Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis

The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.     

临床皮炎外瓶霉荧光定量PCR检测方法的建立

目的 建立基于TaqMan探针技术的皮炎外瓶霉荧光定量PCR检测方法.方法 通过对皮炎外瓶霉ITS区域基因组序列（GenBank：JN675373.1）进行分析,设计合成特异性引物和荧光标记探针,优化荧光定量PCR反应条件.以临床标本中分离的皮炎外瓶霉为阳性菌株,及其他种类真菌和细菌作为阴性对照菌株,从特异性、敏感性及重复性方面对该方法检测效果进行评价.结果 该研究设计的引物和探针能扩增皮炎外瓶霉特异性序列.临床分离得到的皮炎外瓶霉在反应中有明显扩增曲线,而甄氏外瓶霉、棘状外瓶霉、烟曲霉、白色念珠菌、新生隐球菌、马内菲青霉等20株菌在CT值≤38范围内均未有扩增;利用基因重组构建的标准品完成了标准曲线的绘制,在1.0×103～1.0×107拷贝数（Cp）内具有良好的线性关系（R2=1.000）,最低可检出量为10 Cp/μL.结论 成功建立了荧光定量PCR检测皮炎外瓶霉方法,该法特异度强、敏感度高、重复性好,将有助于临床皮炎外瓶霉感染的早期诊断和针对性治疗.     

Isolation of Exophiala jeanselmei from bat guano

Exophiala jeanselmei was isolated from two out of 75 samples of bat guano. The isolates hydrolysed urea and tyrosine, assimilated sodium nitrate and produced a zone of haemolysis on blood agar, They were pathogenic for laboratory mice when injected subcutaneously, intraperitoneally or intravenously.     

Studies on the phylogenesis of pathogenic ‘black yeasts’

This paper deals mainly with the conidium ontogenesis and phylogenesis of black yeasts such as E. jeanselmei, E. gougerotii, E. dermatitidis and E. spinifera. The conidium ontogenesis of E. jeanselmei, E. gougerotii and E. dermatitidis was almost the same. One to five annellated tips were observed through a scanning microscope at the apices of conidiogenous cells, which were bottle- or jar-shaped. Annellations on the tips looked like fringes and the conidiogenous cells of these three species were annellides. Annellated projections occurred on hyphae and annelloconidia were also produced from them. Occasionally, secondary annellides occurred from primary ones. They looked like moniliform hyphae. Daughter conidia sometimes budded directly from mother cells. The shapes and sizes of the conidia of these species were very similar to each other. The conidium ontogenesis of E. spinifera was annellidic as well. However, a single annellated tip usually occurred on an annellide. The annellated tips of the fungus were long and more than 20 annellations were observed on their walls. The conidiogenesis of the four species of Exophiala is only annellidic. There were no differences in the biological examinations except KNO3 assimilation among these four species. The growth of E. jeanselmei and E. gougerotii was poor at 37 degrees C. The GC contents of E. jeanselmei 1171, E. gougerotii B-1800, E. dermatitidis MM-7 and E. spinifera DU-3342 were 54.6, 54.6, 56.6 and 59.7%, respectively.     

Steroid transformations with Exophiala jeanselmei var. lecanii-corni and Ceratocystis paradoxa

The fungi Exophiala jeanselmei var. lecanii-corni [IMI (International Mycological Institute) 312989, UAMH (University of Alberta Microfungus Collection and Herbarium) 8783] and Ceratocystis paradoxa (IMI 374529, UAMH 8784) have been examined for their potential in steroid biotransformation. The study has determined that E. jeanselmei var. lecanii-corni effected overall anti-Markovnikov hydration on dehydroisoandrosterone, and side-chain degradation on a variety of pregnanes. Both ascomycetes were found to carry out redox reactions of alcohols and ketones as well as 1,4 reduction of alpha,beta-unsaturated carbonyl systems.     

全球皮炎外瓶霉感染流行现状的回顾性分析

目的分析皮炎外瓶霉感染的流行病学和临床特征,为提高皮炎外瓶霉感染的诊治水平提供科学依据。方法采用文献回顾和荟萃分析的方法,分析全球范围内已报道的皮炎外瓶霉感染病例的国籍、性别、年龄分布、危险因素、发病部位、临床表现、诊疗方法及预后等流行病学和临床特征。结果皮炎外瓶霉感染在免疫功能正常和免疫功能缺陷患者中均可发生,患者的男女性别比为1.10∶1.00,最常发病年龄段为51~60岁,肺(27.90%,17/61)为最常受累的器官,但不同地域的病例感染器官存在差异。约半数(47.54%,29/61)病例伴有各种免疫抑制的基础疾病或危险因素。皮炎外瓶霉感染的临床确诊主要依赖培养和分子鉴定,系统感染患者推荐联合伊曲康唑和特比萘芬作为抗真菌治疗方案。结论近年来皮炎外瓶霉感染的发病率在全球范围内呈上升趋势,肺部为系统感染患者最常受累的器官,诊断主要依赖于真菌培养。加强皮炎外瓶霉菌株的药敏监测和分子流行病学研究,对于提高皮炎外瓶霉感染的临床诊治水平非常必要。     

棘状外瓶霉核糖体基因的初步研究

外瓶霉可致人类感染 ,不同生物群落的菌种 ,其致病性、药敏性等特征具有差异性。通过对 1 0株棘状外瓶霉核糖体基因及其转录间隔区进行序列测定 ,并与GeneBank中 9株同种真菌对比分析 ,揭示了不同生物群落的棘状外瓶霉虽然形态学差异性小 ,但在基因学上具有差异性 ;原属于甄氏外瓶霉变种的BMU 0 0 0 45 7(ATCC 2 41 5 2E .jeanselmeivar.hetoromorpha)与 2株棘状外瓶霉具有 1 0 0 %的同源性。研究提示了形态学特征相似的棘状外瓶霉在基因水平上具有差异性 ,核糖体基因及其转录间隔区对于研究菌群特性具有一定意义。     

Purification and characterization of cyclohexanone 1,2-monooxygenase from Exophiala jeanselmei strain KUFI-6N

Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.     

致病性暗色霉的浆膜超微结构分类学意义初探

对致病性暗色霉中的着色霉(Fonsecaea Negroni)外瓶霉(Exophiala Charmichael)瓶霉(Phialophoro Medlar)中的五种真菌浆膜超微结构进行了冰冻蚀刻研究,发现裴氏着色霉(Fonsecaea pedrosoi)和紧密着色霉(F.compacta)的内折长而宽,较深,略有弯曲,数量少,多呈平行或垂直排列。皮炎外瓶霉(Exophiala dermatitidis)的内折数量多,密集而分布均匀,呈圆点状或圆棒状。棘状外瓶霉(Exophiala spinifera)的内折少而表浅,多为圆形。疣状瓶霉(Phialophora verrucosa)的内折数量,排列,形状无一定规律。据上述特征,着色霉可以与外瓶霉,疣状瓶霉区别开来,皮炎外瓶霉也可与棘状外瓶霉区分。浆膜超微结构的性状有一定的分类学意义。     

Taxonomy of Exophiala werneckii and its relationship to Microsporum mansonii

Study of the neotype culture of Exophiala werneckii (Cladosporium werneckii), as well as additional isolates, has shown that the yeast-like and hyphal conidiogenous cells are annellides. E. werneckii cannot be accommodated in Cladosporium, a genus characterized by catenulate holoblastic coanidia. E. werneckii and the dematiaceous hyphomycete that Castellani misidentified as Microsporum mansonii (Cladosporium mansonii) are two entirely different fungi. M. mansonii is considered to be a synonym of the Malassezia furfur since both names were used to describe in tissue the etiologic agent of pityriasis versicolor.     

Application of a colorimetric DNA-DNA hybridization method for identification ofAeromonas genomic species,isolated from diarrheal stools

The colorimetric DNA-DNA hybridization method for the identification of 18 strains ofAeromonas spp. isolated from human stools was used. Bacterial isolates were also examined by phenotypic characteristics. On the basis of biochemical tests 13 strains were included in phenogroupA. caviœ and 5 strains inA. sobria. Identification to the species level was obtained by colorimetric hybridization method. DNA-DNA similarity values showed that isolates ofA. caviœ group belong to hybridization group (HG) 4 whereas isolates ofA. sobria belong to HG 8/10. DNA relatedness results obtained by the colorimetric method showed good agreement with values detected by the spectrophotometric method. The background in the colorimetric method is lower than in the spectrophotometric one. Results of this study indicate the usefulness of the colorimetric DNA-DNA hybridization in microplates method for the identification ofAeromonas genomic species, isolated from human diarrheal stools.     

Relationship between genome similarity and DNA-DNA hybridization among closely related bacteria

DNA-DNA hybridization has been established as an important technology in bacterial species taxonomy and phylogenetic analysis. In this study, we analyzed how the efficiency with which the genomic DNA from one species hybridizes to the genomic DNA of another species (DNA-DNA hybridization) in microarray analysis relates to the similarity between two genomes. We found that the predicted DNA-DNA hybridization based on genome sequence similarity correlated well with the experimentally determined microarray hybridization. Between closely related strains, significant numbers of highly divergent genes (<55% identity) and/or the accumulation of mismatches between conserved genes lowered the DNA-DNA hybridization signal, and this reduced the hybridization signals to below 70% for even bacterial strains with over 97% 16S rRNA gene identity. In addition, our results also suggest that a DNA-DNA hybridization signal intensity of over 40% indicates that two genomes at least shared 30% conserved genes (>60% gene identity). This study may expand our knowledge of DNA-DNA hybridization based on genomic sequence similarity comparison and further provide insights for bacterial phylogeny analyses.     

In Vitro Susceptibility of Environmental Isolates of Exophiala dermatitidis to Five Antifungal Drugs

Several dematiaceous fungi frequently isolated from nature are involved in cases of superficial lesions to lethal cerebral infections. Antifungal susceptibility data on environmental and clinical isolates are still sparse despite the advances in testing methods. The objective of this study was to examine the activities of 5-flucytosine, amphotericin B, itraconazole, voriconazole and terbinafine against environmental isolates of Exophiala strains by minimum inhibition concentration (MIC) determination. The strains were obtained from hydrocarbon-contaminated soil, ant cuticle and fungal pellets from the infrabuccal pocket of attine gynes. Broth microdilution assay using M38-A2 reference methodology for the five antifungal drugs and DNA sequencing for fungal identification were applied. Terbinafine was the most active drug against the tested strains. It was observed that amphotericin B was less effective, notably against Exophiala spinifera, also studied. High MICs of 5-flucytosine against Exophiala dermatitidis occurred. This finding highlights the relevance of studies on the antifungal resistance of these potential opportunistic species. Our results also contribute to a future improvement of the standard methods to access the drug efficacy currently applied to black fungi.     

Phylogenetic analysis of ten black yeast species using nuclear small subunit rRNA gene sequences

The nuclear small subunit rRNA genes of authentic strains of the black yeastsExophiala dermatitidis, Wangiella dermatitidis, Sarcinomyces phaeomuriformis, Capronia mansonii, Nadsoniella nigra var.hesuelica, Phaeoannellomyces elegans, Phaeococcomyces exophialae, Exophiala jeanselmei var.jeanselmei andE. castellanii were amplified by PCR and directly sequenced. A putative secondary structure of the nuclear small subunit rRNA ofExophiala dermatitidis was predicted from the sequence data. Alignment with corresponding sequences fromNeurospora crassa andAureobasidium pullulans was performed and a phylogenetic tree was constructed using the neighbor-joining method. The obtained topology of the tree was confirmed by bootstrap analysis. Based upon this analysis all fungi studied formed a well-supported monophyletic group clustering as a sister group to one group of the Plectomycetes (Trichocomaceae and Onygenales). The analysis confirmed the close relationship postulated betweenExophiala dermatitidis, Wangiella dermatitidis andSarcinomyces phaeomuriformis. This monophyletic clade also contains the teleomorph speciesCapronia mansonii thus confirming the concept of a teleomorph connection of the genusExophiala to a member of the Herpotrichiellaceae. However,Exophiala castellanii did not belong to this clade. Therefore, this species is not the anamorph ofCapronia mansonii as it was postulated.     

Use of ribosomal introns as new markers of genetic diversity in Exophiala dermatitidis

Exophiala dermatitidis is one of the prevalent black yeasts found as opportunistic pathogens or colonizers in humans. In the tropics its natural habitat is thought to be fruit surfaces and it is also found in the digestive system of fruit-eating animals. However, it has recently been abundantly isolated from human-made environments (steam baths, railway ties, dishwashers) in tropical and temperate climates. Two genotypes have been distinguished within this species: genotype A, mostly corresponding to strains isolated from patients, and genotype B, to strains isolated from the natural environment. In human-made environments, both genotypes A and B occur. A previous study suggested that one genotype had been selected for in the human host. In our study, the distribution of ribosomal insertions agrees with an ecological specialization of E. dermatitidis genotypes by showing a significantly higher frequency of ribosomal insertions in clinical strains in comparison to environmental ones. The characterization of these insertions shows that they correspond to introns of group IC or IE, the most frequent types within the fungal kingdom. These ribosomal group I introns could be used as new markers for populations of E. dermatitidis.     

Exoantigen tests for the immunoidentification of fungal cultures

Exoantigen tests for the immunoidentification of fungal pathogens are playing a new and significant role in the diagnostic laboratory. Properly performed and controlled exoantigen tests lead to rapid, accurate identification of cultures of many fungal pathogens. The tests are particularly valuable in identifying dimorphic pathogens that are difficult to convert or with atypical cultures. We review the value of exoantigen tests for identifying mycelial form fungi: Aspergillus spp. Blastomyces dermatitidis, Coccidioides immitis, Exophiala jeanselmei, Histoplasma spp., Paracoccidioides brasiliensis, Penicillium marneffei, Pseudallescheria boydii, Sporothrix schenckii, Wangiella dermatitidis and certain dermatophytes. We discuss procedures for performing the tests and sources of error.     

Taxonomy and morphology of dematiaceous fungi isolated from nature

The Hughes system of classification was applied to the identification of dematiaceous fungi isolated from nature. The majority of the isolates encountered could be identified with this system using brightfield microscopy. However, phase contrast microscopy was needed to determine the mode of conidial ontogeny with some species of Phialophora, Exophiala and Wangiella. The identifications based upon phase contrast microscopy were confirmed using the scanning electron microscope (SEM). Scanning electron photomicrographs are presented for Bispora betulina, Cladosporium trichoides, Exophiala jeanselmei, Phialophora revens, P. verrucosa and Wangiella dermatitidis. SEM observations of W. dermatitidis support the present classification of this organism as proposed by other workers. It is suggested that the Hughes system of classification and phase contrast microscopy be used to facilitate identification of isolates of dematiaceous fungi encountered in the clinical laboratory.     

Coccidioidomycosis and blastomycosis: advances in molecular diagnosis

Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.     

Isolation and Identification of Black Yeasts by Enrichment on Atmospheres of Monoaromatic Hydrocarbons

Black yeast members of the Herpotrichiellaceae present a complex ecological behavior: They are often isolated from rather extreme environments polluted with aromatic hydrocarbons, while they are also regularly involved in human opportunistic infections. A selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology. Samples from natural ecological niches and man-made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons. Benzene, toluene, and xylene were provided separately as the sole carbon and energy source via the gas phase. The assayed isolation protocol was highly specific toward mesophilic Exophiala species (70 strains of this genus out of 71 isolates). Those were obtained predominantly from creosote-treated railway ties (53 strains), but isolates were also found on wild berries (11 strains) and in guano-rich soil samples (six strains). Most of the isolates were obtained on toluene (43 strains), but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi (17 and 10 isolates, respectively). Based upon morphological characterizations and DNA sequences of the full internal transcriber spacers (ITS) and the 8.5S rRNA genes, the majority of the obtained isolates were affiliated to the recently described species Exophiala xenobiotica (32 strains) and Exophiala bergeri (nine strains). Members of two other phylogenetic groups (24 and two strains, respectively) somewhat related to E. bergeri were also found, and a last group (three strains) corresponded to an undescribed Exophiala species.