一株南海海洋真菌Sporothrix sp.#4335的甾体类代谢产物研究

目的研究分离自香港近海岸的红树林样品Kandelia candel的树皮中,种属已鉴定为Sporothrixsp的海洋真菌Sporothrixsp.#4335的代谢产物。方法用硅胶柱层析法分离代谢产物。结果分离得到3种甾体类化合物。结论化合物的结构通过解析波谱数据分别鉴定为麦角甾醇,过氧化麦角甾醇,麦角甾-7,22(E)-二烯-3β,5α,6β-三醇。

红树林内源生真菌Sporothrix sp.#4335代谢产物的研究

Diaporthin(1),松胞素4(2)是由一株红树林共生真菌Sporothrix sp.#4335中分离的两个化合物,其结构通过光谱分析确定。并首次对化合物1进行了抗人的肝癌细胞hepG 2及抑制人的DNA拓扑异构酶I(hTopoⅠ)的活性测试,结果显示,化合物1有一定的抗肿瘤活性(IC50为23μg/mL),及很强地抑制拓扑异构酶I的活性。

Limphocutaneous Sporotrichosis Due to Sporothrix schenckii var schenckii: A Relapsing Case after Treatment with Potassium Iodide in Elderly

Sporotrichosis is a subcutaneous mycosis caused by the Sporothrix schenckii complex. It has three classic clinical variants: fixed, lymphangitic, and systemic. Treatment in most cases has been itraconazole or potassium iodide. The aim of this paper is to communicate an unusual relapsing case treated with IK. We report a 73-year-old woman with lymphangitic sporotrichosis, adequate response to treatment with potassium iodide, and recurrence 15 months after. A molecular test was performed through the amplification of a 331 bp fragment of the calmodulin gene. In both infections, the same specie was isolated. The effects of potassium iodide are briefly discussed, and we conclude that the same treatment can be prescribed, if no side effects are observed.

Molecular identification of Sporothrix clinical isolates in China

In this study, we investigated the molecular phylogeny of 64 clinical isolates which were identified as Sporothrix schenckii sensu lato by morphological identification. All of the strains were isolates from patients from several provinces in China. The phylogeny was inferred by DNA sequence analyses based on datasets of the ribo- somal internal transcribed spacer （ITS） and a combined ITS and partial 13-tubulin region. Reference sequences were retrieved from GenBank. Results showed that all of the isolates were clustered in a distinct clade with a type of Spo- rothrix globosa. Our analysis showed that S. globosa is the causal agent of the tested sporotrichosis in China, rather than S. schenckii that was generally believed to be the case. The existence of S. schenckii in China remains to be confirmed. This study improved our understanding of the distribution of the species in S. schenckii complex.

Differential mRNA expression of Sporothrix schenckii catalase gene in two growth phases and effect factors

Sporothrix schenckii （S. schenckii）, a dimorphic fungus, is the etiological agent of sporotrichosis. After entrance of microconidia or mycelial fragments into a mammalian host, the fungus differentiates into the parasitic yeast form. Meanwhile, several defensive signals would be triggered when the innate immune system was stimulated by S. schenckii invasion and microbe-specific phagocytosis. The success of S. schenckii infection partly depends on its ability to avoid oxidative damage from reactive oxygen species （ROS） released by polymorphonuclear leukocytes and activated macrophages. However, the antioxidant defense mechanisms of S. schenckii remains unknown. Catalases, one of the central enzymes involved in scavenging ROS via converting hydrogen peroxide （H2O2） to water and molecular oxygen, play an essential role in protecting intracellular pathogenic fungi against ROS and regulating growth and development in some fungi. No catalase in S. schenckii has been identified and characterized previously. Recently we have cloned a catalase homologous gene from the yeast form of S. schenckii and designated it Sscat. In this report,

Characterization of Sporothrix schenckii by random amplification of polymorphic DNA assay

s To investigate the DNA polymorphism of Sporothrix schenckii ( S schencki i ) and to find the relationship between DNA patterns and geographic areas and clinical manifestations Method The total DNA was extracted with hexadecyltrimethyl ammonium bromide Random A mplification of Polymorphic DNA (RAPD) assay was used to study DNA typing of 24 strains of S schenckii collected from different areas and isolated from di fferent clinical types Results Of seven random primers used, three primers (OPAA11, OPD18 and OPB07) gave good reactions, the sequences of which were 5' ACCCGACCTG 3', 5' GAGAGCCAAC 3', 5 ' GGTGAC^GCAG 3' respectively The RAPD patterns of the 24 isolates were not completely identical, showing certain degrees of hereditary variability Differ ent isolates showed a common conserved DNA band with the same primer Different clinical types showed different genotypes Conclusion RAPD analysis is useful in DNA typing of S schenckii , the DNA band type of which is related to geographic origin and Clinical manifestation

Global ITS diversity in the Sporothrix schenckii complex

Molecular phylogeny has revealed that sporotrichosis is caused by several Sporothrix species which differ in clinical behavior.The complex is embedded within Ophiostoma,a genus mainly comprising fungi that live in association with bark beetles,but differs by a high virulence towards humans and other mammals.The different ecology is corroborated by phylogenetic separation.The aim of the present study was to determine the validity of the rDNA internal transcribed spacer(ITS)region as a marker for diagnostics of species in the clinical group,using betatubulin sequences to calibrate species delimitations.The topology of the two gene trees was concordant,and all clinically relevant Sporothrix species could easily be recognized by means of the ITS region.An increased geographic sampling did not affected delimitation success in the clinical clade of the S.schenckii complex.

Construction and analysis of the c DNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes

Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the di- morphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization （SSH） was used to prepare a complementary DNA （cDNA） subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between dif- ferently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction （qRT-PCR）. It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

Morphological and physiological comparison of taxa comprising the Sporothrix schenckii complex

Based on recent molecular data, it has been suggested that Sporothrix globosa is the main causal agent of sporotrichosis in China. The objective of this study was to compare the morphology, growth characteristics, patterns of carbon source usage, and susceptibility to antifungal agents among Sporothrix strains. A total of 15 clinical strains confirmed to be S. globosa, from three different regions of China, and 11 ex-type strains from the CBS-KNAW biodiversity center were obtained. The elongated conidia of S. pal/ida, S. variecibatus, S. schenckii, and S. schenckii luriei were clearly different from the subglobose and globose conidia of S. globosa strains. S. schenckii is able to assimilate sucrose, raffinose, and ribitol. Susceptibility profiles of these Sporothrix species were evaluated by measuring minimum inhibitory concentrations （MICs）. Fluconazole, itraconazole, terbinafine, and amphotericin B showed good activity against most S. globosa clinical isolates from China. Potassium iodide also showed a low MIC against S. pal/ida, while fluconazole showed a high MIC for S. mexicana, S. humicola, S. g/obosa, S. schenckii, and S. inflata; these strains might be considered tolerant. The species showed differences in susceptibility to antifungal drugs and should therefore be properly identified during diagnosis prior to designing therapeutic strategies.

临床分离99株孢子丝菌病原体的分子和表型鉴定

目的对99株来自我国十个省市孢子丝菌病患者组织分离的病原体进行分子鉴定和表型分析。方法对99株孢子丝菌核糖体基因ribosomal internal transcribed spacer(ITS)、钙调蛋白基因(Calmodulin,CAL)PCR多位点扩增、测序以及种系进化分析,并对其进行包括温度试验(30℃,35℃,37℃)、糖同化试验、不同培养基生长试验、双相转换试验等表型研究。结果依据ITS\CAL基因序列分析显示96株孢子丝菌被鉴定为球形孢子丝菌;37℃时有90.91%(90/99)生长受抑,9株不生长;99株同化蔗糖、96株不同化棉籽糖,3株同化棉籽糖;申克孢子丝菌的孢子形态多样。结论中国十省市的孢子丝菌以球形孢子丝菌为主,也存在申克孢子丝菌。

微量稀释法检测申克孢子丝菌酵母相体外抗真菌药物敏感性

目的用液基微量稀释法观察双相真菌申克孢子丝菌酵母相体外抗真菌药物敏感性。方法将54株申克孢子丝菌临床株于脑心浸液琼脂培养基连续传代获得酵母相,参考美国临床实验室标准化委员会(CLSI)的微量稀释法M27-A2检测菌株酵母相对碘化钾、氟康唑、伊曲康唑和特比萘芬的体外敏感性,并观察碘化钾对伊曲康唑和特比萘芬体外抑菌作用的影响。质控株为克柔念珠菌ATCC6258。结果碘化钾体外无抑菌作用;氟康唑最小抑菌浓度(MIC)几何均数大于64μg/mL;伊曲康唑和特比萘芬MIC几何均数分别为0.98μg/mL和0.17μg/mL。伊曲康唑及特比萘芬的MIC值分别高于伊曲康唑+碘化钾及特比萘芬+碘化钾(P均<0.05)。来源皮肤固定型的菌株与来源皮肤淋巴管型菌株相比MIC值差异无统计学意义(P>0.05)。结论改良的M27-A2方法适用于检测申克孢子丝菌酵母相体外敏感性;酵母相时碘化钾对伊曲康唑、特比萘芬体外抑菌作用有一定的加强效应。

山东“10·21”辐射事故2例放射病病人真菌感染的实验鉴定和动态观察

目的分析引起山东“10·21”辐射事故2例放射病人真菌感染的种类和药敏试验结果。方法用形态学方法结合VITEK(2全自动微生物分析仪以及API 20C AUX生化鉴定方法对临床标本进行微生物学鉴定,真菌药敏试验采用微量稀释法。结果在病例A的大便标本中培养出近平滑念珠菌,中段尿、血液、胸水、腹水、大便、骨髓血、两次咽拭子和脑脊液等标本中培养出申克孢子丝菌,痰液标本中培养出热带念珠菌。病例B的鼻腔、口腔、尿道口分泌物和腹腔引流液等标本中培养出阿萨丝孢酵母菌,痰标本中除阿萨丝孢酵母菌外,还培养出了土曲霉菌。随着抗真菌药物使用时间的延长,上述真菌对抗真菌药物的敏感性逐渐下降,最小抑菌浓度(MIC)逐渐增加。结论大多数抗真菌药物的体外药敏试验敏感,但患者的一般情况很差,常用剂量乃至较大剂量的用药只能抑菌而不能杀菌,且因临床预防性抗生素和抗真菌制剂的使用,常规的微生物培养方法很难培养出细菌或真菌。

香鳞毛蕨有效部位对申克孢子丝菌酵母相的体外抗真菌作用研究

目的探讨香鳞毛蕨有效部位对申克孢子丝菌的体外抑菌作用,进一步明确香鳞毛蕨有效部位的抗菌谱。方法取4株申克孢子丝菌为受试菌株,培养至酵母相,分别采用琼脂扩散法测定抑菌圈直径和微量稀释法测定最低抑菌浓度(MIC)与最低杀菌浓度(MFC)。结果香鳞毛蕨有效部位对4株申克孢子菌酵母相的抑菌圈平均直径为25.5 mm,对4株申克孢子丝菌酵母相的MIC几何均数为生药0.18 mg·mL-1,但MFC>生药20mg·mL-1。结论香鳞毛蕨有效部位对申克孢子丝菌酵母相有较强抑菌作用,但无杀菌作用。

类几丁质酶3蛋白1在申克孢子丝菌刺激巨噬细胞的表达和作用研究

目的探讨巨噬细胞类几丁质酶3蛋白1在申克孢子丝菌刺激后的表达和作用研究。方法通过测定巨噬细胞与申克孢子丝菌共孵育时杀菌率,使用实时荧光定量PCR检测不同时间点巨噬细胞的类几丁质酶3蛋白1mRNA表达水平,同时体外实验检测类几丁质酶3蛋白1对申克孢子丝菌的抑菌作用,同时采用空白对照和白念珠菌阳性对照比较两者的差异。结果巨噬细胞可吞噬杀灭申克孢子丝菌分生孢子,也可吞噬阳性对照白念珠菌酵母相细胞。巨噬细胞类几丁质酶3蛋白1mRNA表达水平在申克孢子丝菌组和空白对照组无明显差别,作用于阳性对照的白念珠菌组的巨噬细胞类几丁质酶3蛋白1mRNA表达水平明显上调。体外实验显示类几丁质酶3蛋白1对申克孢子丝菌分生孢子的无明显抑制作用,对白念珠菌繁殖却有较强的抑制作用。结论申克孢子丝菌未能诱导巨噬细胞表达类几丁质酶3蛋白1抑制其增殖从而逃逸清除,而白念珠菌感染可上调巨噬细胞类几丁质酶3蛋白1表达从而抑制其增殖减轻感染,这可能是申克孢子丝菌特有的致病作用机制之一。

伊曲康唑联合特比萘芬治疗孢子丝菌病疗效及其对病原真菌体外抗菌活性研究

目的初步探讨伊曲康唑和特比萘芬联合治疗孢子丝菌病的疗效,评价两药体外联合对申克孢子丝菌菌丝相和酵母相的抗菌活性。方法口服伊曲康唑200 mg/d和特比萘芬250 mg/d治疗孢子丝菌病;体外联合药敏试验采用棋盘微量稀释法,计算分数抑菌浓度(FIC)指数判定两药相互作用具有协同、拮抗或无关作用。结果伊曲康唑和特比萘芬联合成功治愈3例经伊曲康唑治疗无效的孢子丝菌病患者。两药体外联合对申克孢子丝菌菌丝相具有75%的协同作用,对酵母相为70%(P=0.50)。结论初步研究显示伊曲康唑和特比萘芬联合治疗孢子丝菌病有效安全,体外联合药敏试验显示两药具有较高的协同作用。

温度和培养基在申克孢子丝菌酵母相转化中的作用研究

【目的】探讨不同温度和培养基在申克孢子丝菌酵母相转化中的作用。【方法】将4株临床分离菌株分别接种于SDA、PDA、BHI、50mg/kg羊血BHI及5mg/kg GSBHI5种培养基平皿上,每种培养基接种5个平皿,分别置于25℃、35℃、36℃、37℃及38℃含体积百分数为5%CO2的恒温箱中培养。观察菌落生长情况并计算酵母细胞转化率。【结果】在38℃,平皿转化率为25%(只有1株菌发生了转化);在35℃和37℃时,平皿转化率分别是60%和85%;而在36℃时平皿转化率仅为45%。使用50mg/kg羊血BHI和0.5%GSBHI培养基培养,65%培养皿上的菌株发生了酵母相转化,使用BHI培养基培养,45%培养皿上的菌株发生了酵母相转化,而使用SDA和PDA培养基培养,只有20%培养皿上的菌株发生了酵母相转化。37℃时,有3株菌在BHI、50mg/kg羊血BHI和5mg/kg GSBHI培养基上的酵母细胞转化率均达到90%,1株菌在SDA、PDA和BHI培养基上的转化率均低于10%。【结论】温度是影响申克孢子丝菌酵母相转化的关键因素,培养基的营养成分亦是影响转化效果的重要因素。

中国南北方地区临床孢子丝菌菌种鉴定

目的对分离自我国南、北方地区50株临床孢子丝菌进行菌种鉴定。方法分离菌株分别进行25℃恒温培养和玻片小培养,肉眼和镜下观察形态特征;同时提取菌丝相基因组DNA,用PCR分别扩增部分微管蛋白(β-tubulin)基因和核糖体内部转录间隔区(Ribosomal Internal Transcribed Spacer,ITS),扩增产物进行测序,并采用最大似然法(maximum likelihood,ML)和邻接法(neighbor-joining,NJ)构建联合β-tubulin基因和ITS区域的系统发育树。结果结合形态学及系统发育分析,50株菌均鉴定为球形孢子丝菌(Sporothrix globosa,S.globosa)。结论球形孢子丝菌是目前我国南、北方地区孢子丝菌病的主要致病菌种,为进一步明确我国孢子丝菌病致病菌种的分布提供了依据。

伊曲康唑联合卡泊芬净或特比萘芬对球形孢子丝菌体外抑菌作用的研究

目的探讨伊曲康唑联合卡泊芬净或特比萘芬时对球形孢子丝菌酵母相和菌丝相的体外抑菌作用。方法参照标准的微量液基稀释法及棋盘微量稀释法对33株球形孢子丝菌行药敏试验,结果使用抑菌浓度指数(FICI)判定"协同""不相关"和"拮抗"。结果伊曲康唑与卡泊芬净联合对球形孢子丝菌酵母相菌株的协同率为93.94%(31/33),与特比萘芬联合的协同率为60.61%(20/33);伊曲康唑与卡泊芬净或特比萘芬联合对菌丝相菌株协同率分别为96.97%(32/33)、84.5%(28/33)。对所有受试菌株均未观察到上述药物的拮抗作用。结论体外联合药敏试验结果显示伊曲康唑与卡泊芬净或特比萘芬联合有较好的协同作用。

13种中草药对申克孢子丝菌抑菌实验研究

目的：在马齿苋等13种中草药中,观察筛选出对申克孢子丝菌有抑制作用的中草药。方法：通过微量液基稀释法,测定各药液对申克孢子丝菌的抑制作用。结果：最小抑菌浓度（MIC）由小到大排列分别为：黄连MIC 183.47μg/m L、黄柏MIC 273.07μg/m L、蛇床子MIC 512.00μg/m L、苦参MIC 546.13μg/m L、马齿苋MIC 563.20μg/m L、丹参MIC 1058.13μg/m L、金银花MIC 1297.07μg/m L、陈皮MIC 1433.60μg/m L、地榆MIC 1638.40μg/m L。来源皮肤固定型的菌株与来源皮肤淋巴管型菌株相比MIC值差异无统计学意义。结论：黄连、黄柏、蛇床子、苦参、马齿苋有较强地抑制申克孢子丝菌的作用,可以为临床治疗提供依据。

聚合酶链反应技术检测申克孢子丝菌的实验研究

目的研究申克孢子丝菌的种特异性引物聚合酶链反应鉴定方法,从而为临床孢子丝菌病的分子诊断奠定基础。方法采用根据申克孢子丝菌几丁质合成酶基因1序列设计合成的一对寡核苷酸引物,分别对26株申克孢子丝菌(包括1株标准株,7株实验室保存株,18株临床分离株)及6种6株普通真菌(分属于3个菌属)的基因组DNA进行PCR扩增。结果扩增产物选择性的从26株申克孢子丝菌基因组DNA中获得,而对念珠菌属、毛癣菌属、小孢子菌属的6株普通真菌基因组DNA均为阴性。结论采用以申克孢子丝菌几丁质合成酶基因1序列为基础的聚合酶链反应法鉴定申克孢子丝菌特异、简便、快捷,可用于临床诊断。