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.

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.

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.

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.

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.

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.

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.

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.

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.