Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF)

Cystic fibrosis (CF) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. The black yeast, Exophiala dermatitidis (synonym Wangiella dermatitidis) has been found with increasing frequency in sputum specimens of CF patients, with reported isolation rates ranging from 1.1 to 15.7%. At present, no diagnostic PCR exists to aid with the clinical laboratory detection and identification of this organism. A novel species-specific PCR-based assay was developed for the detection of E. dermatitidis, based on employment of rDNA operons and interspacer (ITS) regions between these rDNA operons. Two novel primers, (designated ExdF & ExdR) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of Exophiala, as well as with other commonly described yeasts and filamentous fungi within CF sputum, including Candida. Aspergillus and Scedosporium. An amplicon of approximately 455 by was generated, spanning the partial ITS I region - the complete 5.8S rDNA region - partial ITS2 region, employing ExdF (forward primer [16-mer], 5'-CCG CCT ATT CAG GTC C-3' and ExdR (reverse primer [16-mer], 5'-TCT CTC CCA CTC CCG C-3', was employed and optimised on extracted genomic DNA from a well characterised culture of E. dermatitidis, as well as with high quality genomic DNA template from a further 16 unrelated fungi, including Candida albicans, C. dubliniensis, C. parapsilosis, C. glabrata, Scedosporium apiospermum, Penicillium sp., Aspergillus fumigatus, Aspergillus versicolor, Pichia guilliermondii, Rhodotorula sp., Trichosporon sp., Aureobasidium pullulans, Fusarium sp., Mucor hiemalis, Bionectria ochroleuca, Gibberella pulicaris. Results demonstrated that only DNA from E. dermatitidis gave an amplification product of the expected sire, whilst none of the other fungi were amplifiable. Subsequent employment of this primer pair detected this yeast from mycological cultures from 2/50 (4%) adult CF patients. These two patients were the only patients who were previously shown to have a cultural history of E. dermatitidis from their sputum. E. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan- and multiresistant bacterial pathogens from sputum. namely Pseudomonas aeruginosa and Burkholderia cepacia complex organisms, hence this species-specific PCR assay may help detect this organism from CF sputum more specifically and rapidly. Overall, employment of this novel assay nay help in the understanding of the occurrence. aetiology and epidemiology of E. dermatitidis, as an emerging fungal agent in patients with CF. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Facile biocatalytic reduction of the carbon-carbon double bond of 5-benzylidenethiazolidine-2,4-diones. Synthesis of ( )-5-(4-{2-[methyl(2-pyridyl)amino]ethoxy}benzyl)thiazolidine-2,4-dione (BRL 49653), its (R)-(+)-enantiomer and analogs.

Cantello, Barrier C. C.; Eggleston, Drake S.; Haigh, David; Haltiwanger, R. Curtis; Heath, Catherine M.; Hindley, Richard M.; Jennings, Keith R.; Sime, John T.; Woroniecki, Stefan R. SmithKline Beecham Pharmaceuticals, Surrey, UK. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1994), (22), 3319-24. Publisher: Royal Society of Chemistry, CODEN: JCPRB4 ISSN: 0300-922X. Journal written in English. CAN 122:105736 AN 1995:237497 CAPLUS (Copyright (C) 2009 ACS on SciFinder (R)) Abstract A novel biotransformation system for the redn. of carbon-carbon double bonds in 5-benzylidenethiazolidine-2,4-diones to give the corresponding 5-benzylthiazolidine-1,4-diones, using whole cells of red yeasts, is described. These reduced compds., which are recovered in good yield, are of potential use in the treatment of non-insulin dependent diabetes mellitus. The mild reaction conditions developed allow redn. of 5-benzylidenethiazolidine-2,4-diones contg. other functionalities which are not compatible with alternative redn. methods. The biocatalytic redn. is enantioselective and the synthesis of R-(+)-5-(4-{2-[methyl(2-pyridyl)amino]ethoxy}benzyl)thiazolidine-2,4-dione by Rhodotorula rubra CBS 6469 and structure confirmation by X-ray crystallog. is detailed. Optimization of reaction conditions (including immobilization) for these whole cell redn. system is described.

The biology of habitat dominance; can microbes behave as weeds?

Competition between microbial species is a product of, yet can lead to a reduction in, the microbial diversity of specific habitats. Microbial habitats can resemble ecological battlefields where microbial cells struggle to dominate and/or annihilate each other and we explore the hypothesis that (like plant weeds) some microbes are genetically hard-wired to behave in a vigorous and ecologically aggressive manner. These 'microbial weeds' are able to dominate the communities that develop in fertile but uncolonized - or at least partially vacant - habitats via traits enabling them to out-grow competitors; robust tolerances to habitat-relevant stress parameters and highly efficient energy-generation systems; avoidance of or resistance to viral infection, predation and grazers; potent antimicrobial systems; and exceptional abilities to sequester and store resources. In addition, those associated with nutritionally complex habitats are extraordinarily versatile in their utilization of diverse substrates. Weed species typically deploy multiple types of antimicrobial including toxins; volatile organic compounds that act as either hydrophobic or highly chaotropic stressors; biosurfactants; organic acids; and moderately chaotropic solutes that are produced in bulk quantities (e.g. acetone, ethanol). Whereas ability to dominate communities is habitat-specific we suggest that some microbial species are archetypal weeds including generalists such as: Pichia anomala, Acinetobacter spp. and Pseudomonas putida; specialists such as Dunaliella salina, Saccharomyces cerevisiae, Lactobacillus spp. and other lactic acid bacteria; freshwater autotrophs Gonyostomum semen and Microcystis aeruginosa; obligate anaerobes such as Clostridium acetobutylicum; facultative pathogens such as Rhodotorula mucilaginosa, Pantoea ananatis and Pseudomonas aeruginosa; and other extremotolerant and extremophilic microbes such as Aspergillus spp., Salinibacter ruber and Haloquadratum walsbyi. Some microbes, such as Escherichia coli, Mycobacterium smegmatis and Pseudoxylaria spp., exhibit characteristics of both weed and non-weed species. We propose that the concept of nonweeds represents a 'dustbin' group that includes species such as Synodropsis spp., Polypaecilum pisce, Metschnikowia orientalis, Salmonella spp., and Caulobacter crescentus. We show that microbial weeds are conceptually distinct from plant weeds, microbial copiotrophs, r-strategists, and other ecophysiological groups of microorganism. Microbial weed species are unlikely to emerge from stationary-phase or other types of closed communities; it is open habitats that select for weed phenotypes. Specific characteristics that are common to diverse types of open habitat are identified, and implications of weed biology and open-habitat ecology are discussed in the context of further studies needed in the fields of environmental and applied microbiology.