Milada Chudíčková

Rare and new etiological agents revealed among 178 clinical Aspergillus strains obtained from Czech patients and characterized by molecular sequencing

A collection of 178 Aspergillus isolates, recovered from Czech patients, mostly from 2007-2011, was subjected to multilocus DNA sequence typing using the ITS region, β-tubulin, and calmodulin genes. An unusually wide spectrum of etiologic agents that included 36 species of Aspergillus is discussed in the context of recent taxonomic and clinical reports. Invasive aspergillosis (IA), onychomycosis, and otitis externa were the predominant clinical entities. Five cases due to species newly proven as etiologic agents of human mycoses, as well as cases with unique clinical manifestations caused by unusual agents are discussed in more detail. Three species (i.e., A. insulicola, A. westerdijkiae and A. tritici) were identified as the confirmed etiologic agents of non-dermatophytic onychomycosis. Emericella rugulosa was recovered from a premature newborn with a fatal necrotising disseminated infection and is reported for only the second time as the cause of IA. Furthermore, we document the first infection due to A. calidoustus in a patient with chronic granulomatous disease. The infection manifested as a latent brain aspergilloma with an unusual clinical-laboratory finding. In addition to the well-known agents of human mycosis, several rarely isolated or poorly documented species were identified. An undescribed cryptic species related to A. versicolor was found to be common among isolates linked to proven and probable onychomycosis. An isolate representing A. fresenii, or an unnamed sister species, were causal agents of otomycosis. Three well defined, and tentative new species belonging to section Cervini, Candidi and Aspergillus (Eurotium spp.), were associated with cases of probable onychomycosis.

Diversity of fungal communities in saline and acidic soils in the Soos National Natural Reserve, Czech Republic

During 2003–2005, the diversity of culturable filamentous soil microfungi in saline and acidic soils of the Soos National Natural Reserve (Czech Republic) was studied. Altogether, 28 soil samples were collected from four sampling sites and were processed by various approaches. In total, 92 fungal taxa were identified using classical and molecular markers. Several detected species were known from similar substrata worldwide; however, the overall fungal spectrum was distinct, as shown by comparison to similar studies. All methodological approaches increased the observed fungal diversity. The different fungal communities observed on the four sampling sites were influenced by the complex effects of environmental factors. The growth response of selected strains to different salinities and pH values was determined. The results of the growth tests showed high adaptability of all tested species to the extreme conditions of the studied substrate. Two acidophilic species (Acidomyces acidophilus, Sporothrix sp.) were isolated.

Delimitation of cryptic species inside Claviceps purpurea

Claviceps purpurea is an ovarian parasite infecting grasses (Poaceae) including cereals and forage plants. This fungus produces toxic alkaloids and consumption of contaminated grains can cause ergotism in humans and other mammals. Recent molecular genetics studies have indicated that it included three cryptic species (G1, G2, G3). In this study, reproductive isolation amongst these groups and among material from Phragmites and Molinia was tested using gene flow statistics for five polymorphic loci, and to support these data, phylogenetic affiliations based on gene trees and a multigene phylogeny were used. The four recognized species are characterized based on morphology and host spectrum and formal taxonomic names are proposed. Claviceps purpurea sensu stricto (G1 group) represents a typical rye ergot, but infects various other grasses. Typical hosts of Claviceps humidiphila (new name for G2 species), like Phalaris arundinacea, belong to grasses preferring humid locations. Claviceps spartinae (G3) is specific to chloridoid grasses from salt barches. The material from Phragmites and Molinia can be authenticated with the species Claviceps microcephala for which the new name Claviceps arundinis is proposed here. The divergence time between species was estimated and the tools for species identification are discussed.

Hydroxylated Anthraquinones Produced by Geosmithia species

Geosmithia fungi are little known symbionts of bark beetles. Secondary metabolites of lilac colored species G. lavendula and other nine Geosmithia species were investigated in order to elucidate their possible role in the interactions of the fungi with environment. Hydroxylated anthraquinones (yellow, orange, and red pigments), were found to be the most abundant compounds produced into the medium during the submerged cultivation. Three main compounds were identified as 1,3,6,8-tetrahydroxyanthraquinone (1), rhodolamprometrin (1-acetyl-2,4,5,7-tetrahydroxyanthraquinone; 2), and 1-acetyl-2,4,5,7,8-pentahydroxyanthraquinone (3). Compounds 2 and 3 (representing the majority of produced metabolites) inhibited the growth of G+-bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentration of 64–512 μg/mL. Anti-inflammatory activity detected as inhibition of cyclooxygenase-2 was found only for compound 3 at 1 and 10 μg/mL. Compound 2 interfered with the morphology, compound 3 with cell-cycle dynamics of adherent mammalian cell lines.

Vitamin B2 as a virulence factor in Pseudogymnoascus destructans skin infection.

Pathogenic and non-pathogenic related microorganisms differ in secondary metabolite production. Here we show that riboflavin overproduction by a fungal pathogen and its hyperaccumulation in affected host tissue exacerbates a skin infection to necrosis. In white-nose syndrome (WNS) skin lesions caused by Pseudogymnoascus destructans, maximum riboflavin concentrations reached up to 815 μg ml−1, indicating bioaccumulation and lack of excretion. We found that high riboflavin concentrations are cytotoxic under conditions specific for hibernation, affect bats’ primary fibroblasts and induce cell detachment, loss of mitochondrial membrane potential, polymerization of cortical actin, and cell necrosis. Our results explain molecular pathology of WNS, where a skin infection becomes fatal. Hyperaccumulation of vitamin B2 coupled with reduced metabolism and low tissue oxygen saturation during hibernation prevents removal of excess riboflavin in infected bats. Upon reperfusion, oxygen reacts with riboflavin resulting in dramatic pathology after arousal. While multiple molecules enable invasive infection, riboflavin-associated extensive necrosis likely contributes to pathophysiology and altered arousal pattern in infected bats. Bioaccumulation of a vitamin under natural infection represents a novel condition in a complex host-pathogen interplay.

Liberomyces gen. nov. with two new species of endophytic coelomycetes from broadleaf trees

During a study of endophytic and saprotrophic fungi in the sapwood and phloem of broadleaf trees (Salix alba, Quercus robur, Ulmus laevis, Alnus glutinosa, Betula pendula) fungi belonging to an anamorphic coelomycetous genus not attributable to a described taxon were detected and isolated in pure culture. The new genus, Liberomyces, with two species, L. saliciphilus and L. macrosporus, is described. Both species have subglobose conidiomata containing holoblastic sympodial conidiogenous cells. The conidiomata dehisce irregularly or by ostiole and secrete a slimy suspension of conidia. The conidia are hyaline, narrowly allantoid with a typically curved distal end. In L. macrosporus simultaneous production of synanamorph with thin filamentous conidia was observed occasionally. The genus has no known teleomorph. Related sequences in the public databases belong to endophytes of angiosperms. Phylogenetic analysis revealed a position close to the Xylariales (Sordariomycetes), but family and order affiliation remained unclear.

Auxarthron ostraviense sp. nov., and A. umbrinum associated with non-dermatophytic onychomycosis

Auxarthron is a genus within the Onygenales encompassing keratinophilic species with typical ascomata (gymnothecia) consisting of anastomosing network of thick-walled hyphae and small globose or oblate ascospores. No association of this genus with clinically relevant cases of human or animal infection has been reported. This paper describes the isolation of an undescribed Auxarthron species as an agent of proven onychomycosis affecting almost all fingernails in a man with psoriasis. The causality of the isolated fungus was verified by repeated sampling and direct microscopy revealing irregular septate hyphae. Based on micro- and macromorphological features and unique sequence data (ITS region, benA and RPB2 gene), the isolated fungus is proposed as the new species A. ostraviense. The sibling species of A. ostraviense, A. umbrinum, was isolated from three patients with suspected onychomycosis and a detailed clinical history is provided for one of these patients. All four isolates were tested for susceptibility to selected antifungal agents. Terbinafine and clotrimazole appear to be effective in vitro. The morphological identification of Auxarthron spp. is non-trivial, time-consuming and requires cultivation media other than Sabouraud glucose agar which is routinely used in dermatomycology.

New polyene macrolide family produced by submerged culture of Streptomyces durmitorensis.

A new polyene macrolide family, closely related to the pentaene macrolide antibiotic roflamycoin, was isolated from the both fermentation broth and biomass of Streptomyces durmitorensis wild-type strain MS405. The main compound was identified by NMR and Fourier transform ion cyclotron resonance mass spectrometry as 32,33-didehydroroflamycoin (1; DDHR). Additional four structurally related compounds were determined solely by MS analysis. DDHR induces cell death by apoptosis in various cancer cell lines as demonstrated by DNA fragmentation. Striking feature of DDHR is its internal fluorescence allowing visualization of labeled plasma membranes and internal membrane structures.

Claviceps nigricans and Claviceps grohii : Their Alkaloids and Phylogenetic Placement

Claviceps purpurea, C. grohii, C. zizaniae, C. cyperi, and C. nigricans are closely related ergot fungi and form a monophyletic clade inside the genus Claviceps. Analysis of alkaloid content in C. nigricans sclerotia using UPLC detected ergocristine (1), ergosine (2), alpha-ergocryptine (3), and ergocristam (4). Alkaloids 1, 3, and 4 were found in the sclerotia of C. grohii. The content of 4 in the mixture of alkaloids from C. nigricans and C. grohii (over 8% and over 20%, respectively) was unusually high. Submerged shaken cultures of C. nigricans produced no alkaloids, whereas C. grohii culture formed small amounts (15 mg L (-1)) of extracellular clavines and 1. In the previously used HPLC method the ergocristam degradation product could have been obscured by the ergosine peak. Therefore sclerotia of a C. purpurea habitat-specific population G2 with the dominant production of 1 and 2 have been reanalyzed, but no 4 was detected. The phylogeny of the C. purpurea-related species group is discussed with regard to alkaloid-specific nonribosomal peptide synthetase duplication leading to the production of two main ergopeptines instead of a single product.

Three new genera of fungi from extremely acidic soils

Extremely acidic soils (pH < 3) harbour poorly diversified mycobiota that are very different from less acidic habitats. During investigations of the mycobiota from several highly acidic soils in the Czech Republic and a coastal site in the Antarctic Peninsula, a group of hyaline fungal isolates was obtained. Based on phenotype and nuclear ribosomal DNA sequences (ITS region, SSU, LSU), the isolates belonged to three phylogenetic lineages within two different classes, Sordariomycetes and Leotiomycetes (Pezizomycotina, Ascomycota). The first lineage is described here as a new genus and species Acidothrix acidophila gen. nov. et sp. nov. (Amplistromataceae, Sordariomycetes, Ascomycota). The most closely related species to this new clade are wood-inhabiting fungi. The isolates belonging to the second and the third lineages are also described as two new genera and species Acidea extrema gen. nov. et sp. nov. and Soosiella minima gen. nov. et sp. nov. (Helotiales, Leotiomycetes, Ascomycota). Their position and the relationships within Helotiales are discussed. Soosiella minima was acidotolerant, Acidothrix acidophila and Acidea extrema exhibited both acidotolerant and acidophilic characteristics. All the species were slightly halophilic. The adaptation of hyaline fungi from mesophilic lineages to highly acidic environments has been revealed. The association between highly acidic and Antarctic habitats is discussed.