Serological Characterization of Trichosporon cutaneum and Related Species

Recent molecular biological, chemical, physiological and morphological studies indicate that Trichosporon cutaneum and related species should be reclassified. In this study, antigenic characteristics of the species were determined. The results of adsorption experiments revealed that there were at least three serological types: I, II and III. Specific factor sera I, II and III were prepared on the basis of adsorption experiments and isolates were serotyped by cell slide agglutination (CSA). Since the CSA test was difficult to read in some strains, the results of the CSA test were compared with the findings from an enzyme-linked immunosorbent assay (ELISA). For the ELISA, crude polysaccharide antigens prepared from the culture supernatant were used as the antigen. The types determined by ELISA correlated well with those determined by the CSA test. These data suggest that T. cutaneum and related species have at least three serological types, and that the typing can be done by either CSA or ELISA.     

Partial Sequences of Large Subunit Ribosomal DNA of a New Yeast Species,Trichosporon domesticum and Related Species

We determined the partial sequences of large subunit rDNA of a new yeast species, Trichosporon domesticum, which was isolated from the house of a summer-type hypersensitivity pneumonitis patient. Phylogenetically, T. domesticum was positioned in the taxonomic group containing T. montevideense and T. brassicae, which indicated an identical serotype. A phylogenetic relationship among all species of the genus Trichosporon which belong to the basidiomycetous yeast is clarified.     

响应面法优化产酰胺酶发酵培养基

采用响应面分析方法,对阿萨希丝孢酵母（Trichosporon asahii）ZZB-1产酰胺酶的发酵培养基进行了优化。运用单N子试验筛选出麦芽糖和酵母浸膏为最适碳源、氮源,金属离子Ca^2＋、Mn^2＋可提高发酵酰胺酶产量;通过最陡爬坡实验逼近以上4个因子的最大响应区域后,采用Box—Behnken响应面分析法,确定产酰胺酶最佳发酵培养基为麦芽糖18．84g／L、酵母浸膏9．55g／L、NaC15g／L、KH2PO41g／L、MgSO4·7H2O0．2g／L、FeS040．001g／L、CaC0370．84μmol／L、MnS0465．39肚mo[／L（1％丙烯酸诱导）,NH4·H2O调节pH至7．0。培养基优化后酰胺酶产量由初始2554U／L提高到4156U／L,为原始发酵培养基配方酶活产量的1．63倍。     

First Report of Trichosporon ovoides Isolated from the Home of a Summer-Type Hypersensitivity Pneumonitis Patient

Trichosporon species have been known to cause summer-type hypersensitivity pneumonitis (SHP). During the isolation of yeasts from an SHP patient's house, we recovered a strain belonging to the genus Trichosporon. Morphologically, the isolate produced rectangular arthroconidia when grown on corn meal agar. DNA-DNA hybridization experiments identified the isolate as T. ovoides. A slide agglutination test using specific factor sera demonstrated that the serotype of the strain was type II. Previously, T. asahii, a serotype II species, was considered to be the major antigen of SHP, but it is possible that T. ovoides may also be responsible for SHP. This is the first report of T. ovoides isolated from an SHP patient's home environment.     

Phylogenetic and taxonomic heterogeneity of Cryptococcus humicolus by analysis of the sequences of the internal transcribed spacer regions and 18S rDNA, and the phylogenetic relationships of C. humicolus, C. curvatus, and the genus Trichosporon

The phylogenetic and taxonomic heterogeneity of a rare opportunistic yeast pathogen, Cryptococcus humicolus, was revealed by analysis of the sequence of the internal transcribed spacer (ITS) region. Sixteen strains of C. humicolus showed a wide diversity in their ITS sequences. In addition, their 18S rDNA sequences were determined and used to analyze the phylogenetic relationships among C. humicolus and related yeasts. On trees constructed by the Neighbor-Joining and Maximum Parsimony methods, C. humicolus strains were phylogenetically closely related to each other with the exception of one strain, and they clustered with C. curvatus and Trichosporon species with high bootstrap values. Three C. humicolus strains obtained from humans belonged to the group of Trichosporon serotype I species. The results suggest that C. humicolus is a genetically heterogeneous species which should be reclassified on the basis of DNA sequence data.     

抑制阿萨希毛孢子菌顶体对其增殖及致病性的影响

目的探讨抑制阿萨希毛孢子菌(Trichosporon asahii,T.asahii)顶体(Spitzenkrper)形成后对其细胞增殖及致病性的影响。方法经不同浓度细胞松弛素D(0.5、1.0及2.0μmol/L)处理阿萨希毛孢子菌,以抑制其顶体形成,体外测定生长曲线及生长菌落变化;小鼠体内接种上述菌悬液后,统计小鼠3周内的死亡率,并取内脏进行组织培养和病理检查,统计感染率。结果经细胞松弛素D处理抑制顶体形成后,T.asahii细胞生长明显延迟8~16 h,生长菌落直径明显变小;小鼠死亡率及感染率亦出现不同程度降低,并随着药物浓度的升高,其致病性明显降低。结论抑制阿萨希毛孢子菌顶体形成后,细胞增殖明显受到抑制,且致病性也随细胞松弛素D浓度的增加而降低。提示顶体可以作为研制新型抗真菌药物的新靶点。     

Real-time PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis

Deep-seated trichosporonosis due to Trichosporon asahii is life-threatening and has high mortality. A real-time PCR assay to detect T. asahii DNA in sera for diagnosis of this fungal infection was developed. The assay showed a higher sensitivity than polysaccharide antigen detection method. Our new real-time PCR assay may be used for diagnosing deep-seated trichosporonosis due to T. asahii.     

两株耐碱酵母的pH耐受实验观察

对分离鉴定的两株酵母菌用比浊法和Bradford法进行pH耐受范围检测。Trichosporon asahiiXJU-1的pH耐受范围为2.0~13.0,最适pH值为8.0。Rhodotorula mucilaginosaXJU-1的pH耐受范围为3.0~12.0,最适pH值为8.5。比浊法在pH4.0~10.0测出数值比较可靠,而在pH<4.0和pH≥10.0产生了较大误差。在最适pH时,Bradford法间接测定两酵母的生长量,T.asahii总溶解蛋白为290μg/mL,Rh.mucilaginosa总溶解蛋白为164μg/mL。Bradford法在pH2.0~13.0范围均能较准确地反映出菌株生长状况,数据表明两株酵母有广阔的pH耐受性,它们是耐碱酵母菌的新成员。     

几种不同基质对阿萨希毛孢子菌形成生物膜影响的初步观察

目的观察不同基质对阿萨希毛孢子菌生物膜形成能力的影响。方法在聚芳脂、聚苯乙烯、聚氯乙烯上构建阿萨希毛孢子菌生物膜,在生物膜形成过程中采用XTT法对其活性进行定量分析,倒置显微镜和扫描电镜下观察不同基质上阿萨希毛孢子菌生物膜形态特征。结果 3种基质上均能形成阿萨希毛孢子菌生物膜,且形成广泛的的生物膜。比较成熟期不同基质上形成生物膜的活性有差别(F=14.743,P0.01),活性由高到低为聚芳脂=聚氯乙烯聚苯乙烯。倒置显微镜和扫描电镜下观察发现聚芳脂、聚氯乙烯形成的生物膜可见孢子、菌丝、假菌丝结构,聚苯乙烯上形成以孢子为主要结构的微生物群落。结论阿萨希毛孢子菌可在聚芳脂、聚苯乙烯、聚氯乙烯上形成生物膜,但形成生物膜的能力不同。聚芳脂、聚氯乙烯比聚苯乙烯更易于真菌的黏附;且以菌丝、假菌丝为主要结构的微生物群落活力比单纯孢子的活力强。     

A nested PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis

Deep-seated trichosporonosis caused by Trichosporon asahii has a high mortality rate and a very poor prognosis. New species-specific oligonucleotide primers for T. asahii were developed from a sequence analysis of rRNA genes that included the internal transcribed spacer regions. A nested PCR assay with specific primers was used to examine 11 serum samples from 7 patients, who were diagnosed with deep-seated trichosporonosis histologically at autopsy. In addition, Trichosporon cell wall polysaccharide (PS) was detected by a latex agglutination (LA) test. Of 11 samples, seven had a positive LA test, and T. asahii DNA was also detected with the nested PCR assay. Of the four samples in which PS antigen was not detected, the nested PCR of two samples was positive. Our new nested PCR assay may be used as an adjunct to conventional methods for diagnosing T. asahii infection.