Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications.

BACKGROUND: Group 1 introns (ribozymes) are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA) regions of pathogenic dematiaceous Phialophora verrucosa were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of P. verrucosa and seven strains of Phialophora americana as the allied species. RESULTS: Intron-F's (inserted at L798 position) were found in 88% of P. verrucosa strains, while intron-G's (inserted at L1921) at 12% and intron-H's (inserted at L2563) at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE. CONCLUSION: The three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of P. verrucosa and P. americana into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species.

Systemic nocardiosis caused by Nocardia concava in China.

A 42-year-old man with polychondritis and a 2-year history of using low-dose prednisone and other immunosuppressive drugs was admitted to our hospital due to persistent high fever of 10 days duration. A strain of Nocardia was twice isolated from his blood and subsequently identified to be N. concava. The patient was initially treated with sulphadiazine sodium, vancomycin and imipenema for 7 days but the symptoms persisted. Consequently, the regimen was changed to sulphadiazine sodium, ciprofloxacin and amikacin sulfate based on the antibiotic susceptibility tests of the Nocardia isolate. The fever disappeared and the patient's condition improved after 10 days of this treatment to the extent that he was discharged. However, 7 days later, the patient's condition deteriorated and he died due to multiple organ failure. This is the first report of N. concava causing systemic nocardiosis in China.

Novoamauromine and ent-Cycloechinulin: two new diketopiperazine derivatives from Aspergillus novofumigatus.

Two new diketopiperazine metabolites, novoamauromine (1) and ent-cycloechinulin (2) have been isolated from Aspergillus novofumigatus CBS117520. The structures of 1 and 2 were established on the basis of spectroscopic and chemical investigation, including a detailed comparison of the spectroscopic and physico-chemical data of amauromine (3) and cycloechinulin (4).

Identification of Trichosporon spp. strains by sequencing D1/D2 region and sub-typing by sequencing ribosomal intergenic spacer region of ribosomal DNA.

To re-identify and further group 25 isolates of Trichosporon spp. identified morphologically previously, sequences of D1/D2 region of large subunit (LSU) of ribosomal DNA (rDNA) of 25 tested strains for identification and those of ribosomal intergenic space 1 (IGS1) region of 11 strains for subgrouping were detected. The identifications of tested strains were changed except 6 strains. According to the alignment of the IGS1 region, 6 T. asahii isolates tested fell into 4 groups and 5 T. faecale isolates into 3 groups. Polymorphism of 2 T. japonicum isolates was found in 10 positions. With the alignments obtained in this research compared with the relative GenBank entries, it was found that T. asahii, T. faecale and T. japonicum species were divided into 7, 3 and 2 subtypes respectively. Morphological and biophysical methods are not sufficient for Trichosporon spp. identification. Sequencing becomes necessary for Trichosporon diagnosis. There is obvious diversity within a species.

Molecular diversity of Fonsecaea (Chaetothyriales) causing chromoblastomycosis in southern China.

Chromoblastomycosis is a chronic, cutaneous and subcutaneous infection caused by members of the order Chaetothyriales and commonly found in China. Among the etiologic agents, members of the genus Cladophialophora are predominant in northern China. Alternatively, Fonsecaea spp. are particularly common in southern China. However, the identification of Fonsecaea isolates recovered in China is difficult due to the fact that different species lack distinctive morphological characters. Therefore, the identification of 24 Fonsecaea isolates from symptomatic patients were re-evaluated by using morphology, ITS rDNA sequence diversity and partly through the use of randomly amplified polymorphic DNA (RAPD) typing. Twenty strains, including a morphological mutant were found to be Fonsecaea monophora, while four strains corresponded to F. pedrosoi. We have demonstrated that Fonsecaea monophora is the predominant etiologic agent of chromoblastomycosis in southern China and populations showed marked geographic structuring.

A new indoloditerpene derivative, penijanthine A, isolated from Penicillium janthinellum.

In a screen searching for new bioactive agents, a new indoloditerpene, penijanthine A (1), was isolated from Penicillium janthinellum IFM 55557. The structure of 1 was established on the basis of spectroscopic and chemical investigation, as well as detailed comparison with the spectroscopic and physico-chemical data of paxilline (2), which was isolated along with 1.

Identification of Trichosporon spp. Strains by Sequencing D1/D2 Region and Sub-typing by Sequencing Ribosomal Intergenic Spacer Region of Ribosomal DNA / 华中科技大学学报（医学）（英德文版）

To re-identify and further group 25 isolates of Trichosporon spp. identified morphologically previously, sequences of D1/D2 region of large subunit (LSU) of ribosomal DNA (rDNA) of 25 tested strains for identification and those of ribosomal intergenic space 1 (IGS1) region of 11 strains for sub-grouping were detected. The identifications of tested strains were changed except 6 strains. According to the alignment of the IGS1 region, 6 T. asahii isolates tested fell into 4 groups and 5 T. faecale isolates into 3 groups. Polymorphism of 2 T.japonicum isolates was found in 10 positions. With the alignments obtained in this research compared with the relative GenBank entries, it was found that T. asahii, T.faecale and T.japonicum species were divided into 7, 3 and 2 subtypes respectively. Morphological and biophysical methods are not sufficient for Trichosporon spp. identification. Sequencing becomes neces-sary for Trichosporon diagnosis. There is obvious diversity within a species.

Basidiomycosis: Schizophyllum commune osteomyelitis in a dog.

A six-year-old female Labrador retriever dog was suffering from osteomyelitis in her hindlimb. A puncture wound caused by a rotted bamboo stick was presumed as the source of infection. The dog suffered from pre-existing aortic stenosis, but otherwise exhibited no significant abnormality in her systemic conditions excluding claudication of the left hindlimb. The results of cytology and pathological examinations of biopsy samples revealed the diagnosis of mycotic osteomyelitis in this dog. Mycological and DNA tests showed the pathogen as the mushroom Schizophyllum commune. Antibiotic sensitivity testing also revealed susceptibility to itraconazole, which was used to successfully treat the dog. This is a rare case of canine basidiomycosis with S. commune as the etiologic agent.

First report of oral colonization by Debaryomyces nepalensis in a dog.

A stray, young male, wire-haired pointing griffon dog, found in a street of Perugia (Italy), was examined in order to check his health status. Two oropharyngeal swabs were collected in 24 h and streaked onto Sabouraud agar and after 6 days the yeasts colonies were transferred onto Malt agar. Ascospores were observed on Potato Dextrose Agar medium. The major ubiquinone of an isolated yeast was identified as ubiquinone-9 (Q-9), and genetical analyses were performed together with the type strains of Debaryomyces hansenii (var. hansenii and var. fabry), C. psychrophila and D. nepalensis type strain. The base sequences of ITS1 and ITS2, and D1/D2 domains of LSU rDNA completely coincided with those of D. nepalensis. From these results, the isolated yeast was identified as D. nepalensis. RAPD patterns between the two strains were found to be identical. The results indicate the first colonization of D. nepalensis in a dog.

A new antifungal macrolide, eushearilide, isolated from Eupenicillium shearii.

In screening for antifungal substances, a new macrolide, eushearilide (1), was isolated from Eupenicillium shearii IFM54447. The structure of 1 was established to be 24-membered macrolide having a non-conjugated diene and a choline phosphate ester moetiy on the basis of detailed investigation of NMR, UV, IR and MS spectral data. Compound 1 showed antifungal activity against various fungi and yeasts, including human pathogens Aspergillus fumigatus, Trichophyton spp. and Candida spp.

Two new meroterpenoids, penisimplicin A and B, isolated from Penicillium simplicissimum.

In the course of our research for new antifungal agents, two new meroterpenoids, penisimplicin A (1) and B (2), were isolated from Penicillium simplicissimum. The absolute structures of 1 and 2 were established by spectroscopic and chemical investigation. Penisimplicin A (1) and B (2) are rare examples of D-ring seco-meroterpenoids. Since compounds 1 and 2 showed no antifungal activities, the isolation of the compounds with antifungal activity will be attempted.

A new nonadride derivative, dihydroepiheveadride, as characteristic antifungal agent against filamentous fungi, isolated from unidentified fungus IFM 52672.

In the screening of searching for new antifungal agents, a new nonaride compound, dihydroepiheveadride (1), was isolated from unidentified fungus IFM 52672 as the most potent antifungal principle from this organism. The structure of 1 was established on the basis of spectroscopic and chemical investigation, as well as detailed comparison of the spectroscopic and physico-chemical data of the oxidized derivative (3) from 1 with those of heveadride (2). Compound 1 showed strong antifungal activity against various filamentous fungi including human pathogens Aspergillus fumigatus, Penicillium marneffei and Trichophyton spp. It also showed the growth inhibition activity against certain human pathogenic yeasts such as Trichosporon species, while it had weak or no antifungal activity against Candida spp. and Cryptococcus neoformans, and no antibacterial activity against Bacillus subtilis nor against Escherichia coli. The antifungal potencies of compounds 2 and 3 were found to be weaker than that of 1.

First case of Arthrographis kalrae ethmoid sinusitis and ophthalmitis in the People's Republic of China.

We present here the first case in the People's Republic of China of human disease caused by the fungus Arthrographis kalrae. The male patient had fungal panophthalmitis and invasive sinusitis involving the maxillary and ethmoid sinuses. He was an apparently healthy man before receiving trauma to his left eye. He complained of pain and loss of visual acuity in the injured eye, which displayed redness and edema and eventually discharged pus. His symptoms became more severe after he was treated with steroids and several antibacterial agents. A computed tomography scan of the left eye revealed that the maxillary and ethmoid sinuses were involved. A smear of purulent material from the left eye orbit revealed fungal elements, and cultures of the material grew a fungus. The isolate was identified as A. kalrae based on gross and microscopic morphologies, biochemical assays, and DNA sequence analysis. The patient received amphotericin B intravenously, itraconazole orally, and atomized allitridum by nebulizing allitridum therapy. The patient's wound healed following surgical intervention, but the patient lost the use of his left eye. This case should remind ophthalmologists and other clinicians to consider the possibility of infections being fungal when antibacterial agents have no effect and the patient's condition worsens.

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.

Specific oligonucleotide primers for identification of Cladophialophora carrionii, a causative agent of chromoblastomycosis.

Cladophialophora carrionii is one of the relatively common causative agents of chromoblastomycosis. We have developed the specific oligonucleotide primer set based on the internal transcribed spacer regions of ribosomal DNA for the rapid identification of this pathogen. PCR with this primer set amplified a 362-bp amplicon from C. carrionii strains. From other relevant dematiaceous species, including medically important dematiaceous fungi, such as Fonsecaea pedrosoi, Phialophora verrucosa, and Exophiala dermatitidis, and eight species of medically important yeasts, such as Candida albicans and Cryptococcus neoformans var. neoformans, the primer set did not produce any amplicon. PCR with this primer set may be a useful tool for the identification of C. carrionii.

Specific oligonucleotide primers for identification of Hortaea werneckii, a causative agent of tinea nigra.

Hortaea werneckii, a black yeast-like hyphomycete that is widely distributed in tropical and subtropical environments, can cause superficial mycotic infection in humans. This fungus was recently isolated from superficial infectious lesions of a guinea pig in Japan. An oligonucleotide primer set specific for Hortaea werneckii was designed on the basis of the internal transcribed spacer regions of the ribosomal DNA (rDNA). Polymerase chain reaction (PCR) with this primer set yielded a 306 bp PCR amplicon from only H. werneckii. This primer set did not amplify DNAs of 42 other related dematiaceous species, including the medically important dematiaceous fungi Cladophialophora carrionii, Fonsecaea pedrosoi, Phialophora verrucosa, and Exophiala dermatitidis, and eight species of medically important yeasts, including Candida (C.) albicans, C. dublinensis, C. glabrata, C. parapsilosis, C. tropicalis, Cryptococcus neoformans var. neoformans, Malassezia furfur, and Trichosporon asahii var. asahii. PCR with this primer set may be a useful technique for rapid identification of H. werneckii.

The epidemiology and mating behavior of Arthroderma benhamiae var. erinacei in household four-toed hedgehogs (Atelerix albiventris) in Japan.

An epidemiological survey of Trichophyton mentagrophytes var. erinacei in the household hedgehog and other rodents was made between January 17, 2002 and February 28, 2002 in Japan. Quills and hairs were collected from sources identified via the internet. The fungus was isolated only from the quills of four-toed hedgehogs (7/18; 39%) from Kanto to Kyushu regions. Isolates were examined morphologically, physiologically and genetically, and identified as T. mentagrophytes var. erinacei anamorph. The isolates were also genetically compared with European hedgehog (Erinaceus europeus)-borne T. mentagrophytes var. erinacei and Kenyan hedgehog (Aterelix albiventris)-borne Arthroderma benhamiae, and their genotypes of the ITS1-5.8S-ITS2 rDNA were all identical. The isolates were crossed with A. benhamiae Americano-European race and African race, A. vanbreuseghemii and A. simii, with the result that they mated only with African race (+) or (-). Mating types of the isolates were (+) in 6 isolates and (-) in one. An intra-isolate mating between one of the 6 plus isolates and the minus one formed abundant mature gymnothesia, the mating type ratio of the F1 progeny was approximately 1:1, and the sib crossings of F1 progeny produced abundant fertile gymnothesia. The present study revealed that the intra-Japanese hedgehog-borne isolate crossing showed complete fertility and that the sexual degeneration pointed out by Takashio (Mycologia 71: 968-976, 1979) did not exist. Two pairs of mating, (+) and (-) mating types of Japanese isolates with (-) and (+) tester strains of A. benhamiae African race formed less gymnothesia, mating type ratios were unbalanced, and sib crossings of F1 progeny produced small gymnothesia containing a low number of asci, pseudogymothesia, or none, respectively. These results show that A. benhamiae var. erinacei, the teleomorph of T. mentagrophytes var. erinacei, belongs to a different mating group (e.g. hedgehog race) than the Americano-European and African races in A. benhamiae.

Rapid identification of the genus fonsecaea by PCR with specific oligonucleotide primers.

An oligonucleotide primer set based on internal transcribed spacer regions of ribosomal DNA for PCR which gives the amplicon for only the DNA from Fonsecaea species was designed. This set yielded an amplicon with 333 bp for all strains of Fonsecaea pedrosoi and Fonsecaea compacta examined but no amplicons for related dematiaceous fungi and pathogenic yeasts. PCR using this primer set was considered to be a useful method for the rapid identification of the genus FONSECAEA:

A DNA Genotyping Study of Candida Isolates from Mothers and Their Neonates / 中华皮肤科杂志

Objectives To identify the route and time of transmission by Candida species from mothers' vagina to their neonates' mouth.Methods Specimens for fungal cultures were obtained from vaginal discharge of mothers just before delivery and also from the mouth of their offspring just after birth.Eleven mother-infant pairs were investigated.Candida species was identified based on morphology,biochemical analysis,and sequencing of D1/D2 domain of the large subunit of ribosomal DNA (LSUrDNA).Electrophoretic karyotyping (EK) and random amplified polymorphic DNA analysis (RAPD) were performed to search for DNA homology.Results Candida isolates (16 strains) from 8 mother-infant pairs were identified as Candida albicans by 100% homology of their D1/D2 sequences with reference strain C.albicans Y-12983 (GenBank access No.U45776).Similarly,4 strains from two mother-infant pairs and 2 isolates from the other pair were identified as Candida glabrata and Candida krusei,respectively,by 100% homology in sequences alignment of the domains with reference strains,C.glabrata Y-65(U44808) and C.krusei Y-5396 (U76347).The same EK profiles were found for each C.albicans or C.krusei strain pair from both mother and her neonate.Although different EK bands with various molecular size were generated for each C.glabrata isolate pair,they were still considered to be homologous based on the fact that main EK bands were identical.Each isolate pair from mother and her infant presented almost the same RAPD profile,except for one pair,isolates F7n and F7m,which showed minor diverse DNA bands.Conclusion Eleven Candida isolates from neonates have identical molecular characteristics with their mother's isolates.Vertical transmission may be the main pathway of Candida spp.from mothers to their neonates.