Novel Taxa Associated with Human Fungal Black-Grain Mycetomas: Emarellia grisea gen. nov., sp. nov., and Emarellia paragrisea sp. nov.

Eumycetoma is a debilitating, chronic, fungal infection that is endemic in India, Indonesia, and parts of Africa and South and Central America. It remains a neglected tropical disease in need of international recognition. Infections follow traumatic implantation of saprophytic fungi and frequently require radical surgery or amputation in the absence of appropriate treatment. Several fungal species can cause black-grain mycetomas, including Madurella spp. (Sordariales), Falciformispora spp., Trematosphaeria grisea, Biatriospora mackinnonii, Pseudochaetosphaeronema larense, and Medicopsis romeroi (all Pleosporales). We performed phylogenetic analyses based on five loci on 31 isolates from two international culture collections to establish the taxonomic affiliations of fungi that had been isolated from cases of black-grain mycetoma and historically classified as Madurella grisea Although most strains were well resolved to species level and corresponded to known agents of eumycetoma, six independent isolates, which failed to produce conidia under any conditions tested, were only distantly related to existing members of the Pleosporales Five of the six isolates shared >99% identity with each other and are described as Emarellia grisea gen. nov. and sp. nov; the sixth isolate represents a sister species in this novel genus and is described as Emarellia paragrisea. Several E. grisea isolates were present in both United Kingdom and French culture collections and had been isolated independently over 6 decades from cases of imported eumycetoma. Four of the six isolates involved patients that had originated on the Indian subcontinent. All isolates were all susceptible in vitro to the azole antifungals, but had elevated MICs with caspofungin.

Investigating the insecticidal potential of Geomyces (Myxotrichaceae: Helotiales) and Mortierella (Mortierellacea: Mortierellales) isolated from Antarctica.

Fungi isolated from environmentally challenging habitats can have adaptations of potential value when developed as insect pest-controls. Fungal isolates collected from Antarctica, Geomyces sp. I, Geomyces sp. II, Mortierella signyensis and M. alpina, were investigated for (i) growth characteristics at 0-35°C, (ii) spore production at 10 and 20°C, (iii) viability following exposure to freezing temperatures, and (iv) insecticidal activity against waxmoths (Galleria mellonella L.), houseflies (Musca domestica L.), mealworms (Tenebrio molitor L.) and black vine weevils (Otiorhynchus sulcatus Fabricius). All isolates showed growth between 5-20°C, with some showing growth outside this range. Geomyces isolates sporulated over a wider range of conditions than the Mortierella isolates. Spore germination at 10°C was higher for Geomyces sp. II when this isolate was produced at 10 compared to 20°C (greatest difference 74.6 vs 32.7%). All isolates grew, with the exception of M. alpina, following exposure to -20°C for 4 weeks. Insecticidal investigations showed M. alpina and M. signyensis caused significant mortality of waxmoth and housefly larvae via injection and soil inoculation, and M. alpina caused significant mortality of housefly larvae via baiting; the Geomyces isolates had little lethal effect.

Deposit of microbial strains in public service collections as part of the publication process to underpin good practice in science.

Despite recommendations to release microbial resources to the community post-publication, the reality is far from satisfying. A workshop discussed the need for a coordinated and effective deposition policy for 'key' microbial strains and proposes a set of criteria to facilitate their deposition into public service collections. The majority of authors either contacted directly or during submission of manuscripts to several international, mainly European bacteriology journals agreed to this set of 'key strain' criteria and to the voluntarily deposition of resources into public resource centres.

Sebacinales are associates of the leafy liverwort Lophozia excisa in the southern maritime Antarctic.

The leafy liverwort Lophozia excisa, which is colonised by basidiomycete fungi in other biomes and which evidence suggests may be colonised by mycorrhizal fungi in Antarctica, was sampled from Léonie Island in the southern maritime Antarctic (67 degrees 36' S, 68 degrees 21' W). Microscopic examination of plants indicated that fungal hyphae colonised 78% of the rhizoids of the liverwort, apparently by entering the tips of rhizoids prior to growing into their bases, where they formed hyphal coils. Extensive colonisation of stem medullary cells by hyphae was also observed. DNA was extracted from surface-sterilised liverwort tissues and sequenced following nested PCR, using the primer set ITS1F/TW14, followed by a second round of amplification using the ITSSeb3/TW13 primer set. Neighbour-joining analyses showed that the sequences obtained nested in Sebacinales clade B as a 100% supported sister group to Sebacinales sequences from the leafy liverworts Lophozia sudetica, L. incisa and Calypogeia muelleriana sampled from Europe. Direct PCR using the fungal specific primer set ITS1F/ITS4 similarly identified fungi belonging to Sebacinales clade B as the principal colonists of L. excisa tissues. These observations indicate the presence of a second mycothallus in Antarctica and support the previous suggestion that the Sebacinales has a wide geographical distribution.

Minimal influence of water and nutrient content on the bacterial community composition of a maritime Antarctic soil.

Bacterial community composition was determined by culture-independent PCR-based methods in two soils differing markedly in their water, C, N and P contents sampled from Mars Oasis on Alexander Island, western Antarctic Peninsula. 16S rRNA sequences of the phyla Actinobacteria, Cyanobacteria, α-Proteobacteria and Acidobacteria were commonly (> 8% frequency) obtained from soil. Those of ß-, γ- and δ-Proteobacteria, Chloroflexi, Bacteroidetes, Verrucomicrobia, Planctomycetes, Gemmatimonadetes and Firmicutes were less frequent. Comparisons of slopes of collector's curves and the Shannon-Weiner diversity index indicated no difference in overall bacterial diversity between the two soils, although sequences of δ-Proteobacteria and the cyanobacterial genus Leptolyngbya were more commonly derived from the soil with the higher water and nutrient content. The data suggest that different levels of soil water, C, N and P have only a minor effect on the bacterial community composition of maritime Antarctic soils.

Microorganisms in the atmosphere over Antarctica.

Antarctic microbial biodiversity is the result of a balance between evolution, extinction and colonization, and so it is not possible to gain a full understanding of the microbial biodiversity of a location, its biogeography, stability or evolutionary relationships without some understanding of the input of new biodiversity from the aerial environment. In addition, it is important to know whether the microorganisms already present are transient or resident - this is particularly true for the Antarctic environment, as selective pressures for survival in the air are similar to those that make microorganisms suitable for Antarctic colonization. The source of potential airborne colonists is widespread, as they may originate from plant surfaces, animals, water surfaces or soils and even from bacteria replicating within the clouds. On a global scale, transport of air masses from the well-mixed boundary layer to high-altitude sites has frequently been observed, particularly in the warm season, and these air masses contain microorganisms. Indeed, it has become evident that much of the microbial life within remote environments is transported by air currents. In this review, we examine the behaviour of microorganisms in the Antarctic aerial environment and the extent to which these microorganisms might influence Antarctic microbial biodiversity.

Candida nivariensis, an emerging pathogenic fungus with multidrug resistance to antifungal agents.

In 2005, Candida nivariensis, a yeast species genetically related to Candida glabrata, was described following its isolation from three patients in a single Spanish hospital. Between 2005 and 2006, 16 fungal isolates with phenotypic similarities to C. nivariensis were submitted to the United Kingdom Mycology Reference Laboratory for identification. The strains originated from various clinical specimens, including deep, usually sterile sites, from patients at 12 different hospitals in the United Kingdom. PCR amplification and sequencing of the D1D2 and internal transcribed spacer 1 (ITS1) regions of the nuclear ribosomal gene cassette confirmed that these isolates from the United Kingdom are genetically identical to C. nivariensis. Biochemically, C. glabrata and C. nivariensis are distinguished by their differential abilities to assimilate trehalose. However, in contrast to the original published findings, we found that C. glabrata isolates, but not C. nivariensis isolates, are capable of assimilating this substrate. Antifungal susceptibility tests revealed that C. nivariensis isolates are less susceptible than C. glabrata isolates to itraconazole, fluconazole, and voriconazole and to have significantly higher flucytosine MICs than C. glabrata strains. Finally, C. nivariensis could be rapidly distinguished from the other common pathogenic fungus species by pyrosequencing of the ITS2 region. In the light of these data, we believe that C. nivariensis should be regarded as a clinically important emerging pathogenic fungus.

Molecular identification of unusual pathogenic yeast isolates by large ribosomal subunit gene sequencing: 2 years of experience at the United kingdom mycology reference laboratory.

Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species.

Tolerance of Antarctic soil fungi to hydrocarbons.

Little is known about the effects of hydrocarbons and fuel oil on Antarctic filamentous fungi in the terrestrial Antarctic environment. Growth of fungi and bacteria from soils around Rothera Research Station (Adelaide Island, Antarctic Peninsula) was assessed in the presence of ten separate aromatic and aliphatic hydrocarbons [marine gas oil (MGO), dodecane, hexadecane, benzoic acid, p-hydroxybenzoic acid, toluene, phenol, biphenyl, naphthalene and m- and p-xylenes with ethylbenzene]. Aromatic hydrocarbons inhibited soil microbial growth more than aliphatic hydrocarbons. Soil microorganisms from a moss patch, where little previous impact or hydrocarbon contamination had occurred, were less tolerant of hydrocarbons than those from high impact sites. Fungal growth rates of Mollisia sp., Penicillium commune, Mortierella sp., Trichoderma koningii, Trichoderma sp. and Phoma herbarum were assessed in the presence of hydrocarbons. Generally, aromatic hydrocarbons inhibited or stopped hyphal extension, though growth rates increased with some aliphatic hydrocarbons. Hyphal dry weight measurements suggested that Mortierella sp. may be able to use dodecane as sole carbon and energy source. Hydrocarbon-degrading Antarctic fungi may have use in future hydrocarbon spill bioremediation.

Arthroderma olidum, sp. nov. A new addition to the Trichophyton terrestre complex.

In 1981, four fungal isolates from hair of the European badger (Meles meles) were examined by Dr Phyllis Stockdale at the Commonwealth Mycological Institute, Kew, and deposited in the UK National Collection of Pathogenic Fungi as an undescribed member of the Trichophyton terrestre complex. The present paper formalizes the complete description of a new ascomycete taxon, Arthroderma olidum following successful recent attempts to re-isolate the same fungus from the soil of Badger holes in South West England. Furthermore, using ribosomal RNA gene sequencing, we show that the asexual form of A. olidum is conspecific with the recently described Trichophyton eboreum1 isolated from a human skin specimen in Germany.

Polycytella hominis is a mutated form of Scedosporium apiospermum.

PCR amplification and sequencing of two separate regions of the nuclear ribosomal repeat region revealed that Polycytella hominis, a hyphomycete isolated from a human case of mycetoma, was genetically indistinguishable from Scedosporium apiospermum (the anamorph of Pseudallescheria boydii). These organisms also exhibited remarkably similar susceptibility profiles to common antifungal agents. P. hominis is thus likely to be a mutant of S. apiospermum showing abnormalities of sporulation, for which a possible mechanism is discussed. Polycytella hominis should thus be regarded as a synonym of Scedosporium apiospermum.

Molecular analysis of geographic patterns of eukaryotic diversity in Antarctic soils.

We describe the application of molecular biological techniques to estimate eukaryotic diversity (primarily fungi, algae, and protists) in Antarctic soils across a latitudinal and environmental gradient between approximately 60 and 87 degrees S. The data were used to (i) test the hypothesis that diversity would decrease with increasing southerly latitude and environmental severity, as is generally claimed for "higher" faunal and plant groups, and (ii) investigate the level of endemicity displayed in different taxonomic groups. Only limited support was obtained for a systematic decrease in diversity with latitude, and then only at the level of a gross comparison between maritime (Antarctic Peninsula/Scotia Arc) and continental Antarctic sites. While the most southerly continental Antarctic site was three to four times less diverse than all maritime sites, there was no evidence for a trend of decreasing diversity across the entire range of the maritime Antarctic (60 to 72 degrees S). Rather, we found the reverse pattern, with highest diversity at sites on Alexander Island (ca. 72 degrees S), at the southern limit of the maritime Antarctic. The very limited overlap found between the eukaryotic biota of the different study sites, combined with their generally low relatedness to existing sequence databases, indicates a high level of Antarctic site isolation and possibly endemicity, a pattern not consistent with similar studies on other continents.

Dihydroisocoumarins and a tetralone from Cytospora eucalypticola.

Two dihydroisocoumarins, 3,5-dimethyl-8-hydroxy-7-methoxy-3,4-dihydroisocoumarin and 3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin were isolated from a culture filtrate of Cytospora eucalypticola, together with three known dihydroisocoumarins and a tetralone derivative. Their structures were determined by spectroscopic methods. These isocoumarins are mildly antifungal, and antibacterial towards gram positive bacteria. A known compound, 5-hydroxymethylmellein, showed mild antifeedant activity towards Spodoptera littoralis.

On the unreliability of published DNA sequences.

• Here, the reliability of published fungal nucleic acid sequences is tested by the critical re-evaluation of 206 named sequences obtained from public-access databases. • Sequences from the ribosomal RNA (rRNA) gene cluster were examined as these are commonly used to establish fungal phylogeny and evolution, and are also increasingly employed in the identification of fungi from nonculture based studies. • Fifty-one rRNA internal transcribed spacer (ITS) sequences were obtained for species of Amanita, 55 ITS sequences were obtained for species of Phoma and 100 rRNA small subunit sequences were obtained from representative genera of the order Helotiales. In each case, the fungal group was selected partly on the basis of sequences deposited by three or more laboratories in order to avoid sample bias. The results suggest that up to 20% of the sequences available for each group may be unreliable, and this proportion is supported by additional informal observations.

Genetic relatedness among Filobasidiella species.

The three accepted species of Filobasidiella, F. neoformans, F. depauperata, and F. lutea, are compared morphologically and by molecular analysis. Sequences of the internally transcribed spacer (ITS) and the small subunit (SSU) gene of the ribosomal RNA (rRNA) gene cluster were obtained, and analysed by Neighbor-joining and Maximum parsimony methods. The three species of Filobsidiella are shown to form a single monophyletic clade, rooted by Tremella mesenterica. F. lutea was recovered as a distinct, but closely related taxon with the Filobasidiella clade. This is the first report of DNA sequences from herbarium specimens of F. lutea.