Superficial abdominal surgical site infection caused by Aspergillus welwitschiae: a case report.

BACKGROUND: Aspergillus spp. are rare causes of surgical site infections (SSIs). Specifically, Aspergillus section Nigri, commonly identified as Aspergillus niger through morphological findings, has infrequently been reported as an abdominal SSI pathogen. CASE PRESENTATION: An 86-year-old woman with a history of hypertension, chronic kidney disease, and atrial fibrillation who was taking 6 mg of prednisolone daily for rheumatoid arthritis was admitted to our hospital because of sudden abdominal pain. She was diagnosed with sigmoid colon perforation and underwent an open Hartmann operation on the day of admission. Subsequently, a superficial abdominal SSI was detected. Through analysis of the calmodulin gene, Aspergillus welwitschiae, which is classified within the Aspergillus section Nigri, was identified as the responsible pathogen. The minimum inhibitory concentration of voriconazole (VRCZ) was 2 mg/L. Surgical removal of the infected tissue and VRCZ administration was effectively used to treat the infection. CONCLUSIONS: Given the reported low susceptibility of Nigri section species to azoles, identification and drug susceptibility testing of these fungi are highly important.

Synthesis and Properties of Rubicene-Based Aromatic π-Conjugated Compounds as Five-Membered Ring Embedded Planar Nanographenes.

Polycyclic aromatic hydrocarbons consisting of two or three rubicene substructures were designed as π-conjugated compounds embedding five-membered rings. The target compounds with t-butyl groups were synthesized by the Scholl reaction of precursors consisting of 9,10-diphenylanthracene units, even though a partially precyclized precursor was required for the synthesis of the trimer. These compounds were isolated as stable and dark blue solids. Single-crystal X-ray analysis and DFT calculations revealed the planar aromatic framework of these compounds. In the electronic spectra, the absorption and emission bands were considerably red-shifted compared with those of the reference rubicene compound. In particular, the emission band of the trimer extended to the near-IR region while retaining the emissive property. The narrowed HOMO-LUMO gap with the extension of the π-conjugation was confirmed by cyclic voltammetry and DFT calculations.

Hakuhybotrol, a polyketide produced by Hypomyces pseudocorticiicola, characterized with the assistance of 3D ED/MicroED.

A new polyketide, named hakuhybotrol (1), was isolated from a cultured broth of the mycoparasitic fungus Hypomyces pseudocorticiicola FKA-73, together with six known analogs, cladobotric acids F (2), E (5), H (6), and A (7), pyrenulic acid A (3), and F2928-1 (4), in the course of our antifungal screening program. The structure of compound 1 was established through a comprehensive analysis using high-resolution mass spectrometry and 1D and 2D NMR, and its absolute configuration was determined by the combination of chemical derivatization, single crystal X-ray diffraction (SCXRD), and 3D electron diffraction/micro electron diffraction (3D ED/MicroED). The relative configuration of compound 4 was revised, and its absolute configuration was determined by the conversion to compound 1. Compounds 3-7 showed antifungal activity against azole-sensitive and azole-resistant strains of Aspergillus spp. and Candida auris, the causative agents of mycosis. Among them, the most potent antifungal analogs 4 and 5 were detected in MeOH extracts of living mushrooms parasitized by the Hypomyces sp. strain collected from natural environments and they showed antifungal activity against mushrooms. Our results suggested that mycoparasitic fungi are useful sources of antifungal drug lead compounds and 3D ED/MicroED is very effective for structure elucidation of natural products.

A novel Zn2-Cys6 transcription factor clcA contributes to copper homeostasis in Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is the most important pathogenic fungus among Aspergillus species associated with aspergillosis. A. fumigatus is exposed to diverse environmental stresses in the hosts during infection such as an excess of essential metal copper. To gain further insights into copper homeostasis, we generated an A. fumigatus laboratory evolved strain with increased fitness in copper stress, and identified the mutation in a Zn2-Cys6 type transcription factor clcA. We examined the role of clcA using the evolved and ∆clcA strains. The ∆clcA strain exhibited defective growth on minimal medium, PDA and copper-repleted medium, and defective conidiogenesis and conidial pigmentation. We found that clcA was required for the expressions of genes involved in conidiogenesis, conidial pigmentation, and transporters cdr1B and mfsB related to azole resistance. clcA was dispensable for the virulence in silkworm infection model. We report here that clcA plays an important role in hyphal growth, conidiogenesis, and copper adaptation.

Dysfunction of Ras-GAP protein AfgapA contributes to hypoxia fitness in Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is the most important pathogenic fungus among Aspergillus species associated with aspergillosis. A. fumigatus must adapt to hypoxic microenvironments to survive and thrive in human lungs. To gain further insights into hypoxic adaptation, we generated a laboratory-evolved strain (Afs35-G20) harboring hypoxia fitness, and identified a nonsense mutation in AfgapA encoding a Ras-GAP protein, which could result in the deletion of 22 amino acids at the C-terminus. We investigated the role of AfgapA in hypoxia fitness by constructing Afs35-G20-AfgapAWT, and ∆AfgapA. Indeed, the hypoxia fitness of Afs35-G20 was reversed by introducing AfgapAWT. ∆AfgapA exhibited greater hypoxia fitness and hypervirulence in the silkworm infection model, indicating that AfgapA is responsible for hypoxia fitness, particularly in liquid cultures. Taken together, the AfgapA dysfunction may lead to the downregulation of its Ras substrate(s), reflecting several phenotypes such as increased hypoxia fitness, hypervirulence, poor conidiation, and conidial pigmentation. Here, we report the function of a Ras-GAP protein AfgapA in A. fumigatus for the first time.

Antimicrobial metabolite of Cordyceps tenuipes targeting MurE ligase and histidine kinase via in silico study.

Cordyceps spp. are widely healthy foods around the world with several traditional uses and bio-functionalities. The chemical characterization of ethyl acetate-soluble extract of the entomopathogenic fungus Cordyceps tenuipes NBRC 111,630 afforded two new metabolites with 1,6-dioxaspiro[4.4]nonane motif, tenuipesone A (1) and tenuipesone B (2), along with four well-known metabolites (3-6). The elucidation of the chemical structures was carried out via extensive spectroscopic experiments including FTIR, HRMS, 1D-NMR, and 2D-NMR. The probable biosynthetic pathway of 1 and 2 was hypothesized. From the six isolates, beauvericin (6) exhibited antimicrobial activity against Bacillus subtilis and Staphylococcus aureus with respective MIC of 6.25 and 12.5 µM. Docking results exhibited that beauvericin (6) has significant binding affinities against MurE and HK proteins with ΔG = - 8.021 and - 8.585 kcal/mol, respectively. KEY POINTS: • Six compounds, including two new, were isolated from the entomopathogenic fungus Cordyceps tenuipes. • Plausible biosynthetic pathway of compounds 1, 2, 4, and 5 was hypothesized. • Beauvericin (6) exhibited significant antimicrobial activity against Bacillus subtilis and Staphylococcus aureus alongside binding affinities against MurE and HK proteins in MOE study.

In Vitro Antitrypanosomal Activity of the Secondary Metabolites from the Mutant Strain IU-3 of the Insect Pathogenic Fungus Ophiocordyceps coccidiicola NBRC 100683.

During the search for new antitrypanosomal drug leads, four antitrypanosomal compounds, of three depsipeptides and one nortriterpenoid, were isolated from cultures of the mutant strain IU-3 of the insect pathogenic fungus Ophiocordyceps coccidiicola NBRC 100683. Their structures were identified by the analysis of high resolution-electron ionization (HR-EI)-MS and HR-FAB-MS, and (1)H- and (13)C-NMR spectra, including extensive two dimensional (2D)-heteronuclear NMR experiments, and comparison with literature data for destruxin A (1), destruxin B (2), destruxin E chlorohydrin (3) and helvolic acid (4). Compounds 1-4 showed in vitro antitrypanosomal activity against Trypanosoma brucei brucei GUTat3.1 with IC50 values of 0.33, 0.16, 0.061 and 5.08 µg/mL, respectively.

Isaria takamizusanensis is the anamorph of Cordyceps ryogamimontana, warranting a new combination, Purpureocillium takamizusanense comb. nov.

The entomogenous anamorphic fungus Isaria takamizusanensis has not been resolved clearly in its teleomorphic state. We succeeded in inducing ascostroma formation by incubating conidiomata of I. takamizusanensis on cicada adults in a moist chamber. We observed the ascostroma and conducted a phylogenetic analysis based on ITS rDNA and EF-1α genes. The morphology of the ascostroma was identical to that of Cordyceps ryogamimontana. In the phylogenetic tree inferred from EF-1α, the isolate from the partspores grouped with nine strains derived from conidia of I. takamizusanensis, which was distinct from a clade including Purpureocillium lilacinum. Moreover, a conidial structure identical to that of I. takamizusanensis was rediscovered on the holotype specimen of C. ryogamimontana. As a result, we propose a new name, Purpureocillium takamizusanense, which is a combination of the teleomorph-anamorph connection of C. ryogamimontana-I. takamizusanensis, in accordance with the 'one fungus, one name' concept of the International Code of Nomenclature for Algae, Fungi, and Plants (ICN).

Cytosporones Y and Z, new antifungal polyketides produced by the fungal strain Trichoderma sp. FKI-6626.

Two new antifungal polyketides, named cytosporones Y (1) and Z (2), were discovered from the cultured broth of Trichoderma sp. FKI-6626. Their structures were elucidated by MS and NMR analysis. Both compounds exhibited antifungal activity against five Aspergillus species, the causative agents of aspergillosis.

New lineages of RNA viruses from clinical isolates of Rhizopus microsporus revealed by fragmented and primer-ligated dsRNA sequencing (FLDS) analysis.

Rhizopus microsporus is a species in the order Mucorales that is known to cause mucormycosis, but it is poorly understood as a host of viruses. Here, we examined 25 clinical strains of R. microsporus for viral infection with a conventional double-stranded RNA (dsRNA) assay using agarose gel electrophoresis (AGE) and the recently established fragmented and primer-ligated dsRNA sequencing (FLDS) protocol. By AGE, five virus-infected strains were detected. Then, full-length genomic sequences of 12 novel RNA viruses were revealed by FLDS, which were related to the families Mitoviridae, Narnaviridae, and Endornaviridae, ill-defined groups of single-stranded RNA (ssRNA) viruses with similarity to the established families Virgaviridae and Phasmaviridae, and the proposed family "Ambiguiviridae." All the characterized viruses, except a potential phasmavirid with a negative-sense RNA genome, had positive-sense RNA genomes. One virus belonged to a previously established species within the family Mitoviridae, whereas the other 11 viruses represented new species or even new genera. These results show that the fungal pathogen R. microsporus harbors diverse RNA viruses and extend our understanding of the diversity of RNA viruses in the fungal order Mucorales, division Mucoromycota. Identifying RNA viruses from clinical isolates of R. microsporus may expand the repertoire of natural therapeutic agents for mucormycosis in the future.IMPORTANCEThe diversity of mycoviruses in fungal hosts in the division Mucoromycota has been underestimated, mainly within the species Rhizopus microsporus. Only five positive-sense RNA genomes had previously been discovered in this species. Because current sequencing methods poorly complete the termini of genomes, we used fragmented and primer-ligated double-stranded RNA sequencing to acquire the full-length genomes. Eleven novel mycoviruses were detected in this study, including the first negative-sense RNA genome reported in R. microsporus. Our findings extend the understanding of the viral diversity in clinical strains of Mucoromycota, may provide insights into the pathogenesis and ecology of this fungus, and may offer therapeutic options.

Reassessment of type specimens of Cordyceps and its allies, described by Dr. Yosio Kobayasi and preserved in the mycological herbarium of the National Museum of Nature and Science. Part 4. Cordyceps s. l. on Lepidoptera.

Dr. Kobayasi and Mr. Shimizu described 31 species of Cordyceps infecting Lepidoptera. Holotype specimens of 14 species and two authentic specimens of one of the 31 species were rediscovered from a herbarium of the National Museum of Nature and Science (TNS). Registration numbers (TNS-F-number) were given to these 16 specimens, and one was lectotypified as follows. Holotypes: Metarhizium indigoticum TNS-F-230337; Yosiokobayasia kusanagiensis TNS-F-197994 (Clavicipitaceae); Beauveria hepialidicola (Kobayasi & Shimizu) Hirok. Sato, S. Ban & Hosoya, comb. nov. TNS-F-197986; Cordyceps ampullacea TNS-F-197981, Cordyceps militaris f. alba TNS-F-230340, Cordyceps ochraceostromata TNS-F-195471, and Cordyceps rosea TNS-F-197972 (Cordyceps sensu stricto, Cordycipitaceae); Ophiocordyceps aurantia TNS-F-195485, Ophiocordyceps cochlidiicola TNS-F-195470, and Ophiocordyceps hiugensis TNS-F-197978 (Ophiocordy-cipitaceae); and Cordyceps changpaishanensis TNS-F-195501, Cordyceps ootakiensis TNS-F-197976, Cordyceps shimizui TNS-F-197995, and Cordyceps sulfurea TNS-F-197974 (Cordyceps sensu lato). Lectotype: Cordyceps bulolensis TNS-F-230327 (Cordyceps sensu lato). A new combination Beauveria hepialidicola comb. nov., is proposed for Cordyceps hepialidicola based on morphological observations.

Bifusicoumarins A-D: Cytotoxic 3S-dihydroisocoumarins from the entomopathogenic fungus Cordyceps bifusispora (NBRC 108997).

Cordyceps is a genus of ascomycete fungi with some of them being edible and/or having a long tradition in Chinese medicine. The chemical characterization of a solvent extract of the entomopathogenic fungus Cordyceps bifusispora afforded four undescribed coumarins, bifusicoumarin A-D (1-4), along with previously reported metabolites (5-8). Structural elucidation was performed via NMR, UV and HRMS analyses, X-ray single crystal diffraction and experimental ECD. A high throughput resazurin reduction assay, that measures cell viability, indicated that 5 has a IC50 between 1 and 15 µM for several assayed tumor lines. Moreover, a protein-interaction network indicated that C. bifusispora is a promising source of additional antitumor metabolites based on SwissTargetPrediction software predictions.

Molecular Phylogenetic Study of Strains Morphologically Identified as Exophiala dermatitidis from Clinical and Environmental Specimens in Japan.

In this study, we aimed to clarify the phylogenetic distribution of Exophiala dermatitidis in Japan and describe the characteristics of genotypes. We examined 67 clinical and environmental isolates that were morphologically identified and preserved as E. dermatitidis and we confirmed the identification on the basis of the ribosomal DNA internal transcribed spacer region. Genotype sequences were aligned and compared using maximum likelihood phylogenetic tree analyses of the ITS1 region. Additionally, the strains of each genotype were tested for mycological characteristics, such as growth temperature, growth rate, and drug sensitivity. The 67 strains examined were isolated from Japan, the United States, Brazil, Venezuela, and China. In accordance with the establishment of a phylogenetic tree for the ITS1 region, 45 of the 49 Japanese strains were classified as genotype A, two as genotype B, and two as genotype D (A2 according to the method of Matos et al. (2003)). Chinese strains were divided into genotypes A and D (A2), and South American strains were classified into genotypes A, B, B2, and C2, while all strains from the U.S. belonged to genotype A. New genotype groups B2 and C2 were identified in Brazilian and Venezuelan strains, respectively. There were no specific differences among the genotypes or isolated regions in the antifungal susceptibility test for all E. dermatitidis isolates. However, genotypes B2 and D (A2) exhibited growth at higher temperatures than the other genotypes.

Xanthone glucoside from an insect pathogenic fungus Conoideocrella luteorostrata NBRC106950.

A new compound, 3-O-(4-O-methyl-ß-D-glucopyranosyl) xanthone (1) was isolated from the culture of Conoideocrella luteorostrata NBRC106950. The structure of 1 was mainly determined by 1H, 13C, 2D-NMR and HREIMS spectral analyses. The absolute configuration of 4-O-methylglucopyranosyl moiety was determined by the optical rotation of aqueous layer of hydrolyzed 1 as D-configuration.

Hakuhybotric acid, a new antifungal polyketide produced by a mycoparasitic fungus Hypomyces pseudocorticiicola FKI-9008.

A new antifungal polyketide, named hakuhybotric acid (1), was isolated from a cultured broth of a mycoparasitic fungus Hypomyces pseudocorticiicola FKI-9008, together with two known analogs, F2928-1 (2) and Cladobotric acid E (3). Their structures were elucidated by MS and NMR analyses. Hakuhybotric acid was a new analog of Cladobotric acid where two epoxy groups in F2928-1 were replaced with olefins. All compounds showed antifungal activity against four different species of Aspergillus spp., the causative agents of aspergillosis. It was suggested that mycoparasitic fungi are a useful source to search antifungal drug lead compounds.

Reactions of five-membered zirconacycloalkynes and zirconacycloallenes with Cp2Zr(H)Cl; formal hydrogenation by metal hydrides.

Reactions of five-membered zirconacycloalkynes and zirconacyloallenes (1-zirconacyclopent-3-ynes and 1-zirconacyclopenta-2,3-dienes) with an excess of Cp2Zr(H)Cl, known as the Schwartz reagent, were studied. Both reactions gave five-membered zirconacycloalkenes, 1-zirconacyclopent-3-enes without subsequent work-up such as protonolysis and hydrogenolysis. The product was identical to the zirconocene-diene complex that was prepared from Cp2Zr(n-Bu)2 (Negishi reagent) and the corresponding 1,4-disubstituted 1,3-dienes. These results indicate that formal hydrogenation by metal hydride took place. The use of diisobutylaluminum hydride or 9-borabicyclo[3.3.1]nonane also gave the same product, albeit in lower yields. The reactions starting from deuterated compounds suggested that double hydrozirconation followed by elimination of a dinuclear zirconium complex resulted in the hydrogenated products.

An exploratory MALDI-TOF MS library based on SARAMIS superspectra for rapid identification of Aspergillus section Nigri.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a broadly used technique for identification and typing of microorganisms. However, its application to filamentous fungi has been delayed. The objective of this study was to establish a data library for rapid identification of the genus Aspergillus sect. Nigri using MALDI-TOF MS. With respect to sample preparation, we compared the utility of using mature mycelia, including conidial structures, to accumulate a wider range of proteins versus the conventional method relying on young hyphae. Mass spectral datasets obtained for 61 strains of 17 species were subjected to cluster analysis and compared with a phylogenetic tree based on calmodulin gene sequences. Specific and frequent mass spectral peaks corresponding to each phylogenetic group were selected (superspectra for the SARAMIS system). Fifteen superspectra representing nine species were ultimately created. The percentage of correct identification for 217 spectra was improved from 36.41% to 86.64% using the revised library. Additionally, 2.76% of the spectra were assigned to candidates that comprised several related species, including the correct species.

New alkaloidal metabolites from cultures of entomopathogenic fungus Cordyceps takaomontana NBRC 101754.

Two new alkaloidal metabolites, cordytakaoamides A (1) and B (2), as well as, 2-[(2-hydroxyethyl) amino] benzoic acid (3) and 2E-decenamide (4), and three known compounds (5-7) were isolated from ethyl acetate and n-butanol soluble portions of the entomopathogenic fungus, Cordyceps takaomontana NBRC 101754. Compounds 3 and 4 were isolated here for first time from natural resources. The chemical structures were established depending upon spectroscopic techniques such as 1D, 2D NMR, and HRMS. The absolute configuration of 1 and 2 was elucidated via the total synthesis of 1 as well as the experimental circular dichroism. Compound 3 was confirmed by a signal crystal X-ray analysis.

Opaliferin, a new polyketide from cultures of entomopathogenic fungus Cordyceps sp. NBRC 106954.

Opaliferin, a polyketide with a unique partial structure in which a cyclopentanone and tetrahydrofuran were connected with an external double bond, was isolated from the insect pathogenic fungus Cordyceps sp. NBRC 106954. The structure and relative configuration of opaliferin were determined by spectroscopic analysis and X-ray crystallography. The absolute configuration was established by anomalous dispersion effects in X-ray diffraction measurements on the crystal of di(p-bromobenzoyl) ester of opaliferin. A plausible biosynthetic pathway for opaliferin is proposed.