矢口 貴志

侵襲性コニディオボルス症は致死率の高い新興真菌症として注目されている.ホルマリン固定パラフィン(FFPE)組織切片上でコニディオボルス症起因菌(Conidiobolus sp.)をムーコル症起因菌(Rhizopus microsporus)およびアスペルギルス症起因菌(Aspergillus fumigatus)から判別する手法の確立を目的とし,各症例のFFPE組織切片を用いて18S rRNAを標的としたin situ hybridization(ISH)法について検討した.18S rRNAの中でも特に変異に富んだV4領域の可変領域に着目し,Conidiobolus sp.,R.microsporusおよびA.fumigatusのそれぞれの培養株についてコンセンサスプライマーを用いて相互に塩基数および配列位置の異なる3つずつのアンチセンスプローブ(プローブA,BおよびC)を作製し,ISHを行った.その結果,相互に相同性が60%以下となるそれぞれのプローブC(Conidiobolus sp.189b,R.microsporus 155bおよびA.fumigatus 154b)を用い,ストリンジェンシーの条件を最適化することによりコニディオボルス症起因菌(Conidiobolus sp.),ムーコル症起因菌(R.microsporus)およびアスペルギルス症起因菌(A.fumigatus)を高感度に判別することができた.これらの起因菌をFFPE組織切片上で迅速かつ確実に検出,同定することが可能となり,真菌症治療戦略に大きく寄与することが期待される.(著者抄録)

A 73-year-old healthy woman noticed black pigmentation on both thumbnails for 6 years. Upon her visit to our clinic, she had pigmented onychomycosis with onycholysis in the distal area. There was no evidence of paronychia. Direct microscopy using Zoomblue™ and histopathological examination showed aggregated blastoconidia. Fontana-Masson staining confirmed fungal melanin production. A combination of morphological features and genetic testing identified the isolates as Candida parapsilosis. Fungal melanonychia due to C. parapsilosis is rare, with only six cases reported since 1979. The minimum inhibitory concentration of the isolates was 0.25 μg/mL for itraconazole, less than 0.03 μg/mL for ravuconazole and 2.0 μg/mL for terbinafine. Both oral terbinafine treatment and itraconazole pulse therapy performed for 6 months were unsuccessful. The disease was ultimately cured with a 3-month treatment of oral fosravuconazole.

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© 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases Here, we present a case of disseminated nocardiosis, involving pneumonia, percutaneous abscess, and bacteremia, in a 67-year-old Japanese woman. She had also been treated for rheumatoid arthritis with prednisolone, methotrexate, and tocilizumab (interleukin-6 receptor inhibitor). Based on the 16S rRNA sequence analysis and a blast search, we identified the isolate as Nocardia brasiliensis. We discontinued methotrexate and tocilizumab on admission, and administered intravenous antimicrobial combination therapy for 6 weeks, followed by oral trimethoprim-sulfamethoxazole for 12 months, in total. Nocardia bacteremia is rare, often difficult to diagnose, and substantially fatal. However, due to our prompt diagnosis within one day of the onset of symptoms, and administration of appropriate treatment based on antimicrobial susceptibilities, this patient succeeded in surviving the infection. Not only microbiologists but also clinicians should be aware of the characteristic bacterial form of Gram/Kinyoun staining for early recognition of nocardiosis.

皮膚クリプトコックス症はCryptococcus neoformansをおもな原因菌とし,クリプトコックス症の10〜15%にみられ,その皮膚症状は多彩である.まれな一型として蜂窩織炎様皮膚症状を呈することがあり,さらに進行すると時に壊死性筋膜炎にいたる.皮膚クリプトコックス症のうち,皮膚のみならず肺や脳など他臓器に感染が及ぶ全身型は免疫能が低下した患者に多くみられる日和見感染症である.蜂窩織炎様皮膚症状を呈する症例は,全身型であることが多く,皮膚が初発症状となり得る.細菌性蜂窩織炎として初期加療されたが,抗生剤への反応が乏しく,潰瘍形成など非特異的な臨床像を呈し,培養・病理組織学的検査を通して初めて診断されることが多い.今回われわれは,前立腺癌骨転移に対しデカドロンを長期内服中,蜂窩織炎様皮膚症状を呈し,抗生剤を投与・変更するも亜急性の壊死性筋膜炎にいたった,高齢の全身型皮膚クリプトコックス症の1例を経験した.壊死は潰瘍部のみならず,潰瘍周囲皮下織深部にも広く及び,最終的に筋膜直上までの潰瘍となった.デブリードマンと抗真菌薬投与を継続し,皮膚症状は改善したが,全身状態の悪化に伴い逝去された.日和見感染症を起こし得る患者の蜂窩織炎様皮膚症状の場合には,本疾患も鑑別にいれ,早期デブリードマンと抗真菌薬の全身投与が肝要と考えた.(著者抄録)

© 2019 Shimizu et al. Species of the Aspergillus section Nigri are taxonomically very complex. The taxonomic assignment of Aspergillus awamori is unclear. Here, we present the draft genome sequence of A. awamori strain IFM 58123 NT .

© 2019, Japanese Society for Medical Mycology. All rights reserved. We present a 17-year-old Japanese male high school student, who had applied steroid ointment for atopic dermatitis, with fingernail onychomycosis due to Trichophyton tonsurans. He was found positive for T. tonsurans infection based on hairbrush culture performed due to an epidemic of T. tonsurans infection in his judo club. The hairbrush culture method is very important in screening for this infection, and dermatologists should examine the entire body of athletes who are found positive using this method. For the diagnosis of T. tonsurans infection, other than the skin and hair, the nails should also be checked by dermoscopy because the fingernail may be the origin of this fungus.

© 2018 Naturalis Biodiversity Center &amp; Westerdijk Fungal Biodiversity Institute. Although Aspergillus fumigatus is the major agent of invasive aspergillosis, an increasing number of infections are caused by its cryptic species, especially A. lentulus and the A. viridinutans species complex (AVSC). Their identification is clinically relevant because of antifungal drug resistance and refractory infections. Species boundaries in the AVSC are unresolved since most species have uniform morphology and produce interspecific hybrids in vitro. Clinical and environmental strains from six continents (n = 110) were characterized by DNA sequencing of four to six loci. Biological compatibilities were tested within and between major phylogenetic clades, and ascospore morphology was characterised. Species delimitation methods based on the multispecies coalescent model (MSC) supported recognition of ten species including one new species. Four species are confirmed opportunistic pathogens; A. udagawae followed by A. felis and A. pseudoviridinutans are known from opportunistic human infections, while A. felis followed by A. udagawae and A. wyomingensis are agents of feline sino-orbital aspergillosis. Recently described human-pathogenic species A. parafelis and A. pseudofelis are synonymized with A. felis and an epitype is designated for A. udagawae. Intraspecific mating assay showed that only a few of the heterothallic species can readily generate sexual morphs in vitro. Interspecific mating assays revealed that five different species combinations were biologically compatible. Hybrid ascospores had atypical surface ornamentation and significantly different dimensions compared to parental species. This suggests that species limits in the AVSC are maintained by both pre-and post-zygotic barriers and these species display a great potential for rapid adaptation and modulation of virulence. This study highlights that a sufficient number of strains representing genetic diversity within a species is essential for meaningful species boundaries delimitation in cryptic species complexes. MSC-based delimitation methods are robust and suitable tools for evaluation of boundaries between these species.

We describe a case of a 23-year-old female patient with no apparent underlying diseases. She showed a discoloration of the proximal portion of the left big toenail with paronychia. Direct microscopy revealed septate hyphae with conidiophores, and a periodic acid-Schiff-stained nail specimen revealed septate hyphae branching at angles of approximately 45°. On the basis of phylogenetic analysis, we finally arrived at the diagnosis of ungual aspergillosis caused by Aspergillus subramanianii. After p.o. administration of terbinafine and topical application of 10% efinaconazole solution, the disease resolved in 6 months. A. subramanianii is one of the new species in the genus Aspergillus section Circumdati. Reported clinical isolates have been isolated from lung tissue, wounds and feet. This is the first documented case of onychomycosis caused by A. subramanianii. Onychomycosis due to Aspergillus species is uncommon. We summarized the reported cases of ungual aspergillosis in Japan.

The occurrence and bioactivities of marine-derived fungi are evaluated in this paper. A total of 16 morphospecies of marine-derived fungi (MDF) were isolated from four host macroalgae and two seagrasses and identified as belonging to the genera Aspergillus, Fusarium, Paecilomyces, Penicillium, Sclerotinia, Thamnidium and Trichoderma, including five mycelia sterilia. Among these host organisms, the rhodophyte Laurencia inter-media harboured the highest number of isolated MDF. Selected MDF were then assayed and showed to inhibit Pseudomonas aeruginosa (8-19 mm zone of inhibition) and Staphylococcus aureus (6-19 mm zone of inhibition), and were cytotoxic against the brine shrimp Artemia salina nauplii (LD50: 201.56-948.37 mu g mL(-1)). The screening led to the selection of five of the most bioactive morphospecies, all belonging to the genus Aspergillus. These marine aspergilli were subjected to beta-tubulin gene sequence analysis for species identification, and to mass production in different culture media with or without marine salts, and screening of the crude culture extracts for their cytotoxic and trypanocidal activities. Aspergillus tubingensis cultivated in potato dextrose broth with marine salt proved to be the most cytotoxic against P388 (IC50: 1028 ng mL(-1)) and HeLa (IC50: 1301 ng mL(-1)) cancer cells. On the other hand, A. fumigatus cultivated in malt extract broth without marine salt was shown to be the most potent against Trypanosoma congolense (IC50: 298.18 ng mL(-1)). Our study therefore showed that salinity may influence the bioactivities of some species of MDF.

Copyright © 2018 American Chemical Society. A coculture method with a pathogenic actinomycete of the genus Nocardia and an animal cell line was designed to reconstruct and emulate the initial infection state, and a new cyclic nonapeptide, named nocarjamide (1), was obtained by coculture of Nocardia tenerifensis IFM 10554T and the mouse macrophage-like cell line J774.1 in a modified Czapek-Dox medium. Nocarjamide (1) exhibited Wnt signal-activating effects.

Purpose: To report a difficult-to-identify case of keratitis due to Fusarium solani, diagnosed with the help of exhaustive gene analysis. Case: A 47-year-old woman attended our hospital with a refractory corneal ulcer associated with contact lens wear in her left eye that had appeared two weeks earlier. On her initial visit, slit lamp examination revealed a small double-ringed irregular hyphate ulcer in the center of the cornea, which had no epithelial defect, suggesting fungal infection. Microscopic visualization and culture of corneal scrapings were performed repeatedly, but the results were negative. Despite intensive antifungal treatment, infiltration of the cornea worsened and therapeutic keratoplasty was performed. Specimens from a corneal button were microbiologically and histologically negative for microorganisms. During irrigation of the anterior chamber with amphotericin B, aspirated corneal infiltrations were submitted for culture and metagenomic analysis. Genes belonging to F. solani were identified by metagenomic analysis, and an isolate presumed to be a Fusarium species was cultured. Genotypic identification of the isolates confirmed F. solani. Conclusion: The detection of microorganisms, especially fungi, can be extremely difficult. In difficult-to-culture cases, metagenomic analysis seems to be a promising technique for the identification of microbial pathogens.

Background: Kirschsteiniothelia is a saprophytic fungus that is abundantly present in the environment. To date, there have been no reports of human infection caused by this fungus. We report a case of Kirschsteiniothelia infection superimposed on a pre-existing non-infectious bursitis of the ankle. Case presentation: An 81-year-old immunocompetent female local farmer noticed the presence of a nodule on her right ankle 5 years before her first visit to our hospital. A cystic mass of approximately 45 mm × 30 mm was present at the tip of the right lateral malleolus. Culture of the aspirated fluid revealed visibly black colonies and characteristic blackish hyphae nucleotide sequence of the internal transcribed spacer region was determined and compared in a GenBank database. The results indicated Kirschsteiniothelia infection. Conclusions: We described the first case of Kirschsteiniothelia infection manifested as ankle bursitis. The disease seemed to be localized and systemic antibiotics had not been used in this case. However, continued observation is needed because of the possibility of disease progression with the pathogen.

The presence of yeasts at different steps of Vietnamese soy paste production was studied. Yeast growth occurred during primary soybean fermentation, with the cell density reaching 4.106 c.f.u. ml-1, and terminated during brine fermentation. The dominant species were Pichia kudriavzevii and Millerozyma farinosa. Over the span of 14 years, nine strains of Moniliella were isolated. The strains had identical PCR fingerprints generated with primer (GAC)5 and identical D1/D2 and internal transcribed spacer (ITS) sequences. A D1/D2-based phylogeny indicated that the strains were closest to a group of four previously assigned as Moniliella suaveolens strains. Together they form a new lineage that is well separated from all known species, including M. suaveolens (over 12.7 % divergence). ITS sequences indicated the presence of four species differing from each other by 9-57 nt. The name Moniliella sojae sp. nov. is proposed to accommodate the strains isolated from Vietnamese soy paste, Moniliella pyrgileucina sp. nov. is proposed for PYCC 6800 and Moniliella casei sp. nov. is proposed for CBS 157.58. An emended combination Moniliella macrospora is proposed for CBS 221.32 and CBS 223.32. The type strains and MycoBank numbers are: M. sojae sp. nov., SS 4.2T=CBS 126448T=NRRL Y-48680T and MB 822871 M. pyrgileucina sp. nov., PYCC 6800T=CBS 15203T and MB 823030 M. casei sp. nov., CBS 157.58T=IFM 60348T and MB 822872 M. macrospora emend. comb. nov., CBS 221.32T (=MUCL 11527T) and MB 822874.

Cryptic species of Aspergillus fumigatus, including the Aspergillus viridinutans species complex, are increasingly reported to be causes of invasive aspergillosis. Their identification is clinically relevant, as these species frequently have intrinsic resistance to common antifungals. We evaluated the susceptibilities of 90 environmental and clinical isolates from the A. viridinutans species complex, identified by DNA sequencing of the calmodulin gene, to seven antifungals (voriconazole, posaconazole, itraconazole, amphotericin B, anidulafungin, micafungin, and caspofungin) using the reference European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. The majority of species demonstrated elevated MICs of voriconazole (geometric mean [GM] MIC, 4.46 mg/liter) and itraconazole (GM MIC, 9.85 mg/liter) and had variable susceptibility to amphotericin B (GM MIC, 2.5 mg/ liter). Overall, the MICs of posaconazole and the minimum effective concentrations of echinocandins were low. The results obtained by the EUCAST method were compared with the results obtained with Sensititre YeastOne (YO) panels. Overall, there was 67% agreement (95% confidence interval [CI], 62 to 72%) between the results obtained by the EUCAST method and those obtained with YO panels when the results were read at 48 h and 82% agreement (95% CI, 78 to 86%) when the results were read at 72 h. There was a significant difference in agreement between antifungals agreement was high for amphotericin B, voriconazole, and posaconazole (70 to 86% at 48 h and 88 to 93% at 72 h) but was very low for itraconazole (37% at 48 h and 57% at 72 h). The agreement was also variable between species, with the maximum agreement being observed for A. felis isolates (85 and 93% at 48 and 72 h, respectively). Elevated MICs of voriconazole and itraconazole were cross-correlated, but there was no correlation between the other azoles tested.

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K-means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high-adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin-like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.

© 2018 The Pharmaceutical Society of Japan A new aminocyclitol derivative, designated nabscessin C (1), was isolated from Nocardia abscessus IFM 10029T. Nabcessin C is an isomer of nabscessins A (2) and B (3) with different positioning of the acyl group. Absolute configuration of nabscessin A was determined by conversion into the 2-deoxy-scyllo-inosamine pentaacetyl derivative (4) by hydrolysis and acetylation of 2. The biosynthetic pathway of nabscessins is proposed based on gene expression analysis.

© 2018 The Japan Institute of Heterocyclic Chemistry. – Four new cyclodipeptides, asnovolenins A (1) and B (2), and asnovozines A (3) and B (4), were isolated from the fungus Aspergillus novofumigatus CBS 117520. The structures of 1-4 were determined by the detailed analysis of mainly 1D- and 2D-NMR and MS data. Compounds 1 and 2 are composed of epi-aszonalenin (5) and dihydroterrein (6), and they are 2’-epimers of each other. Compounds 3 and 4 consist of D-alanine and tryptophan attached to a 3-methyl-1-butene group. The stereochemistry of 1 and 2 was determined from ROESY spectra and the exciton chirality method from CD spectra, and that of 3 and 4 was determined from NOE or NOESY spectra using the modified Marfey’s method.

Allergic bronchopulmonary mycosis (ABPM) is a pulmonary hypersensitivity disease mainly caused by Aspergillus fumigatus. The mainstay treatment for ABPM is systemic corticosteroid therapy. A 25-year-old man presented with pulmonary infiltrates. His peripheral eosinophil, total serum IgE, and serum Aspergillus-specific IgE levels were elevated. The patient tested positive in a skin test for Aspergillus. However, sputum cultures revealed a Curvularia lunata infection. We therefore diagnosed ABPM possibly caused by C. lunata, which is rare in Japan. The clinical state of the patient improved under observation. Identification of the causative fungus is an important aspect of the ABPM diagnosis.

© 2017, The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature. A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from d-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a “co-culture” in which the genus Nocardia was cultured in the presence of an animal cell line.

This research examined the production of fungal metabolites as a biological response to Kampo medicines. Shimbu-to (SMB) is a Kampo medicine composed of five herbal components: peony root (Shakuyaku), ginger (Shokyo), processed aconite root (Bushi), Poria sclerotium (Bukuryo), and Atractylodes lancea rhizomes (Sojutsu). High-performance liquid chromatography (HPLC) analysis of the fungus Aspergillus nidulans CBS 112.46 incubated in potato dextrose broth supplemented with SMB extract revealed emericellin (2) as the major peak and new xanthone analogues 24-hydroxyshamixanthone (1), shamixanthone (3), epishamixanthone (4), pre-shamixanthone (5), and variecoxanthone A (6) as minor peaks. The structure of 1 was determined by detailed analysis of 1D-NMR, 2D-NMR, and MS data. The results suggest that SMB extract regulates the biosynthesis of emericellin and its analogues in A. nidulans. Further investigations revealed that glucose induces the biosynthesis of emericellin and its analogues in A. nidulans in a concentration-dependent manner.

Aspergillus is a diverse fungal genus containing many species of great agricultural, biotechnological and medical relevance. Because of the broad use of the genus name in diverse disciplines, and the importance of individual species names in these areas, the taxonomy and nomenclature of Aspergillus should remain stable. A formal proposal to change the generic type from A. glaucus to A. niger was recently published. Here we present arguments against this proposal. We assert that it should be rejected because it will not ensure nomenclatural stability for Aspergillus, and will put the names of several important species, such as A. flavus, A. fumigatus and A. oryzae at risk of being classified in different genera and being lost.

Aspergillus niger and its related species, known as Aspergillus section Nigri, are ubiquitously distributed across the globe and are often isolated from clinical specimens. In Japan, Aspergillus section Nigri is second most often isolated from clinical specimens following Aspergillus fumigatus. We determined the species of Aspergillus section Nigri isolated in Japan by DNA sequencing of partial beta-tubulin genes and investigated drug susceptibility by the CLSI M38-A2 method. The collection contained 20 Aspergillus niger, 59 Aspergillus welwitschiae, and 39 Aspergillus tubingensis strains. Drug susceptibility testing revealed 30 to 55% of A. niger, 6.8 to 18.6% of A. welwitschiae, and 79.5 to 89.7% of A. tubingensis isolates to be less susceptible (so-called resistant) to itraconazole (ITC) and/or voriconazole (VRC) according to the epidemiologic cutoff values (ECVs) proposed for A. niger previously. MIC distributions of ITC or VRC showed no remarkable differences between clinical and environmental isolates. When the cyp51A sequences were compared between susceptible and resistant strains, 18 amino acid mutations were specific for resistant isolates of A. niger and A. tubingensis; however, none of them were confirmed to be associated with azole resistance. Three nonrelated A. welwitschiae isolates possessed a partial deletion in cyp51A, likely attributable to being more susceptible to azoles than other isolates. One of five ITC-resistant A. tubingensis isolates showed higher expression of cyp51A than did susceptible strains. Our results show that cyp51A point mutations may have no association with azole resistance but that in some cases the overexpression of cyp51A may lead to the azole resistance in these species.

Copper (Cu) is an essential metal for all living organisms, although it is toxic in excess. Filamentous fungus must acquire copper from its environment for growth. Despite its essentiality for growth, the mechanisms that maintain copper homeostasis are not fully understood in filamentous fungus. To gain insights into copper homeostasis, we investigated the roles of a copper transcription factor Afmac1 in the life-threatening fungus Aspergillus fumigatus, a homolog of the yeast MAC1. We observed that the Afmac1 deletion mutant exhibited not only significantly slower growth, but also incomplete conidiation including a short chain of conidia and defective melanin. Moreover, the expressions of the copper transporters, ctrA1, ctrA2, and ctrC, and metalloreductases, Afu8g01310 and fre7, were repressed in Delta Afmac1 cells, while those expressions were induced under copper depletion conditions in wildtype. The expressions of pksP and wetA, which are, respectively, involved in biosynthesis of conidia-specific melanin and the late stage of conidiogenesis, were decreased in the Delta Afmac1 strain under minimal media condition. Taken together, these results indicate that copper acquisition through AfMac1 functions in growth as well as conidiation.

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (approximate to 1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu(393), Phe(397), Phe(415), and His(440)) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

The phylogenetic position of two Aspergillus strains isolated from Australian soil and phenotypically resembling A. unilateralis was investigated by using multigene phylogeny based on beta-tubulin (benA), calmodulin (CaM), actin (act), and RNA polymerase II second largest subunit (RPB2) genes. The analysis supported their placement into a separate lineage within a well-supported clade containing 10 other members of section Fumigati ("A. unilateralis clade''). Comparisons of extrolite profiles, taxonomically informative morphological and physiological characters were made, and it was discovered that the two strains can be differentiated from all relatives by their low maximum growth temperature, short stipes, and ornamentation of conidia. The data justified the proposal of a new species, A. tasmanius sp. nov. Amplification of mating-type genes showed that the A. unilateralis clade contains five heterothallic species. Only the MAT1-1-1 idiomorph was detected among isolates of A. unilateralis, A. tasmanicus, and A. marvanovae, while isolates having both opposite mating types were detected in A. turcosus and A. nishimurae. The sexual state of A. turcosus was induced by mating experiments and is described in this study. Ascospores of this species were unique by their smooth to finely verrucose convex surface and two well-visible equatorial crests. Some exometabolites detected in A. marvanovae and A. tasmanicus are also indicative of a perfect state, thus supporting the hypothesis that these species have cryptic sexual cycles. The epitype and ex-epitype culture is designated for A. nishimurae to facilitate further taxonomic work with this species.

Background: Accurate identification of Aspergillus species is a very important subject. Mass spectral fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is generally employed for the rapid identification of fungal isolates. However, the results are based on simple mass spectral pattern-matching, with no peak assignment and no taxonomic input. We propose here a ribosomal subunit protein (RSP) typing technique using MALDI-TOF MS for the identification and discrimination of Aspergillus species. The results are concluded to be phylogenetic in that they reflect the molecular evolution of housekeeping RSPs. Results: The amino acid sequences of RSPs of genome-sequenced strains of Aspergillus species were first verified and compared to compile a reliable biomarker list for the identification of Aspergillus species. In this process, we revealed that many amino acid sequences of RSPs (about 10-60%, depending on strain) registered in the public protein databases needed to be corrected or newly added. The verified RSPs were allocated to RSP types based on their mass. Peak assignments of RSPs of each sample strain as observed by MALDI-TOF MS were then performed to set RSP type profiles, which were then further processed by means of cluster analysis. The resulting dendrogram based on RSP types showed a relatively good concordance with the tree based on beta-tubulin gene sequences. RSP typing was able to further discriminate the strains belonging to Aspergillus section Fumigati. Conclusions: The RSP typing method could be applied to identify Aspergillus species, even for species within section Fumigati. The discrimination power of RSP typing appears to be comparable to conventional beta-tubulin gene analysis. This method would therefore be suitable for species identification and discrimination at the strain to species level. Because RSP typing can characterize the strains within section Fumigati, this method has potential as a powerful and reliable tool in the field of clinical microbiology.

© 2017 The Author(s). Background: Polymicrobial keratitis with fungus and bacteria can lead to blindness and is challenging to treat. Here, we introduce a case of fungal keratitis caused by two different strains in addition to definite bacterial super-infection caused by an α-Streptococcus sp., and describe the importance of microscopic examination. Case presentation: A 74-year-old woman, who had a past history of infection with leprosy, presented with conjunctival hyperaemia, pain, and corneal opacity in her right eye. Under the presumptive diagnosis of infectious keratitis, corneal scrapings were stained by various reagents and inoculated on several agar plates. Microscopic findings of the scrapings revealed fungi and a small number of Gram-positive cocci. Multiple anti-fungal therapies with levofloxacin ophthalmic solution were administered. Although empiric treatment was initially effective, keratitis recurred 10 days after its initiation. Repeated corneal scraping revealed an abundance of Gram-positive chain cocci and a small amount of fungi, resulting in the switching of an antibiotic medication from levofloxacin to moxifloxacin and cefmenoxime. Keratitis resolved gradually after the conversion. Stemphylium sp., Acremonium sp., and α-Streptococcus sp. were simultaneously isolated from the corneal scrapings. Conclusions: To the best of our knowledge, this is the first case of fungal keratitis caused by Stemphylium sp., and also the first case of super-infection in the cornea caused by two different fungi and one bacterium. Microscopic examination of the corneal scrapings was beneficial in rapid decision of changing to appropriate drug according to the dominancy of pathogenicity.

New two pebrolide type sesquiterpenoids, deacetoxy-1-deoxypebrolide (1) and 7'-hydroxyisoasperphenamate (2) along with asperphenamate (3), mycophenolic acid (4) and 1-deoxypebloride (5) were isolated from Penicillium sp. IFM 62525. Their structures were established from spectroscopic and chemical method. The absolute stereochemistry of 2 was confirmed by Marfey's method. Antifungal activities of these compounds were tested and 4 showed strong activity.

The study of mycobiota remains relatively unexplored due to the lack of sufficient available reference strains and databases compared to those of bacterial microbiome studies. Deep sequencing of Internal Transcribed Spacer (ITS) regions is the de facto standard for fungal diversity analysis. However, results are often biased because of the wide variety of sequence lengths in the ITS regions and the complexity of high-throughput sequencing (HTS) technologies. In this study, a curated ITS database, ntF-ITS1, was constructed. This database can be utilized for the taxonomic assignment of fungal community members. We evaluated the efficacy of strategies for mycobiome analysis by using this database and characterizing a mock fungal community consisting of 26 species representing 15 genera using ITS1 sequencing with three HTS platforms: Illumina MiSeq (MiSeq), Ion Torrent Personal Genome Machine (IonPGM), and Pacific Biosciences (PacBio). Our evaluation demonstrated that PacBio's circular consensus sequencing with greater than 8 full-passes most accurately reconstructed the composition of the mock community. Using this strategy for deep-sequencing analysis of the gut mycobiota in healthy Japanese individuals revealed two major mycobiota types: a single-species type composed of Candida albicans or Saccharomyces cerevisiae and a multi-species type. In this study, we proposed the best possible processing strategies for the three sequencing platforms, of which, the PacBio platform allowed for the most accurate estimation of the fungal community. The database and methodology described here provide critical tools for the emerging field of mycobiome studies.

In Japan, Fusarium species are known etiological agents of human fungal infection however, there has been no report of a large-scale epidemiological study on the etiological agents of fusariosis. A total of 73 Fusarium isolates from patients with invasive fusariosis (IF, n = 36) or superficial fusariosis (SF, n = 37), which were obtained at hospitals located in 28 prefectures in Japan between 1998 and 2015, were used for this study. Fusarium isolates were identified using Fusarium- and Fusarium solani species complex (FSSC)-specific real-time PCR and partial DNA sequences of the elongation factor-1 alpha (EF-1α) gene and the nuclear ribosomal internal transcribed spacer (ITS)region. FSSC was predominately isolated from both patients with IF and SF (IF, 77.8% and SF, 67.6%). Distribution of the phylogenetic species of FSSC isolates from patients with IF and SF exhibited different spectra specifically, F. keratoplasticum (FSSC 2) (25.0%)was the most frequent isolate from patients with IF, whereas F. falciforme (FSSC 3+ 4) (32.4%)was the most frequent isolate from patients with SF. Fusarium sp. (FSSC 5)was the second most frequent isolate from both patients with IF and SF (IF, 22.2% and SF, 24.3%). Notably, F. petroliphilum (FSSC 1)was isolated only from patients with IF. Each species was isolated from a broad geographic area, and an epidemic was not observed. This is the first epidemiological study of Fusarium species causing IF and SF in Japan.

Malformin A(1), a cyclic pentapeptide, was isolated from the marine-derived fungus Aspergillus tubingensis IFM 63452. The identity of the compound was established based on TOFMS and H-1 NMR data. Malformin A(1) exhibited trypanocidal activity against Trypanosoma congolense (IC50: 15.08 ng/mL). Interestingly, the compound was selective for T. congolense rendering a selectivity index value that ranged from 3.33 to 4.67. It also demonstrated cytotoxicity against HeLa (IC50: 50.15 ng/mL) and P388 (IC50: 70.38 ng/mL) cell lines. To further identify the possible mechanism of its cytotoxic effect, immunofluorescence staining was conducted to follow the epigenetic changes induced by the compound in the amino acid lysine of histone H3 and H4 in HeLa. The compound induced repressive levels of H3K27me3, H3K27ac and H4K5ac, and enhanced levels of H3K9me2, H3K9me3 and H4K16ac supporting the compound's chemotherapeutic potential.

A basidiomycetous yeast Cryptococcus neoformans is stained by DBB. We found that the edges of DBB stained cells were specifically detected by using fluorescence microscopy. We also found that the only the edges of cap64 Delta strain cells was not fluorescent among several acapsular mutants, although whose colonies turned to red or light pink by DBB staining. When the vacuoles were stained by FM4-64, those of the cap64 Delta cells showed aberrant morphology. In addition, quinacrine treatment showed that the cap64 Delta strain could not accumulate quinacrine in the vacuole. These data suggest that Cap64 was not only involved in capsule formation, but also in intracellular pH regulation. (C) 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

Background: Candida albicans (C. albicans) can become a pathogen causing superficial as well as life-threatening systemic infections, especially in immunocompromised patients. Many phenotypic attributes contribute to its capacity to colonize human organs. In our study, 93 C. albicans isolates from patients of various candidiasis in a hospital of China were surveyed. We aimed to investigate the white-opaque (WO) switching competence, drug sensitivity, and virulence of mating type-like (MTL) a/alpha isolates. Methods: Internal transcribed spacer (ITS) gene and the MTL configuration were detected in all the isolates by reverse transcription-polymerase chain reaction. White/opaque phenotype and doubling time of cell growth were determined. The minimum inhibitory concentrations of antifungal agent were measured using broth microdilution method. Results: Sixty-four isolates (69.6%) were classified to serotype A, 19 (20.6%) to serotype B, and 9 (9.8%) to serotype C. Moreover, phylogenetic analysis showed that these isolates were divided into four different subgroups of ITS genotypes. Most of our clinical isolates were MTLa/alpha type, while 6.8% remained MTLa or MTL alpha type. The frequency of opaque phenotype was 71.0% (66 isolates). Following the guidelines of Clinical and Laboratory Standards Institute M27-A3, all isolates were susceptible to caspofungin and a few (0.6-3.2%) of them showed resistance against amphotericin B, flucytosine, fluconazole, itraconazole, and voriconazole. Conclusions: From these analyses, there were comparatively more C. albicans strains classified into serotype B, and the frequency of opaque phase strains was significant in the clinical isolates from China. Genetic, phenotypic, or drug susceptibility patterns were not significantly different from previous studies. MTLa/alpha isolates could also undergo WO switching which facilitates their survival.

A nocardioform strain IFM 11456(T) was isolated from the aqueous humor from a patient with endophthalmitis and was characterized to its taxonomic position. IFM 11456(T) contained arabinose, galactose and meso-diaminopimelic acid in whole-cell hydrolysates and mycolic acids that co-migrated with those from the type strain of Nocardia asteroides. The acyl type of muramic acid was N-glycolyl. The diagnostic polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and two unidentified glycolipids and the predominant menaquinone was MK-8 (H-4, omega-cycl.). These characteristics are typical of members of the genus Nocardia. Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the isolate represented a novel species of the genus Nocardia and was most closely related to the type strains of Nocardia mikamii JCM 15508(T) (98.1 %) and Nocardia aobensis IFM 0372(T) (98.1 %). However, analysis of partial gyrB sequences showed that strain IFM 11456(T) had 90.2% similarity to Nocardia concava IFM 0354(T) and 90% to Nocardia niigatensis IFM 0330(T). The DNA-DNA relatedness values for strain IFM 11456(T) compared with N. mikamii JCM 15508(T), N. aobensis IFM 0372(T) and N. concava IFM 0354(T) ranged from 24.4 to 39.9 %. Phenotypic characteristics that differentiated IFM 11456(T) from phylogenetically related species were growth at 45 degrees C, utilization of citrate and growth with inositol as a sole carbon source. On the basis of this polyphasic study, the isolate represents a novel species within the genus Nocardia, for which the name Nocardia shinanonensis sp. nov. is proposed. The type strain is IFM 11456(T) (= NBRC 109590(T) = TBRC 5149(T)).