Jack W. Fell

Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis

The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, Cystofilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.

Hybrid genotypes in the pathogenic yeast Cryptococcus neoformans

Amplified fragment length polymorphism (AFLP) genotyping of isolates of the pathogenic fungus Cryptococcus neoformans suggested a considerable genetic divergence between the varieties C. neoformans var. neoformans and C. neoformans var. grubii on the one hand versus C. neoformans var. gattii on the other. This divergence is supported by additional phenotypic, biochemical, clinical and molecular differences. Therefore, the authors propose the existence of two species, C. neoformans (Sanfelice) Vuillemin and C. bacillisporus Kwon-Chung, which differ in geographical distribution, serotypes and ecological origin. Within each species three AFLP genotypes occur, which differ in geographical distribution and serotypes. Differences in ecological origin (AIDS patients, non-AIDS patients, animals or the environment) were found to be statistically not significant. In C. neoformans as well as in C. bacillisporus one of the genotypes represented a hybrid. The occurrence of hybridization has consequences for the reproductive biology of the species, as new genotypes with altered virulence or susceptibility to antifungal drugs may arise through the exchange of genetic material.

Methods for isolation, phenotypic characterization and maintenance of yeasts

Yeasts are recovered from a wide range of aquatic, marine, atmospheric, and terrestrial habitats. Many yeasts occur widely, whereas some appear to be confined to specific habitats. Yeasts seldom occur in the absence of either molds or bacteria. Consequently, selective techniques are often used for recovery of yeasts, employing media that permit the yeasts to grow, while suppressing molds and bacteria. Under the morphological characterization of yeasts there are several factors that include: texture, color, surface, elevation, and margin. In texture, mucoid growth is frequently associated with encapsulation of cells from production of extracellular polysaccharides; membranous growth generally results from profuse formation of hyphae or pseudohyphae. In color, the presence of red, orange, or yellow nondiffusible carotenoid pigments is characteristic of certain genera, for instance, Phaffia, Rhodosporidium, and Sporidiobolus. In surface, the strains that are smooth when first isolated sometimes become rough when maintained on agar. This change is, in some cases, accompanied by a change in texture from butyrous to membranous. Restreaking generally results, once again, in formation of smooth and rough colonies. In elevation, the growth is flat, depressed in the center, raised and dome-like, or conical. And in margin, the edge of the streak or colony is entire, undulating, lobed, erose, or fringed with hyphae or pseudohyphae.

Polyphasic taxonomy of the basidiomycetous yeast genus Rhodosporidium: Rhodosporidium kratochvilovae and related anamorphic species

The phenotypic and genetic heterogeneity of the basidiomycetous yeast species Rhodosporidium kratochvilovae was investigated in a group of recent isolates and collection strains. A polyphasic taxonomic approach was followed which included micromorphological studies, nuclear staining, determination of sexual compatibility, physiological characterization, comparison of electrophoretic isoenzyme patterns, PCR fingerprinting, determination of mol% G+C, DNA-DNA reassociation experiments and 26S and ITS rDNA sequence analysis. The results allowed a more natural circumscription of the species, both from the genetic and phenotypic perspectives. The relationships with anamorphic species of the genus Rhodotorula were studied and isolates previously identified as Rhodotorula glutinis were found to belong to Rhodosporidium kratochvilovae. Other isolates included in the study were found to represent members of Rhodotorula glutinis var. dairenensis. Rhodosporidium kratochvilovae was found to include heterothallic strains, besides those already known to be self-sporulating. A total of 17 isolates, which were found to belong to this species, were heterothallic, self-sporulating and anamorphic strains. It is anticipated that integrated polyphasic studies of basidiomycetous yeasts will provide a more coherent classification system and the basis for accurate identification schemes, which in turn are essential for detailed ecological studies.

Bandoniozyma gen. nov., a Genus of Fermentative and Non-Fermentative Tremellaceous Yeast Species

Background Independent surveys across the globe led to the proposal of a new basidiomycetous yeast genus within the Bulleromyces clade of the Tremellales, Bandoniozyma gen. nov., with seven new species. Methodology/Principal Findings The species were characterized by multiple methods, including the analysis of D1/D2 and ITS nucleotide sequences, and morphological and physiological/biochemical traits. Most species can ferment glucose, which is an unusual trait among basidiomycetous yeasts. Conclusions/Significance In this study we propose the new yeast genus Bandoniozyma, with seven species Bandoniozyma noutii sp. nov. (type species of genus; CBS 8364T  =  DBVPG 4489T), Bandoniozyma aquatica sp. nov. (UFMG-DH4.20T  =  CBS 12527T  =  ATCC MYA-4876T), Bandoniozyma complexa sp. nov. (CBS 11570T  =  ATCC MYA-4603T  =  MA28aT), Bandoniozyma fermentans sp. nov. (CBS 12399T  =  NU7M71T  =  BCRC 23267T), Bandoniozyma glucofermentans sp. nov. (CBS 10381T  =  NRRL Y-48076T  =  ATCC MYA-4760T  =  BG 02-7-15-015A-1-1T), Bandoniozyma tunnelae sp. nov. (CBS 8024T  =  DBVPG 7000T), and Bandoniozyma visegradensis sp. nov. (CBS 12505T  =  NRRL Y-48783T  =  NCAIM Y.01952T).

High-Throughput Detection of Pathogenic Yeasts of the Genus Trichosporon

ABSTRACT The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 102 genome molecules in PCRs and 107 to 108 molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.

Molecular sequence analyses of the intergenic spacer (IGS) associated with rDNA of the two varieties of the pathogenic yeast, Cryptococcus neoformans

The pathogen Crytococcus neoformans has been traditionally grouped in two varieties, C. neoforrmans var. neoformans (serotypes A, D and AD) and C. neoformans var. gattii (serotypes B and C). A recent taxonomic evaluation of C. neoformans var. neoformans described C. neoformans var. grubii as a new variety represented by serotype A isolates. Despite immunological, biochemical, ecological and molecular differences the three varieties are classified within one species. We examined the genetic variability of one hundred and five clinical and environmental isolates that included all varieties and serotypes. Sequence analysis of the intergenic spacer (IGS) associated with rDNA revealed significant differences in nucleotide composition between and within the varieties. Parsimony analysis showed five different genotypes representing distinct genetic lineages. Although there was a high degree of relatedness between serotype and genotype this relatedness was not exclusive as serotypes were not restricted to one particular genotypic group. Serotyping and sequence analyses indicate that C. neoformans var. grubii (serotype A) should not be recognized as a separate variety. Based on this study we propose to accept two separate species, C. neoformans (serotypes A, D and AD) and C. bacillisporus (serotypes B and C synonymous with C. neoformans var. gattii).

Chapter 90 – Candida Berkhout (1923)

Publisher Summary This chapter studies the genus Candida. In the asexual reproduction it is seen that cells are globose, ellipsoidal, cylindroidal, or elongate and occasionally ogival, triangular, or lunate. Reproduction is by holoblastic budding. Pseudohyphae and septate hyphae may be formed. The cell wall is ascomycetous and two-layered. Ballistoconidia are not formed. Arthroconidia may be formed, but not extensively. Sexual reproduction is absent. The chapter also discusses physiology/biochemistry and phylogenetic placement of the genus in which glucose may be fermented, nitrate may be assimilated, and starch-like compounds are not produced. The diazonium blue B reaction is negative and xylose, rhamnose, and fucose are not present in cell hydrolysates. The type species taken is Candida vulgaris. The chapter also explores the systematic discussion of the species that involves growth on YM agar, growth in glucose-yeast extract broth, and Dalmau plate culture on corn meal agar.

Cryptococcus Vuillemin (1901)

Publisher Summary This chapter discusses the genus Cryptococcus. In the determination of the asexual reproduction it is seen that cells are spheroidal, ovoid, ellipsoidal, or elongate. A polysaccharide capsule is present in most species. Reproduction is by multilateral or polar, enteroblastic budding; pseudohyphae or true hyphae may develop. In species with true hyphae, septa have dolipores with or without parenthesomes. In the sexual reproduction it is found that some species are anamorphic states of teleomorphic genera in the Cystofilobasidiales, Filobasidiales, and Tremellales. The chapter also discusses physiology/biochemistry and phylogenetic placement of the genus. The type species taken is Cryptococcus neoformans. In the systematic discussion of the species phylogenetic placement, growth on malt extract, soytone, yeast extract (MYP) agar, growth in YM broth, Dalmau plate culture on corn meal agar, gene sequence accession numbers, type strain, origin of the strains studied, and systematics are determined.

Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex

ABSTRACT Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immunocompromised patients. The disease, cryptococcosis, is mostly acquired by inhalation and can result in a chronic meningoencephalitis, which can be fatal. Here, we describe a molecular method to identify the varieties and genotypic groups within the C. neoformans species complex from culture-based assays. The method employs a novel flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin R-phycoerythrin. In this study we developed and validated eight probes derived from sequence analysis of the intergenic spacer region of the rRNA gene region. The assay proved to be specific and sensitive, allowed discrimination of a 1-bp mismatch with no apparent cross-reactivity, and detected 101 to 103 genome copies. The described protocol, which can be used directly with yeast cells or isolated DNA, can be undertaken in less than 1 h following PCR amplification and permits identification of species in a multiplex format. In addition to a multiplex capability, the assay allows the simultaneous detection of target sequences in a single reaction. The accuracy, speed, flexibility, and sensitivity of this technology are a few of the advantages that will make this assay useful for the diagnosis of human cryptococcal infections and other pathogenic diseases.