℃恒温箱中培养。 3.4肾脏组织载菌量(CFU)的测定 分别取氟康唑组、伊曲康唑组、特比萘芬组和对照组中的15只小鼠,每组的15只小鼠各取一侧的肾脏,组织研磨器研碎后,用无菌生理盐水稀释至1ml,取10微升组织悬液均匀涂布于平皿培养基表面,27℃恒温箱培养,48h后读取菌落数。 3.5组织病理学检查 取各脏器表面肉眼可见病理改变组织,用10%福尔马林溶液常温固定,常规脱水、透明、包埋、切片,HE和PAS染色,光镜下观察组织病理学变化。 结果： 1形态学观察试验 1.1绘制的生长曲线 在SDA和PDA两种培养基上,短帚霉于培养的第1周生长较快,其生长速度与时间呈正比；在两种温度下,27℃较37℃生长快。在米饭培养基和吐温80两种培养基上生长速度始终较慢,27℃较37℃生长稍快。 1.2菌落直径 同种培养基在不同温度下其菌落直径的比较结果显示,于生长7天和14天时,各培养基在27℃的菌落直径均比37℃菌落直径大,有显著性差异(P0.05)。 27℃不同培养基上,生长第7天和14天时,菌落直径均以PDA最大,SDA次之,米饭培养基和吐温80更小。生长第7天和14天时,PDA与SDA菌落差异无显著性(P0.05),米饭培养基和吐温80菌落差异无显著性(P0.05),其余各培养基之间均有显著性差异(P0.05)。 1.3大体菌落观察培养3-4天,菌落开始生长,初为白色膜样菌落,生长迅速,以后变为灰褐色。 1.4光镜下观察可见分枝分隔菌丝,帚状枝,分生孢子自环孢子梗长出一串,呈球形,壁厚,表面光滑或粗糙。 2生理学实验 2.1尿素酶实验呈阳性。 3生存率观察 氟康唑组在给药后21天的存活只数为17只,第6天开始死亡,第7天为死亡高峰；伊曲康唑组在给药后21天的存活只数为16只,第6天开始死亡,第7天为死亡高峰；特比萘芬组在给药21天的存活只数为15只,第6天开始死亡,第8天为死亡高峰；对照组在给药后21天的存活只数为9只,第2天开始死亡,第7天为死亡高峰。 4真菌逆培养结果 氟康唑治疗组、伊曲康唑治疗组和特比萘芬治疗组小鼠心肺组织真菌逆培养阳性率生理盐水对照组低,有显著性差异(P0.05)。余下各组的真菌逆培养阳性率比较均无差异(P0.05)。 5肾脏组织载菌量 伊曲康唑治疗组、氟康唑治疗组和特比萘芬治疗组中小鼠的肾脏组织载菌量较生理盐水对照组均有显著减少(P0.05)；余下各组的肾脏组织载菌量比较均无显著性差异(P0.05)。 6病理学变化苏木精-伊红染色早期为急性炎细胞浸润,可见组织细胞坏死,后期可见组织细胞及多核细胞反应性增生。PAS染色可见紫红色的菌丝和圆形孢子。 结论： 1沙堡氏琼脂培养基(SDA)、马铃薯葡萄糖琼脂(PDA)均适于短帚霉生长,米饭培养基和玉米吐温80琼脂不适于其生长；与37℃温度相比较,短帚霉更适于在27℃下生长；短帚霉的尿素酶实验为阳性。 2氟康唑、伊曲康唑和特比萘芬在小鼠体内均具有良好的抗短帚霉活性,在小鼠的心、肺组织中有较高的真菌清除率,小鼠的肾脏组织载菌量显著减少,明显提高了小鼠生存率。 3对于短帚霉感染的小鼠来说,氟康唑、伊曲康唑和特比萘芬的治疗效果无明显差异。
[Abstract]:Objective: Sopulariopsis brevicalis is one of the pathogenic fungi of saprochomycosis, which usually causes onychomycosis, and less causes the infection of other parts, but there are reports of deep tissue infection at present. The bacterium was isolated from the outpatients of our family and was identified as Sopulariopsis brevicalis by the Institute of Microbe of the Chinese Academy of Medical Sciences. one aspect of the present study is to find a culture medium and a temperature suitable for its growth, on the one hand, by observing the culture of the broom in four different media; on the other hand, the application The effect of the three common antifungal agents, such as fluconazole, ilaconic and tebiprofen, was used to treat the short-broom system infection in mice and to evaluate the survival rate of the test mice and the positive rate of the fungal reverse culture of the heart, the liver, the spleen, the lung tissue, and the detection of the amount of the tissue of the kidney. The activity of the invention is to provide a laboratory basis for the clinical treatment of the infection of the broom fungus system, and the pathological change of the mouse is provided by the histopathological examination to further understand the pathological change of the mouse caused by the bacteria. chamber basis . Method: 1 form The general morphology of the test 1.1 was first recovered at normal temperature, and three days later, it was transferred to the Sabouraud's agar medium (SDA), and the potato dextrose agar (P (DA), rice culture medium and corn Tween 80 agar, respectively, and incubated at 37 & deg; C and 27 & deg; C for 2 weeks. The colony diameter was measured and recorded daily, and the colony morphology was observed. The specimen is placed on the carrier glass. On-chip, stained with lactic acid phenol and cotton blue, and observed under light microscope. Inoculate the colonies to the urine The culture was cultured at room temperature for 1 week, and the culture was observed at room temperature. Color-raising and color-changing conditions. In-vivo drug sensitivity test: 3.1. 3.1 Production of an animal model of the infection of the short-broom-mold system: the preservation of the strain in the family is taken to restore the strain. transferring, preparing a bacterial suspension by using sterile physiological saline, and over-abdominal injection Inoculated to mice with immunosuppression with cyclic fosfamine, with a suspension of 0.5 ml for each mouse's intraperitoneal injection of bacteria. 3.2 Survival observation The administration was started at 2 h after the inoculation, and the dose of fluor-kang, irqu-kang and the specific ratio of fenofene was 10 mg/ (kg. d). The intraperitoneal injection was carried out once a day. 5 days. Continuous observation The general condition after vaccination in the group and the natural death in 21 days after the inoculation of the mice, the date of death and the number of deaths per day were recorded. The tissue-carrying amount of the tissue was sacrificed in the group, respectively. The mouse, the heart, the liver, the spleen and the lung were placed in the tissues, respectively. In the grinder, a small amount of tissue was taken and a small group of tissue was inoculated at 3 points in the modified Sabdberg solid culture medium and placed in an incubator at 27.degree. C., and the determination of the amount of bacterial carrier (CFU) in the kidney was determined separately 15 of the 15 mice in each group were taken from each group of 15 mice in the group consisting of the group consisting of the group consisting of the group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group of grinding of the kidney and the tissue of the tissue After crushing, dilute to 1 ml with sterile saline, and uniformly coat the 10 microliters of tissue suspension on the surface of the culture medium, culture at 27.degree. C., and read the number of colonies after 48 h. Neo-Confucianism Check and take all the dirty Visual and pathological changes of the surface of the device The tissues were fixed at normal temperature with 10% formalin, and the normal dehydration, transparency, embedding, slicing, HE and PAS staining were used to observe the pathological changes of the tissues under light microscope. Fruit:1 Morphological observation test 1.1 The growth curve plotted in SDA and P DA two media In the first week of culture, the growth rate of the short-broom was proportional to the time, and at the two temperatures, the growth rate was fast at 27 鈩，
本文编号：2487355
[Abstract]:Objective: Sopulariopsis brevicalis is one of the pathogenic fungi of saprochomycosis, which usually causes onychomycosis, and less causes the infection of other parts, but there are reports of deep tissue infection at present. The bacterium was isolated from the outpatients of our family and was identified as Sopulariopsis brevicalis by the Institute of Microbe of the Chinese Academy of Medical Sciences. one aspect of the present study is to find a culture medium and a temperature suitable for its growth, on the one hand, by observing the culture of the broom in four different media; on the other hand, the application The effect of the three common antifungal agents, such as fluconazole, ilaconic and tebiprofen, was used to treat the short-broom system infection in mice and to evaluate the survival rate of the test mice and the positive rate of the fungal reverse culture of the heart, the liver, the spleen, the lung tissue, and the detection of the amount of the tissue of the kidney. The activity of the invention is to provide a laboratory basis for the clinical treatment of the infection of the broom fungus system, and the pathological change of the mouse is provided by the histopathological examination to further understand the pathological change of the mouse caused by the bacteria. chamber basis . Method: 1 form The general morphology of the test 1.1 was first recovered at normal temperature, and three days later, it was transferred to the Sabouraud's agar medium (SDA), and the potato dextrose agar (P (DA), rice culture medium and corn Tween 80 agar, respectively, and incubated at 37 & deg; C and 27 & deg; C for 2 weeks. The colony diameter was measured and recorded daily, and the colony morphology was observed. The specimen is placed on the carrier glass. On-chip, stained with lactic acid phenol and cotton blue, and observed under light microscope. Inoculate the colonies to the urine The culture was cultured at room temperature for 1 week, and the culture was observed at room temperature. Color-raising and color-changing conditions. In-vivo drug sensitivity test: 3.1. 3.1 Production of an animal model of the infection of the short-broom-mold system: the preservation of the strain in the family is taken to restore the strain. transferring, preparing a bacterial suspension by using sterile physiological saline, and over-abdominal injection Inoculated to mice with immunosuppression with cyclic fosfamine, with a suspension of 0.5 ml for each mouse's intraperitoneal injection of bacteria. 3.2 Survival observation The administration was started at 2 h after the inoculation, and the dose of fluor-kang, irqu-kang and the specific ratio of fenofene was 10 mg/ (kg. d). The intraperitoneal injection was carried out once a day. 5 days. Continuous observation The general condition after vaccination in the group and the natural death in 21 days after the inoculation of the mice, the date of death and the number of deaths per day were recorded. The tissue-carrying amount of the tissue was sacrificed in the group, respectively. The mouse, the heart, the liver, the spleen and the lung were placed in the tissues, respectively. In the grinder, a small amount of tissue was taken and a small group of tissue was inoculated at 3 points in the modified Sabdberg solid culture medium and placed in an incubator at 27.degree. C., and the determination of the amount of bacterial carrier (CFU) in the kidney was determined separately 15 of the 15 mice in each group were taken from each group of 15 mice in the group consisting of the group consisting of the group consisting of the group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group consisting of a group of grinding of the kidney and the tissue of the tissue After crushing, dilute to 1 ml with sterile saline, and uniformly coat the 10 microliters of tissue suspension on the surface of the culture medium, culture at 27.degree. C., and read the number of colonies after 48 h. Neo-Confucianism Check and take all the dirty Visual and pathological changes of the surface of the device The tissues were fixed at normal temperature with 10% formalin, and the normal dehydration, transparency, embedding, slicing, HE and PAS staining were used to observe the pathological changes of the tissues under light microscope. Fruit:1 Morphological observation test 1.1 The growth curve plotted in SDA and P DA two media In the first week of culture, the growth rate of the short-broom was proportional to the time, and at the two temperatures, the growth rate was fast at 27 鈩，
本文编号：2487355
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