Miroslav Kolarik

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.

Morphological and molecular characterisation of Geosmithia putterillii, G. pallida comb. nov. and G. flava sp. nov., associated with subcorticolous insects.

Geosmithia putterillii is an anamorphic fungus with connections to bark beetles. Genetic variability of 89 isolates traditionally grouped in G. putterillii and G. lavendula isolated from different geographical regions from subcorticolous insects and from other unspecific substrata was assessed using RAPD, sequencing of the ITS region (ITS1-5.8SrDNA-ITS2) and morphological characters. RAPD analysis revealed eight distinct groups. One group was represented by G. lavendula type strain and showed no relations to any other isolate. Five RAPD-types with similar ITS sequences and phenotype were related to the ex-type strain of Penicillium pallidum (generally given as a synonym of G. putterillii). Because of unique phylogenetic position and a phenotype markedly different from G. putterillii, the new combination G. pallida is made here. For another group of isolates formerly identified as G. putterillii the new species G. fiava is described based on a characteristic RAPD-type, a unique ITS sequences and a different phenotype. These newly recognized species are stable in culture and with worldwide distribution.

A complex of three new white-spored, sympatric, and host range limited Geosmithia species

All hypocrealean species of the genus Geosmithia are anamorphic fungi with connections to bark beetles. G. fassatiae, G. langdonii and G. obscura are described as new sympatric species associated with Scolytus carpini, S. intricatus and S. rugulosus in Central Europe. The species represent a complex of three sister taxa with affinities to G. flava that may be distinguished by differences in morphology, unique RAPD patterns and by sequences of ITS region rDNA. Intraspecific variability and habitat specificity of new species is described and discussed. The high morphological, genetic and ecological uniformity suggest that these Geosmithia spp. are recently derived. A key to all accepted hypocrealean species of the genus is provided.

White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America

A striking feature of white-nose syndrome, a fungal infection of hibernating bats, is the difference in infection outcome between North America and Europe. Here we show high WNS prevalence both in Europe and on the West Siberian Plain in Asia. Palearctic bat communities tolerate similar fungal loads of Pseudogymnoascus destructans infection as their Nearctic counterparts and histopathology indicates equal focal skin tissue invasiveness pathognomonic for WNS lesions. Fungal load positively correlates with disease intensity and it reaches highest values at intermediate latitudes. Prevalence and fungal load dynamics in Palearctic bats remained persistent and high between 2012 and 2014. Dominant haplotypes of five genes are widespread in North America, Europe and Asia, expanding the source region of white-nose syndrome to non-European hibernacula. Our data provides evidence for both endemicity and tolerance to this persistent virulent fungus in the Palearctic, suggesting that host-pathogen interaction equilibrium has been established.

Molecular Characterization of a Heterothallic Mating System in Pseudogymnoascus destructans, the Fungus Causing White-Nose Syndrome of Bats

White-nose syndrome (WNS) of bats has devastated bat populations in eastern North America since its discovery in 2006. WNS, caused by the fungus Pseudogymnoascus destructans, has spread quickly in North America and has become one of the most severe wildlife epidemics of our time. While P. destructans is spreading rapidly in North America, nothing is known about the sexual capacity of this fungus. To gain insight into the genes involved in sexual reproduction, we characterized the mating-type locus (MAT) of two Pseudogymnoascus spp. that are closely related to P. destructans and homothallic (self-fertile). As with other homothallic Ascomycota, the MAT locus of these two species encodes a conserved α-box protein (MAT1-1-1) as well as two high-mobility group (HMG) box proteins (MAT1-1-3 and MAT1-2-1). Comparisons with the MAT locus of the North American isolate of P. destructans (the ex-type isolate) revealed that this isolate of P. destructans was missing a clear homolog of the conserved HMG box protein (MAT1-2-1). These data prompted the discovery and molecular characterization of a heterothallic mating system in isolates of P. destructans from the Czech Republic. Both mating types of P. destructans were found to coexist within hibernacula, suggesting the presence of mating populations in Europe. Although populations of P. destructans in North America are thought to be clonal and of one mating type, the potential for sexual recombination indicates that continued vigilance is needed regarding introductions of additional isolates of this pathogen.

Aspergillus waksmanii sp. nov. and Aspergillus marvanovae sp. nov., two closely related species in section Fumigati.

Two new and phylogenetically closely related species in Aspergillus section Fumigati are described and illustrated. Homothallic Aspergillus waksmanii sp. nov. was isolated from New Jersey soil (USA) and is represented by the ex-type isolate NRRL 179(T) ( = CCF 4266(T) = Thom 4138.HS2(T) = IBT 31900(T)). Aspergillus marvanovae sp. nov. was isolated from water with high boracic acid anions content in Dukovany nuclear power station (Czech Republic). The sexual stage of this species is unknown, but the MAT1-1 locus was successfully amplified suggesting that the species is probably heterothallic and teleomorphic but is represented by only the ex-type isolate CCM 8003(T) ( = CCF 4037(T) = NRRL 62486(T) = IBT 31279(T) = IFM 60873(T)). Both species can be distinguished from all previously described species in section Fumigati based on morphology, maximum growth temperature, sequence data from five unlinked loci and unique secondary metabolites profiles.

Aspergillus baeticus sp. nov. and Aspergillus thesauricus sp. nov., two species in section Usti from Spanish caves

Two novel species of Aspergillus that are clearly distinct from all known species in section Usti were revealed during a study of microfungal communities in Spanish caves. The novel species identified in this study and additional species of Aspergillus section Usti are associated with places and substrates related to human activities in caves. Novel species are described using data from four loci (ITS, benA, caM and rpb2), morphology and basic chemical and physiological analyses. Members of the species Aspergillus thesauricus sp. nov. were isolated from various substrates, including decaying organic matter, cave air and cave sediment of the Cueva del Tesoro Cave (the Treasure cave); the species is represented by twelve isolates and is most closely related to the recently described Aspergillus germanicus. Members of the species Aspergillus baeticus sp. nov. were isolated from cave sediment in the Gruta de las Maravillas Cave (the Grotto of the Marvels); the species is represented by two isolates. An additional isolate was found in the Cueva del Tesoro Cave and in the Demänovská Peace Cave (Slovakia), suggesting a potentially wide distribution of this micro-organism. The species is related to Aspergillus ustus and Aspergillus pseudoustus. Both species were unable to grow at 37 °C, and a weakly positive, light greenish yellow Ehrlich reaction was observed in A. thesauricus. Unique morphological features alone are sufficient to distinguish both species from related taxa.

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.

Discovery of a sexual stage in Trichophyton onychocola, a presumed geophilic dermatophyte isolated from toenails of patients with a history of T. rubrum onychomycosis

Trichophyton onychocola is a recently described geophilic dermatophyte that has been isolated from a toenail of Czech patient with a history of onychomycosis due to T. rubrum and clinical suspicion of relapse. In this study, we report a similar case from Denmark in an otherwise healthy 56-year-old man. The patient had a history of great toenail infection caused by T. rubrum in 2004 and presented with suspected relapse in 2011 and 2013. Trichophyton onychocola was the only microbial agent isolated at the second visit in 2013 and the identification was confirmed by DNA sequencing. Direct microscopic nail examination was positive for hyphae, however the etiological significance of T. onychocola was not supported by repeated isolation of the fungus. This new species may be an overlooked geophilic species due to the resemblance to some common species, for example, zoophilic T. interdigitale or some species of geophilic dermatophytes. We included differential diagnosis with phenotypically similar species; however, it is recommended that molecular methods are used for correct identification. The MAT locus of Danish strain was of opposite mating type than in the previously isolated Czech strain and the two isolates were successfully mated. The mating experiments with related heterothallic species T. thuringiense and Arthroderma melis were negative. The sexual state showed all typical signs of arthroderma-morph and is described by using optical as well as scanning electron microscopy. The sexual state was induced on a set of agar media, however low cultivation temperature and the presence of keratin source were crucial for the success rather than formulation of medium.

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.