应用免疫荧光技术检测肺微小根毛霉的感染

近几年因源性导致患者机体免疫功能低下或受损有所增加,从而使深部丝状真菌感染有逐年上升的趋势,由于检测上的种种原因,大部份是尸检后发现,难以鉴定对属种,对临床治疗带来直接影响,为进一步探讨丝状真菌的快速诊断。我们从肺活组织中分离出的微小根毛霉,注射接种到不同剂量的＾６０Ｃｏ－ｒ辐照鼠体内后,间隔一定时间,用间接免疫荧光法检测,发现３ＧＹ辐照后１４－２８日检出率是４１．８７％（皮下注射）,６６．６６％     

微小根毛霉的感染治疗研究

微小毛霉感染途径、感染部位、感染机理研究的基础上,进一步探讨机体受损不同程度,微小根毛霉感染不同药物治疗效果。本实验选用5GY、7GY等不同剂量的60Co照射18～20g♀LACA小鼠,每只鼠接种六千万个微小根毛霉的孢囊孢子,设对照组。通过氟康唑,野山花治疗,观察其治疗效果和小鼠活存情况及感染部位等。结果证明：5GY、7GY两种不同剂量60Co照射感染后,从活存时间、体重、治疗后的感染只数,感染部位等均有差异。7GY60Co照射后,腹腔注射六千万个微小根毛霉的孢囊孢子,感染后分别以氟康唑、中草药野山花进行治疗,野山花治疗组小鼠体重平均高于氟康唑的治疗组。活存35日后处死,平均每只鼠体重由原来的19.3g增加到24.2g。氟康唑治疗组10目前全部死亡,每只鼠平均体重由原来的19．2g减少到1487g。5GY照射后接种六千万个微小根毛霉孢囊孢子采用野山花、氟康唑进行的治疗效果,可看出两组治疗鼠体重与对照组无明显差异。但是从治疗效果看出,5GY照射感染治疗后,野山花治疗效果优于氟康唑治疗组,治疗后野山花组感染2只,氟康唑组感染5只。野山花治疗组感染了脾、肺、淋巴结等三个组织,氟康唑治疗组感染了肝、脾、肾、淋巴结等五个组织,而检出比例比野山花治疗组高。结果证明微小根毛霉均对脾、肾侵害严重。     

微小根毛霉感染机理研究

本文在首次报告二例肺结核、糖尿病患者合并感染肺微小根毛霉的基础上,进一步探讨免疫功能受损与微小根毛霉感染关系。本文采用不同剂量~(60)Co-γ全身辐照小鼠,然后以不同途径注射同剂量的微小根毛霉的孢囊孢子,观察动物的感染情况和感染后的真菌检出率,结果发现各种辐射剂量均在辐射后7-14日感染菌的检出率最高;各种脏器感染菌的检出率以脾脏最高(66.7—81.8％);淋巴结最低(0.0—25.0％);其他脏器的感染菌检出率也有不同程度的差异。     

低水平辐射诱导的细胞遗传学适应性反应

先用0.01GY x-射线(剂量率:0.01GY/分)体外照射人、兔外周血,经不同时间后再用1.5GY X-射线(0.44GY/分)照射,发现在G_0、G_1、S和G_2期受0.01GY X-射线照射后再给大剂量照射者,其染色体畸变率明显低于单纯受1.5GY X-射线照射组(P<0.01)。这一适应性反应能持续3个细胞周期,在接受小剂量照射后超过3个细胞周期再受大剂量照射者,染色体畸变率未见减少。若在第三细胞周期以后再次给予小剂量照射,可再次诱导适应性反应。用小鼠整体小剂量照射后骨髓细胞和生殖细胞亦出现这种适应性反应。另外也探讨了不同剂量和不同剂量率的预先照射对适应性反应的影响。     

河北地区临床实验室丝状真菌分离鉴定情况分析

目的开展一项由河北地区多所医院参与的临床实验室丝状真菌检测研究,促进实验室丝状真菌检测能力提升。方法共收集丝状真菌菌株,采用沙堡弱培养基和乳酸酚棉兰染色直接镜检进行菌种常规鉴定;中心实验室采用Vitek MS质谱进行复核鉴定,对MS不能有效鉴定的菌种进行核糖体DNA内转录间隔区ITS和/或钙调蛋白CaM测序分析。结果参与研究的15家三级医院2016～2017年共收集到丝状真菌225株。其中烟曲霉133株(59.11%)、黄曲霉/米曲霉28株(12.44%)、黑曲霉复合群18株(8.00%)、聚多曲霉6株(2.67%)、构巢曲霉6株(2.67%)、其他丝状真菌34株(15.11%)。样本类型包括下呼吸道痰标本203株(90.22%),耳道分泌物10株(4.44%),肺泡灌洗液4株(1.78%),其他样本8株(3.56%)。菌株鉴定正确率(148/225)65.78%,错误率(77/225)34.22%。结论丝状真菌感染中最常见的是曲霉菌属,主要以烟曲霉菌、黄曲霉菌和黑曲霉菌为主。传统的鉴定方法错误率高达30%以上,采用微生物质谱鉴定结合ITS/CaM区测序方法可以有效提高丝状真菌的鉴定正确率,为临床丝状真菌的治疗提供病原学依据。     

南极乔治王岛的丝状真菌

本文报道从南极乔治王岛26个样品中分离到60株丝状真菌,其中6个样品未分离到任何真菌,经初步鉴定有8属10个种。根据本次温度实验和该地寒冷的气候,只有桔灰青霉(Penicillium aurantiogriseum Dierckx),大毛霉Mucor mucedo(L.)Fres.)和毡状金孢霉(chrysosporium pannorum(Link)Hughes)在本地能生长繁殖。其中毡状金孢霉占明显优势。     

微小根毛霉感染机理研究

本文在首次报告二例肺结核、糖尿病患者合并肺微小根毛霉的基础上,进一步探讨免疫功能受损与微小根毛霉关系。本文采用不同剂量＾６０Ｃｏ－γ全身辐照小鼠,然后以不同途径注射同剂量的微小根毛霉的孢囊孢子,观察动物的感染情况和感染后的真菌检出率,结果发现各种辐射剂量均在辐射后７－１４日感染菌的检出率最高;各种脏器感染菌的检出率以脾脏最高;淋巴结最低;其他脏器的感染菌检出率也有不同程度的差异。     

铅锌尾矿中耐重金属镉的丝状真菌的分离鉴定

【目的】旨在从重金属污染地分离出耐重金属镉真菌, 获得耐受重金属镉污染的高效菌株, 为重金属污染微生物修复提供菌种资源。【方法】利用稀释平板涂布法, 采用4种培养基对粤东北梅州市梅县铅锌尾矿废弃地9个样品进行分离, 并结合形态学和ITS rDNA基因序列分析, 鉴定分离到的耐镉真菌, 最后对分离到的耐镉真菌进行最小抑制浓度 (MIC) 检测。【结果】从粤东北梅州市梅县铅锌尾矿废弃地分离出72株丝状真菌, 经形态学和分子技术鉴定, 它们主要属于曲霉属(Aspergillus)、青霉属(Penicillium)、枝孢属(Cladosporium)、油瓶霉属(Lecythophora)、拟青霉属(Paecilomyces)、镰刀孢属(Fusarium)等。MIC检测发现有4株丝状真菌耐镉浓度较高, Paecilomyces lilacinus (Thom) Samson (6?20 p), Penicillium pinophilum Hedgcock (6?16 p), Penicillium rolfsii Thom (6?16 m) 和Fusarium oxysporum Schlecht. (8?11 p) 分别为200、40、25和15 mmol/L。【结论】从粤东北梅州市梅县铅锌尾矿废弃地分离到的72株丝状真菌, 不同程度耐受重金属镉, 在重金属污染的治理中有可能发挥作用。本研究为镉污染环境的微生物修复提供了重要菌株。     

用SDS-PAGE和Vorum银染法比较真菌菌丝体破壁效果

目的:研究不同处理方法对丝状真菌Pythium sp.GY1938菌丝体破壁效果的差异。方法:采用7种方法对真菌菌丝体进行破壁处理,将制备的胞内可溶蛋白质样品通过SDS-PAGE和Vorum法银染处理,比较不同破壁方法处理后蛋白质释放效果的优劣。结果:方法VI(石英砂、液氮、研磨)的破壁效果最好,蛋白质的分离效率和提取质量最高。结论:方法VI适合于丝状真菌Pythium sp.GY1938蛋白组学研究中目的蛋白的提取。     

一种快速高效获取丝状真菌PCR反应模板的方法

建立一种快速高效获取丝状真菌PCR反应模板的方法,提高丝状真菌PCR鉴定效率。通过单因素法对机械破壁联合微波法进行条件优化,利用优化后的方法获取13株不同种属丝状真菌的PCR反应模板,同时与Chelex-100法、机械破壁法作对比,以试剂盒抽提法作为阳性对照,进行ITS序列扩增,琼脂糖凝胶电泳检测扩增结果。机械破壁联合微波法获取丝状真菌PCR反应模板的最佳条件为40 Hz机械破壁1 min、微波700 W高温裂解3 min,采取该法与试剂盒抽提法获得的模板均成功扩增13株不同种属丝状真菌ITS序列,且PCR鉴定结果一致;Chelex-100法获得的模板成功扩增6株丝状真菌ITS序列;机械破壁法获得的模板虽成功扩增9株丝状真菌ITS序列,但扩增效果欠佳。机械破壁联合微波法能够有效获取丝状真菌PCR反应模板,与试剂盒抽提法相比具有操作简便、快速高效的优点,提高丝状真菌PCR鉴定效率。     

Passive immunization in murine mucormycosis

Antibody raised in mice against mycelial homogenates of Rhizomucor pusillus was effective in passive immunization against pulmonary and disseminated mucormycosis (phycomycosis) in immunocompromised mice. Mice intranasally inoculated and infected with Rh. pusillus and treated with antisera had a statistically significant increased resistance to infection and a diminished secondary dissemination of viable fungal fragments. Histological examination of infected lung tissues showed that antibody treated animals were apparently able to degrade hyphal fragments.     

Identification and Retting Efficiencies of Fungi Isolated from Dew-Retted Flax in the United States and Europe

Seven strains of filamentous fungi and one yeast were isolated from flax that was dew retted in the United States. These filamentous fungi were subcultured to purity and identified, and six appear not to have been reported earlier as isolates from dew-retted flax. Five of the purified U.S. strains, two fungi isolated from flax that was dew retted in Europe, and a laboratory culture of Aspergillus sojae were tested for their ability to ret flax stems. The monocultures were evaluated for the degree of retting, fiber strength, dry weight loss, and tactile response (i.e., feel of softness) as reflected in the retted fiber. Structural modifications of representative samples of the retted flax were assessed by scanning electron microscopy. All of the filamentous fungi were able to carry out some retting, whereas the isolated yeast could not. All organisms produced pectinases when they were cultivated in shake flasks on ball-milled flax as the sole carbon source. Some fungi also produced cellulases, mannanases, and xylanases. Rhizomucor pusillus and Fusarium lateritium were noteworthy as retting organisms by their high level of pectinase activity, ability to attack noncellulosic cell types without attacking cellulose, capacity to penetrate the cuticular surface of the stem, and efficient fiber release from the core. The results indicated that these organisms deserve further study as potential organisms for retting of bast fibers in industrial applications.     

Biosorption of simulated dyed effluents by inactivated fungal biomasses

Treatment of dyed effluents presents several problems mainly due to the toxicity and recalcitrance of dyestuffs. Innovative technologies, such as biosorption, are needed as alternatives to conventional methods to find inexpensive ways of removing dyes from large volumes of effluents. Inactivated biomasses do not require a continuous supply of nutrients and are not sensitive to the toxicity of dyes or toxic wastes. They can also be regenerated and reused in many cycles and are both safe and environment-friendly. The sorption capacities (SC) of autoclaved biomasses of three Mucorales fungi (Cunninghamella elegans, Rhizomucor pusillus and Rhizopus stolonifer), cultured on two different media, were evaluated against simulated effluents containing concentrations of 1000 and 5000 ppm of a single dye and a mix of 10 industrial textile dyes in batch experiments. SC values of up to 532.8 mg of dye g(-1) dry weight of biomass were coupled with high effluent decolourisation percentages (up to 100%). These biomasses may thus prove to be extremely powerful candidates for dye biosorption from industrial wastewaters. Even better results were obtained when a column system with the immobilised and inactivated biomass of one fungus was employed.     

泊沙康唑对临床来源接合菌的体外抗菌活性研究

目的研究泊沙康唑对临床来源的接合菌体外抗菌活性。方法对临床来源的43株接合菌采用ITS区序列分析进行准确鉴定,采用E-test纸片法研究泊沙康唑对43株菌的体外药物敏感性,观察其MIC值。结果经ITS序列分析鉴定,43株临床来源接合菌中最为常见的是小孢根霉和不规则毛霉(各12株),其次为米根霉(10株),ramosa(3株)。其他菌种分别为ornata、总状共头霉、微小根毛霉、卷曲毛霉、印度毛霉和冻土毛霉各1株。E-test纸片法测得泊沙康唑对毛霉属、根霉属和Lichtheimia spp.的MIC值范围分别为0.19～>32μg/mL、0.19～3μg/mL、0.002～0.38μg/mL,对总状共头霉和微小根毛霉的MIC值分别为1μg/mL和0.19μg/mL。结论泊沙康唑对大部分接合菌有效,不同种属MIC值范围不同,毛霉属真菌的MIC值差异较大。     

Production of saccharogenic and dextrinogenic amylases by Rhizomucor pusillus A 13.36

A newly-isolated thermophilic strain of the zygomycete fungus Rhizomucor pusillus 13.36 produced highly active dextrinogenic and saccharogenic enzymes. Cassava pulp was a good alternative substrate for amylase production. Dextrinogenic and saccharogenic amylases exhibited optimum activities at a pH of 4.0-4.5 and 5.0 respectively and at a temperature of 75 degrees C. The enzymes were highly thermostable, with no detectable loss of saccharogenic or dextrinogenic activity after 1 h and 6 h at 60 degrees C, respectively. The saccharogenic activity was inhibited by Ca(2+) while the dextrinogenic was indifferent to this ion. Both activities were inhibited by Fe(2+) and Cu(2+) Hydrolysis of soluble starch by the crude enzyme yielded 66% glucose, 19.5% maltose, 7.7% maltotriose and 6.6% oligosaccharides.     

Screening microbial strain for improving the nutritional value of wheat and corn straws as animal feed

From 18 strains of cellulolytic microorganisms including bacteria and filamentous fungi, one strain of soft rot fungus identified as Chaetomium cellulolyticum was screened with respect to stronger decomposition ability of cellulose and hemicellulose and its ability for protein synthesis. As it grew on raw corn straw in solid layer fermentation (SLF) for 5 days, the amino acid content in the fermentation product attained 19.29% (w/w) from 6.43% while the total cell wall was reduced by 54%. A toxicity test with mice showed that the fermentation product is not poisonous. The two filamentous fungi, Trichoderma pseudokoningii S-38 and Penicillium decumbens JU-A10 produced large amounts of extracellular cellulase and hemicellulase in the SLF process, but their growth was limited and they sporulated profusely with regard to their value as animal feed products.     

Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments

Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. Halorosellinia sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3?min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15?min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of Halorosellinia sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.     

Experimental cerebral zygomycosis in alloxan-diabetic rabbits: variation in virulence among zygomycetes

We investigated the potential of 33 different zygomycete isolates to cause cerebral disease following the intranasal instillation of their spores into ketotic rabbits with alloxan induced diabetes. The isolates represented six thermotolerant species of Rhizopus (R. arrhizus, R. chinensis, R. microsporus, R. oligosporus, R. oryzae, and R. rhizopodiformis), Absidia corymbifera, Cunninghamella bertholletiae, and Rhizomucor pusillus. All 13 isolates of the thermotolerant Rhizopus species proved to be cerebral pathogens as confirmed by culture and histopathology. One isolate of R. oligosporus and one isolate of R. rhizopodiformis, however, were less pathogenic than isolates of other Rhizopus species tested. Cerebral pathogenicity was noted with 2 of 5 isolates of Rh. pusillus and only 1 of 13 A. corymbifera isolates. Two thermotolerant C. bertholletiae cultures, recovered from human lesions, did not cause either cerebral or pulmonary disease in ketotic rabbits. The incidence of pulmonary zygomycosis caused by the isolates of the species of the four genera under study was as follows: Rhizomucor 24%, Rhizopus 22%, Absidia 9%, and Cunninghamella 0%. This study confirms the pathogenic potential of the thermotolerant species of Rhizopus to cause cerebral zygomycosis in ketotic diabetic rabbits and also revealed the potential of Rh. pusillus and A. corymbifera occasionally to cause the same disease in animals and humans.     

Filamentous fungi found in Atta sexdens rubropilosa colonies after treatment with different toxic bait formulations

Leaf‐cutting ants maintain a symbiotic relationship with basidiomycetous fungi cultivated as food. Here, we profiled the non‐symbiotic filamentous fungi in laboratory nests of Atta sexdens rubropilosa submitted to treatments with different toxic bait formulations (using the insecticide sulfluramide as the active ingredient). After treatment, several filamentous fungi were found in different nest compartments. Culture‐dependent techniques recovered a total of 93 fungal isolates comprising 10 genera, 11 species and four unidentified fungi. The genus Penicillium was prevalent in both control and insecticide treatments. Overall, the majority of fungal isolates obtained in this study are commonly found in soil. Escovopsis spp., the specialized parasite of the ant‐fungus mutualism was only recorded in the fungus gardens of nests submitted to the toxic treatments. Moreover, no correlation was found regarding the presence of fungi in the different nest compartments (chi‐square, P > 0.4182). This study reveals that Escovopsis spp. is not the only fungus to overgrow the fungus garden of debilitated nests, thus adding more evidence on the possible negative impacts of such alien fungi. As suggested by previous studies, fast‐growing filamentous fungi likely overgrow the fungus garden in such conditions.