Search

Aspergillus fumigatus is the major causative agent of aspergillosis in immunosuppressed hosts and patients with chronic pulmonary diseases. Recently, azole resistance of this fungus has become a problem worldwide; thus, rapid detection of resistant strains is desirable in clinical practice. In this study, we established a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) combined with multivariate and discriminant analysis to detect azole-resistant A. fumigatus strains. The score plot from a partial least squares discriminant analysis clearly separated resistant and susceptible strains, regardless of the culture period. The discriminant analysis using the support-vector machine algorithm based on a dataset for strains cultured on potato dextrose agar for 3 days showed the best performance, with a correct answer rate of 84.75–99.85%. We found a MS peak (4359.5 m/z) specific to some azole-resistant strains with a 34-bp tandem repeat in the promoter region of cyp51A. These tandem repeat strains are extremely rare in Japan. Instead, most resistant strains in Japan have point mutations in cyp51A. No specific biomarker for strains with point mutations was found, but it was possible to detect these resistant strains using a model for point-mutation strains.

The introduction of the prophylactic use of antifungal drugs caused the increased occurrence of invasive fungal infections due to previously rare molds, such as fusariosis, after allogeneic hematopoietic stem cell transplantation. We herein report the case of a patient with diffuse large B-cell lymphoma who developed fungemia due to Fusarium solani during liposormal amphotericin B on day 25 after cord blood transplantation (CBT). Because Fusarium species might differ in virulence and drug susceptibility, the sequencing of the internal transcribed spacer region of the ribosomal RNA gene accurately identified Fusarium solani to be the cause of fungemia at the species level. This case highlights Fusarium solani as the cause of fungemia in a patient under liposormal amphotericin B treatment after CBT.

During dismantling and relocation of the Takamatsuzuka Tumulus stone chamber, many Cladophialophora and chaetothyrialean black fungi, such as Exophiala and Phialophora, were isolated from samples taken from the joints between the stone walls. However, inside the stone chamber of the Kitora Tumulus, after intermittent UV irradiation in 2009, these black fungi were also isolated from samples taken from the stone walls. Molecular phylogenetic analyses based on only nrLSU and the concatenated (nrLSU D1/D2 + ITS) sequences revealed that the 35 Takamatsuzuka and Kitora Tumuli isolates of Cladophialophora and the chaetothyrialean black fungi were divergent. Two new species of Cladophialophora are described herein: C. tumulicola from the viscous gels and various substrates on the stone walls of the Takamatsuzuka and Kitora Tumuli and C. tumbae from black substances on the plastic cover over the “thief hole,” soil and plaster pieces between the stone walls, and the exterior of the Takamatsuzuka Tumulus chamber. Also, molecular phylogenetic placements for the remaining eight Takamatsuzuka and Kitora Tumuli isolates of chaetothyrialean black fungi have been determined or suggested.

Phenotypic and genotypic characteristics of five isolates of Cephalotrichum and Sagenomella from various samples collected while dismantling the Takamatsuzuka Tumulus stone chamber were found to represent Cephalotrichum verrucisporum and Sagenomella striatispora. In addition to these, one isolate from the ceiling stone surface in the adjacent small room of the Kitora Tumulus was assignable to Sagenomella griseoviridis. Colonization on biodeteriorated plaster and stone walls as substrata or habitats of these fungi is noteworthy. Physiologically Sagenomella isolates solubilized CaCO3 and may be involved in the deterioration of the plaster walls inside the stone chamber. Cephalotrichum verrucisporum is a new addition to Japan.

Two achlorophyllous microalgal strains were isolated from the soil and white moldy colony collected inside the stone chamber of the Takamatsuzuka Tumulus in Japan. Phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) and Dl/D2 large subunit ribosomal RNA (LSU rRNA) gene sequences, and concatenated gene sequences of the SSU and D1/D2 LSU rRNA genes indicated that our two isolates were the members of the non-photosynthetic, yeast-like microalgal Chlorellaceous genus Prototheca (Chlorellales, Trebouxiophyceae, Chlorophyta) but well distinguished from known species. Based on phenotypic and genotypic characteristics, isolates T6713-13-10T and T61213-7-11 are proposed to represent a novel species in Prototheca, P. tumulicola, with the type strain JCM 31123T (isolate T6713-13-10T).

Microbial outbreaks and related biodeterioration problems have affected the 1300-year-old multicolor (polychrome) mural paintings of the special historic sites Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT). Those of TT are designated as a national treasure. The microbiomes of these tumuli, both located in Asuka village, Nara, Japan, are critically reviewed as the central subject of this report. Using culture-dependent methods (conventional isolation and cultivation), we conducted polyphasic studies of the these microbial communities and identified the major microbial colonizers (Fusarium spp., Trichoderma spp., Penicillium spp., dark Acremonium spp., novel Candida yeast spp., Bacillus spp., Ochrobactrum spp., Stenotrophomonas tumulicola, and a few actinobacterial genera) and noteworthy microbial members (Kendrickiella phycomyces, Cephalotrichum verrucisporum (≡Doratomyces verrucisporus), Sagenomella striatispora, Sagenomella griseoviridis, two novel Cladophialophora spp., Burgoa anomala, one novel species Prototheca tumulicola, five novel Gluconacetobacter spp., three novel Bordetella spp., and one novel genus and species Krasilnikoviella muralis) involved in the biodeterioration of mural paintings, plaster walls, and stone chamber interiors. In addition, we generated microbial community data from TT and KT samples using culture-independent methods (molecular biological methods, including PCR-DGGE, clone libraries, and pyrosequence analysis). These data are comprehensively presented, in contrast to those derived from culture-dependent methods. Furthermore, the microbial communities detected using both methods are analytically compared, and, as a result, the complementary roles of these methods and approaches are highlighted. In related contexts, knowledge of similar biodeterioration problems affecting other prehistoric cave paintings, mainly at Lascaux in France and Altamira in Spain, are referred to and commented upon. Based on substrate preferences (or ecological grouping) and mapping (plotting detection sites of isolates), we speculate on the possible origins and invasion routes whereby the major microbial colonizers invaded the TT stone chamber interior. Finally, concluding remarks, lessons, and future perspectives based on our microbiological surveys of these ancient tumuli, and similar treasures outside of Japan, are briefly presented. A list of the microbial taxa that have been identified and fully or briefly described by us as known and novel taxa for TT and KT isolates since 2008 is presented in Supplementary Materials.

Analysis of D1/D2 large-subunit (LSU) rRNA gene sequences predicted that 17 yeast isolates, mainly from viscous gels (biofilms) taken from the stone chamber interior of the Kitora tumulus in Nara, Japan, were placed in the Yamadazyma and Zygoascus clades. Polyphasic characterization, including morphological, physiological and chemotaxonomic characteristics, multigene sequence divergence and DNA-DNA hybridization, strongly suggested the assignment of one novel species to each of the clades; these are Yamadazyma kitorensis f.a., sp. nov., with the type strain JCM 31005T (ex-type CBS 14158T=isolate K8617-6-8T), and Zygoascus biomembranicola f.a., sp. nov., with the type strain JCM 31007T (ex-type CBS 14157T=isolate K61208-2-11T). Furthermore, the transfer of five known species of the genus Candida as novel combinations to the genera Yamadazyma and Zygoascus is proposed; these are Yamadazyma olivae f.a., comb. nov. (type strain CBS 11171T=ATCC MYA-4568T), Yamadazyma tumulicola f.a., comb. nov. (type strain JCM 15403T=ex-type CBS 10917T=isolate T6517-9-5T), Yamadazyma takamatsuzukensis f.a., comb. nov. (type strain JCM 15410T=CBS 10916T = isolate T4922-1-1T), Zygoascus polysorbophila f.a., comb. nov. (type strain NRRL Y-27161T=CBS 7317T) and Zygoascus bituminiphila f.a., comb. nov. (type strain CBS 8813T=MUCL 41424T).

Integrated analysis of phenotypic and genotypic characteristics of 20 isolates of “black particles” (“Kuro-tsubu” in Japanese), that mainly appeared on the plaster wall of the ceiling tuff stone surface of the Kitora Tumulus stone chamber interior in Asuka village, Nara, Japan, has identified them as the bulbilliferous basidiomycetous anamorphic fungus Burgoa anomala. Our 28S rRNA gene D1/D2 (28S) and internal transcribed spacer (ITS) regions sequence-based phylogenies indicated that the 20 isolates displayed no genetic divergence and firmly clustered with the authentic strain B. anomala CBS 130.38. These isolates and B. anomala CBS 130.38 grouped together with species of the teleomorphic genus Sistotrema and its allies in the Cantharelloid clade. Colonization of biodeteriorated murals, plaster, and stone walls as the substrata or habitats by this fungus colonized, are noteworthy. Additionally, B. anomala is recorded for the first time from Japan.

The Kitora tumulus, which is thought to have been built around the late seventh to early eighth century, has beautiful mural paintings which were executed directly on a very thin layer of plaster in the stone chamber. When the paintings were found, the plaster was partly detached from the stone wall and the murals were therefore considered to be likely to fall off the wall with a casual touch or stimulation. Therefore, it was decided to detach the mural paintings and to store them flat and under controlled environmental conditions. This was initiated immediately after the excavation of the tumulus in 2004. However, fungal growth and biofilm development were observed within the stone chamber during the relocation work. In 2005, small holes containing black substances were observed on certain areas of the ceiling plaster, and following investigation an acetic acid bacterium, Gluconacetobacter sp., was isolated from the black substances. The bacterium was also isolated from the ceiling, floor, and ea...

Using an integrated analysis of phenotypic and genotypic characterizations, a total of 18 isolates of “bristle-like” fungal colonizers of the Kitora and Takamatsuzuka Tumuli, which had been provisionally identified as Phialocephala phycomyces, were here determined to be Kendrickiella phycomyces (Auersw.) K. Jacobs & M. J. Wingf. The 18 isolates consisted of 10 from stone surfaces or viscous gels (biofilms) of the stone chamber interior and adjacent small room, and air in the adjacent small room of the Kitora Tumulus, and 8 from viscous gels on the stone surfaces of the stone chamber interior, plant roots, and soil in the adjacent space or stone wall interspaces (interstices) in the stone chamber of the Takamatsuzuka Tumulus. Plaster and stone walls in both tumuli were recorded as novel substrates of this fungus. Our 18S sequence-based phylogeny indicated that K. phycomyces and species of the leotiomycetous anamorph genera Chaetomella, Pilidium, Sphaerographium, and Synchaetomella formed a monophyletic lineage distant from the core taxa of the Leotiomycetes (Pezizomycotina, Ascomycota). The relationship between the physicochemical characteristics of these isolates on GYC agar plates, i.e., soluble brownish pigments and dissolution of calcium carbonate (CaCO3), and the biodeterioration of the plaster and plaster walls of both tumuli, are briefly discussed.

Unidentified black spots (or stains) appeared on the plaster walls of the Takamatsuzuka and Kitora Tumuli in the village of Asuka, Nara Prefecture, Japan. Public attention was drawn to the biodeterioration of the colorful 1,300-year-old murals. A total of 46 isolates of Acremonium sect. Gliomastix were obtained from various samples (mainly black spots) of the Takamatsuzuka Tumulus (TT) (sampling period, May 2004–December 2006) and the Kitora Tumulus (KT) (June 2004–May 2007). These isolates were assignable to four known taxa and a new species in the ‘series Murorum’ sensu W. Gams as inferred from the integrated analysis of phenotypic and genotypic (i.e., ITS and 28S rDNA-D1/D2 sequences) characters: these were Acremonium masseei, A. murorum, A. felinum comb. nov. with the neotype designation, A. polychromum, and A. tumulicola sp. nov., which have been accommodated in the validated series Murorum in the section Gliomastix. The black spots on the murals of the TT and KT were caused mainly by A. masseei and A. murorum, respectively.

Penicillium appeared as the major dweller in the Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT) stone chambers, both located in the village of Asuka, Nara Prefecture, in relation to the biodeterioration of the 1,300-year-old mural paintings, plaster walls and ceilings. Of 662 Penicillium isolates from 373 samples of the TT (sampling period, May 2004-2007) and the KT (sampling period, June 2004-Sep 2007), 181 were phenotypically assigned as Penicillium sp. 1 which shared similar phenotypic characteristics of sect. Roqueforti in Penicillium subg. Penicillium. Fifteen representative isolates of Penicillium sp. 1, 13 from TT and 2 from KT, were selected for molecular phylogenetic analysis. The 28S rDNA D1/D2, ITS, beta-tubulin, and lys2 gene sequence-based phylogenies clearly demonstrated that the three known species P. roqueforti, P. carneum and P. paneum in sect. Roqueforti, and all TT and KT isolates grouped together. In addition to this, TT and KT isolates formed a monophyletic group with the ex-holotype strain CBS 101032 of P. paneum Frisvad with very strong bootstrap supports. So far, P. paneum has been isolated only from mouldy rye breads, other foods, and baled grass silage. Therefore, this is the first report of P. paneum isolation from samples relating to the biodeteriorated cultural properties such as mural paintings on plaster walls.

In an effort to clarify the cause of the deterioration of the colorfully painted murals that adorn the inner walls of the small stone chambers in the Takamatsuzuka and Kitora Tumuli in Japan, we enumerated the fungi that were isolated from moldy spots on the plaster walls collected between May 2004 and April 2005. The 262 fungal isolates from 79 samples of both tumuli were identified as approximately 100 species based on their phenotypic characters. Fusarium, Trichoderma, and Penicillium species were the predominant colonizers in the stone chamber interior and adjacent areas of both tumuli. In addition to the 28S phylogeny, neighbor-joining and Bayesian phylogenies of partial EF-1-alpha gene sequences revealed 24 genetically diverse fusaria in the Takamatsuzuka and Kitora Tumuli. Most of the fusaria were nested in clade 3 of the Fusarium solani species complex (FSSC); however, a few isolates were members of the F. oxysporum species complex (FOSC) clade or the F. avenaceum/F. tricinctum species complex clade. The FSSC isolates were compared with those detected in the Lascaux cave in France. In addition, a partial EF-1α gene phylogeny indicated that 13 Trichoderma isolates clustered in the Harzianum-Virens clade and 5 isolates in the Viride clade or Trichoderma sect. Longibrachiatum. Our analyses suggest that most of the fungi recovered from both tumuli are typically soil dwellers.

The prophylactic use of antifungal drugs in allogeneic hematopoietic cell transplant recipients has revealed that the rate of non-albicans candidemia has increased. We herein report the case of a patient with adult T-cell leukemia who developed candidemia due to Candida fermentati during micafungin treatment after cord blood transplantation. The isolate was identified on day 47 by sequencing of the internal transcribed spacer region of the ribosomal RNA gene. The sequencing of the hot spot region of fks1p of isolate revealed naturally occurring amino acid substitutions, which conferred reduced echinocandin susceptibility. This case highlights that breakthrough candidemia due to C. fermentati occurred in a patient receiving micafungin treatment. This article is protected by copyright. All rights reserved.

During a survey of the mycobiota in the stone chamber of the Takamatsu-zuka tumulus in the village of Asuka, Nara Prefecture, Japan, we isolated 19 yeast strains assigned to the genus Candida from various samples, taken mainly from mouldy spots where the colour of the murals had changed to black, white or another tone, and from viscous gels (biofilms) on plaster walls. The 26S rDNA D1/D2 domain sequence-based phylogeny clearly indicates two groups of isolates. Polyphasic characterization, including morphological, physiological and chemotaxonomic characteristics, and sequence analysis of the 26S rDNA D1/D2 domain and ITS regions suggest that each group is assignable to one of two novel species within the Candida membranifaciens clade. Proposed herein are the names Candida tumulicola sp. nov. (originally T6517-9-5T; holotype JCM 15403T; isotypes CBS 10917T, NBRC 104392T) and Candida takamatsuzukensis sp. nov. (originally T4922-1-1T; holotype JCM 15410T; isotypes CBS 10916T, NBRC 104391T). The 26S rDNA D1/D2 domain sequence divergence indicates that C. tumulicola differs from Candida friedrichii NBRC 10277T, the type strain of the nearest species, in 15 nucleotides (3 %), whereas C. takamatsuzukensis differs from Candida insectorum NBRC 10283T and Pichia mexicana NBRC 10544T, the type strains of the nearest species, in 20 nucleotides (4 %). Both novel species are also clearly distinguishable from the species closest to them by various physiological characteristics.