临床相关毛孢子菌的鉴定及体外药物敏感性研究

目的探讨临床相关毛孢子菌的鉴定方法及对常见抗真菌药物的体外敏感性。方法对48株临床分离的毛孢子菌分别通过形态学、API20C AUX、Vitek 2 Compact及核糖体rDNA ITS序列分析等方法鉴定到种;采用浓度梯度法（E-test）测定氟康唑、伏立康唑、伊曲康唑、两性霉素B及卡泊芬净对48株毛孢子菌的最低抑菌浓度。结果形态学和API20C AUX、Vitek 2 Compact不能准确区分不同种的毛孢子菌,以核糖体rDNA ITS序列分析将48株毛孢子菌鉴定为8个种：阿萨希毛孢子菌,星型毛孢子菌,皮瘤毛孢子菌,真皮毛孢子菌,皮肤毛孢子菌,赖巴克毛孢子菌,T.domesticum,T.jirovecii。体外药敏结果显示：卡泊芬净对毛孢子菌无体外活性,MIC〉32μg/mL;氟康唑和两性霉素B对毛孢子菌活性差,体外活性最好的药物是伏立康唑和伊曲康唑。结论常规方法不易将毛孢子菌准确鉴定到种的水平,ITS序列分析准确快速,可以辅助临床区分难鉴定毛孢子菌。毛孢子菌药敏谱不同于临床常见其他酵母菌,氟康唑和两性霉素B对其活性差,伏立康唑具有良好的体外抗菌活性。     

三个发表于国内的丝孢酵母种名分类地位的订正*

最新的酵母菌分类系统,丝孢酵母属Trichosporon Behrend仅限于产生节孢子的担子菌酵母的无性型。因此,以前根据该属旧的定义在国内发表的三个新种板仓丝孢酵母T. bancangense、北京丝孢酵母T. beijingense和中国丝孢酵母T. sinense的分类地位,需要进行调整。根据26S rDNA D1/D2区的序列分析和最新标准方法重新测定的生理生化反应,发现上述三个种名分别是子囊菌酵母Saccharomycopsis fibuligera, Pichia burtonii和Issatchenkia orientalis的异名。     

Update on the Genus <Emphasis Type="Italic">Trichosporon</Emphasis>

Trichosporon spp. are widely distributed in nature and can occasionally belong to the human microbiota. For many years, the unique species of the genus, Trichosporon beigelli, was only known as an environmental and saprophytic fungus occasionally found as the etiological agent of white piedra. However, case reports of invasive trichosporonosis have been frequently published and the genus is currently considered the second most common agent of yeasts disseminated infections. Based on molecular analysis, the taxon T. beigelli was replaced by several species and the taxonomy of the genus was progressively modified. Despite the reported increase of Trichosporon infections refractory to conventional antifungal drugs, there are only a few studies investigating in vitro susceptibility of Trichosporon spp. to new compounds. Difficulties on different species identification as well as the lack of standardized sensitivity tests in vitro, contribute to the limited information available on epidemiology, diagnosis and therapeutics of trichosporonosis.     

载有磷酸甘油激酶基因启动子的新表达载体的构建以及在发酵性丝孢酵母中启动外源基因的表达研究

利用简并PCR技术从一株丝孢酵母(Trichosporon sp.)中克隆到磷酸甘油激酶基因的部分序列,然后利用染色体步移的方法克隆到了已知片段的上游序列约950bp。通过启动子序列分析软件分析,发现序列中含有启动子所需的必须元件如TATA BOX和CAAT BOX等,因此确定克隆到的基因片段含有启动子序列。将潮霉素基因置于该启动子下构建了丝孢酵母整合型表达载体pTFPH,并转化发酵性丝孢酵母(Trichosporon fermentans),转化后的酵母能够在含有潮霉素的抗性选择性平板长出,而未进行转化的对照菌株则不能生长。以上试验证明:丝孢酵母的磷酸甘油激酶基因启动子具有启动异源基因在发酵性丝孢酵母中表达的功能,这个结果为油脂酵母工程菌的构建和开发新的酵母表达宿主奠定了基础。     

Trichosporon species isolated from guano samples obtained from bat-inhabited caves in Japan

Yeasts from caves have rarely been examined. We examined yeasts collected from bat guano samples from 20 bat-inhabited limestone and volcanic caves located in 11 prefectures in Japan. Of approximately 700 yeast-like colonies, nine Trichosporon species were recovered from 15 caves. Two of these were known species, and the remaining seven are potentially novel species, based on molecular phylogenetic analyses. In addition to Trichosporon species, identifiable strains of eight ascomycetous yeasts and one basidiomycetous yeast were recovered at frequencies of 5 to 35%. Our findings suggest that Trichosporon spp. are the major yeast species in bat guano in Japan and that bat guano is a potentially rich source of previously undescribed yeast species.     

Characterization of oleaginous yeasts revealed two novel species: Trichosporon cacaoliposimilis sp. nov. and Trichosporon oleaginosus sp. nov

Two new species in the anamorphic basidiomycetous genus Trichosporon (Tremellomycetes, Agaricomycotina) were uncovered in a DNA sequence-based molecular analysis of oleaginous yeasts maintained in the ATCC Mycology Collection. One yeast is named as Trichosporon cacaoliposimilis sp. nov. for its capability of synthesizing and accumulating a large amount of lipids having a composition equivalent to that of natural cacao butter. The type strain is ATCC 20505(T), originally deposited as Trichosporon sp. The other can use food industry wastes and agricultural byproducts as the substrate for growth and accumulation of a high level of oil and accordingly is named Trichosporon oleaginosus sp. nov. The type strain is ATCC 20509(T), previously identified as Cryptococcus curvatus. Molecular phylogenetic analyses indicate that T. cacaoliposimilis is a novel taxon in the Gracile clade of the genus, close to T. gracile and T. dulcitum, and that T. oleaginosus belongs to the Cutaneum clade, with T. jirovecii as the closest sister taxon. Other oleaginous yeasts were identified as new strains of known taxa, T. insectorum, Candida orthopsilosis and C. palmioleophila.     

Trichosporon porosum comb. nov., an anamorphic basidiomycetous yeast inhabiting soil,related to the loubieri/laibachii group of species that assimilate hemicelluloses and phenolic compounds

Several isolates representing the genus Trichosporon were collected over a 6-year period from soils in The Netherlands. Based on classical growth tests with carbon and nitrogen compounds these were identical. Three of these (CBS 8396, CBS 8397 and CBS 8522) were subjected to molecular analysis of the D1/D2 region of the large subunit of rDNA. This confirmed that the three strains were identical, yet distinct from other members of the genus. Conspecificity was demonstrated with the type strain (CBS 2040) of Apiotrichum porosum Stautz (1931), with the exception that A. porosum, which had been isolated from exudate of a yew tree, differed morphologically from the soil strains. Based on the identity of DNA base sequences, morphology was not considered to be an adequate parameter to separate otherwise identical strains into two genera. Therefore, the new combination Trichosporon porosum is presented. Based on molecular sequence analysis, T. porosum may be related to T. sporotrichoides, within a weakly related clade that includes species such as Trichosporon laibachii and Trichosporon loubieri. The strains of T. porosum degrade phenolic compounds and hemicelluloses, which are characteristics with potential ecological importance in soil habitats. Characters distinguishing the nine species of the laibachii/loubieri group of species were listed. These include traditionally used tests as well as assimilation patterns of some aliphatic and phenolic compounds. Based on these tests, species such as Trichosporon multisporum and T. laibachii could be separated.     

阿萨希毛孢子菌的溶血活性及生物膜形成能力与其基因型关系研究

目的 分析临床分离自尿路感染患者的阿萨希毛孢子菌的体外溶血活性及生物膜形成能力与其基因型的关系,为临床诊治提供依据.方法 玻璃珠法提取总DNA,并采用PCR技术利用IGS1区特异性引物确定其基因型别;同时,平板法和XTT还原比色法分别检测阿萨希毛孢子菌的溶血活性及生物膜形成能力,并分析其与基因型的关系.结果 10株分离自尿路感染的阿萨希毛孢子菌基因型分别属于Ⅰ、Ⅲ、Ⅳ,其中以Ⅳ型为主;所分离菌株均具有不同程度的溶血活性;除1例分离株外,其余各分离株均具有在聚苯乙烯表面形成生物膜的能力;基因型Ⅲ型菌株具有较强的溶血活性和生物膜形成能力.结论 分离自尿路感染患者的10株阿萨希毛孢子菌以Ⅳ型为主,而其中Ⅲ型菌株表现出较强的溶血活性和生物膜形成能力.     

Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species.     

Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli.

A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.     

Phenol utilization by filamentous yeast Trichosporon cutaneum

The investigated strain Trichosporon cutaneum shows well expressed capability for metabolizing high concentrations of phenol, up to 1 g/l, utilizing it as the sole carbon source for the growth and development of the population. The data reported, prove the good perspectives for its application in protecting the environment from phenol pollution. No data about modelling the process of cultivation of Trichosporon cutaneum in phenol media is available in scientific literature up to now. The mathematical model, reported here, consists of two nonlinear differential equations, describing cell growth and substrate consumption. The unknown parameters are estimated following the method of Hooke and Jeeves. A number of simulation investigations are carried out. They prove the adequacy of the model and its applicability in further studies on the processes of growth and phenol uptake of Trichosporon cutaneum.     

Biotransformation of furfural by yeast cells covalently bound to cellulose granules

A covalent binding to cellulose granules of two yeast strains Candida tropicalis and Trichosporon cutaneum was achieved. The maximum activity for destroying furfural by the immobilized cells was obtained when the procedure conditions were: reaction medium at pH 5.0, 20°C and cell suspension concentration of 80 mg/ml. The continuous furfural transformation was studied using a growth medium in a fermenter with immobilized Trichosporon cutaneum in which a 84% bioconversion was achieved. The reduced values of furfural remained constant even after 10-fold transformation.     

Yeast flora of plant rhizosphere and phyllosphere]

The species belonging to the genera Cryptococcus and Hansenula with saturnian spores predominate in the rhizosphere of agricultural plants. The sporiferous strains of Debaryomyces, Hanseniaspora apiculata, Metschnikowia pulcherrima, and asporogenic Candida krusei and Trichosporon cutaneum prevail in the rhizosphere of wild plants. Candida krusei and Trichosporon cutaneum prevail in the rhizosphere of wild plants. The cultures of Rhodotorula, Candida krusei and Metschnikowia pulcherrima are typical of the phyllosphere of both cultural and wild plants. The phyllosphere of cultural plants contains also the asporogenic strains of Cryptococcus, Candida tropicalis, Trichosporon pullulans, Tr. cutaneum, and Hansenula, while Hanseniaspora apiculata and Saccharomyces cerevisiae predominate in the phyllosphere of wild plants. The yeast flora of the majority of studied plants is diverse and comprises 10--20 species (in cabbage, potato, linden, aspen, and pear trees). The rhizophere and phyllosphere of some plants contain only 2 to 4 yeast species (onion, hop, wild apple).     

Genome profiling (GP) as an effective tool for monitoring culture collections: a case study with Trichosporon

Species identification and classification of a large number of microbes are essential and heavy workloads in culture collections and relevant laboratories. The identification of species usually requires different methods depending on species. Therefore, the development of a method which is simple and applicable to any organisms will lessen the burdens, increase the reliability of databases and thus enhance the science on microbes. The genome profiling (GP) method, developed previously, was found effective in monitoring authenticities of all strains/species tested in culture collections and expectedly various species, which was shown by applying the GP and the conventional sequencing methods to identifying and classifying species/strains belonging to the genus Trichosporon (38 strains; 16 species). Small differences between strains (11 strains of Trichosporon asahii and 4 strains of Trichosporon coremiiforme) can be reliably discriminated by GP, which was unsuccessful in the conventional sequencing approach. Importantly, seven possible false-assignments contained in the database were all pointed out by the GP method with near-perfect correctness, showing the power of the GP method.GP was shown to be a potent tool for rapidly and correctly monitoring species and strains of fungi in culture collections owing to its simple and informative natures.     

Induction and glucose repression of endo-β-xylanase in the yeast Trichosporon cutaneum SL409

Extracellular endo-1,4-β-xylanase (EC 3.2.1.8) synthesis in the yeast Trichosporon cutaneum SL409 is inducible. The enzyme can be induced in washed glucose-grown cells by xylan or xylose. Methyl β-d-xylopyranoside, a synthetic analogue of xylobiose, however, was not an inducer of xylanase in Trichosporon cutaneum SL409. The induction of xylanase by xylan lasted longer and the final total activities were significantly higher compared to the induction by xylose. Xylanase induction was subject to glucose repression.     

内蒙古西部区酸粥中酵母菌的分离鉴定及优势菌分析

从内蒙古地区采集28份酸粥样品,从中分离出40株酵母菌,并对其进行了分子生物学鉴定和生物多样性分析。26S rDNA D1/D2区域 (600bp左右)碱基序列分析结果表明,酸粥中的酵母菌有Issatchenkia orientalis、Saccharomyces cerevisiae、Geotrichum sp.、Candida pararugosa、 Candida parapsilosis、Trichosporon asahii、Trichosporon coremiiforme、Clavispora lusitaniae和Candida tropicalis。经过分析,Issatchenkia orientalis(75%,Frequency percentage)为酸粥中的优势菌。     

Two new yeasts:Trichosporon aquatile andTrichosporon eriense spp. n.

Trichosporon aquatile sp. n. is described and compared withT. cutaneum andT. inkin, Trichosporon eriense sp. n. is described and compared withT. capitatum andT. penicillatum. A key is presented for all the species in the genusTrichosporon.     

Isolation of proteolytic psychrotrophic yeasts from fresh raw seafoods

A total of 103 cultures of yeasts were isolated from seven kinds of fresh raw seafoods. The isolates comprised six genera, Candida, Cryptococcus, Debaryomyces, Rhodotorula, Sterigmatomyces and Trichosporon , and included 21 different species. All the isolates were psychrotrophic yeasts. Proteolytic activities of 50 psychrotrophic strains were studied by use of skim milk within the temperature range of 0–42°C. All the strains showed various degrees of proteolysis. In particular, Candida lipolytica. Trichosporon pullulans and Candida scottii were active species at low temperatures. Sensory spoilage due to the proteolytic yeasts were observed in mackerel homogenates stored at 10°C. C. lipolytica -inoculated homogenates caused spoilage with ammoniacal odours after 1 week of storage. Values of total volatile basic nitrogen at 10°C were highest with C. lipolytica among 35 strains tested, followed by Tr. pullans. Trichosporon cutaneum, C. scottii, Rhodotorula glutinis and Cryptococcus luteolus. Proteolytic psychrotrophic yeasts were widely distributed in raw seafoods.