Josep Cano

Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region

Abstract In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.

Sporothrix brasiliensis, S. globosa, and S. mexicana, Three New Sporothrix Species of Clinical Interest

ABSTRACT Sporothrix schenckii is the species responsible for sporotrichosis, a fungal infection caused by the traumatic implantation of this dimorphic fungus. Recent molecular studies have demonstrated that this species constitutes a complex of numerous phylogenetic species. Since the delineation of such species could be of extreme importance from a clinical point of view, we have studied a total of 127 isolates, most of which were received as S. schenckii, including the available type strains of species currently considered synonyms, and also some close morphological species. We have phenotypically characterized all these isolates using different culture media, growth rates at different temperatures, and numerous nutritional tests and compared their calmodulin gene sequences. The molecular analysis revealed that Sporothrix albicans, S. inflata, and S. schenckii var. luriei are species that are clearly different from S. schenckii. The combination of these phenetic and genetic approaches allowed us to propose the new species Sporothrix brasiliensis, S. globosa, and S. mexicana. The key phenotypic features for recognizing these species are the morphology of the sessile pigmented conidia, growth at 30, 35, and 37°C, and the assimilation of sucrose, raffinose, and ribitol.

Molecular Phylogeny of the Pseudallescheria boydii Species Complex: Proposal of Two New Species

ABSTRACT Pseudallescheria boydii (anamorph Scedosporium apiospermum) is the species responsible for human scedosporiosis, a fungal infection with a high mortality rate and which is difficult to treat. Recently, it has been demonstrated that high genetic variation exists within this species. We have performed a morphological and molecular study involving numerous strains of clinical or environmental origins and from different countries. The analysis of partial sequences of the β-tubulin (two loci) and calmodulin genes and the internal transcribed spacer region of the rRNA gene has demonstrated that P. boydii is a species complex. The combined analysis of the sequences of the four loci of 60 strains has showed the presence of 44 haplotypes in the ingroup. Three species morphologically related to P. boydii sensu stricto, i.e., Pseudallescheria angusta, Pseudallescheria ellipsoidea, and Pseudallescheria fusoidea, which had previously been considered synonyms, could be differentiated genetically from P. boydii in our study. It is relevant that two of the three strains now included in P. ellipsoidea have caused invasive infections. The species Pseudallescheria minutispora and Scedosporium aurantiacum are clearly phylogenetically separated from the other species studied and are here proposed as new. Morphological features support this proposal. All the strains included in S. aurantiacum species have a clinical origin, while those included in P. minutispora are environmental. Further studies are needed to demonstrate whether all the species included in the P. boydii complex have different clinical spectra and antifungal susceptibility.

Molecular and Phenotypic Data Supporting Distinct Species Statuses for Scedosporium apiospermum and Pseudallescheria boydii and the Proposed New Species Scedosporium dehoogii

ABSTRACT Based on the morphological, physiologic, and molecular (β-tubulin gene) study of 141 isolates of the Pseudallescheria boydii species complex (including several synonyms) and relatives, the new species Scedosporium dehoogii is proposed. Scedosporium apiospermum and P. boydii are considered two different species and the new name Scedosporium boydii is proposed for the anamorph of the latter species. A summary of the key morphological and physiological features for distinguishing the species of Pseudallescheria/Scedosporium is provided.

Isolation and identification of yeasts associated with vineyard and winery by RFLP analysis of ribosomal genes and mitochondrial DNA.

Yeast colonies isolated from vineyard and cellar substrates were analysed in the present study. Yeast species assessment was carried out by amplification and digestion of a region of the ribosomal RNA gene repeat unit. Saccharomyces strains were also characterised using mitochondrial DNA restriction analysis. Oxidative basidiomycetous yeasts without enological potential were predominant in the vineyard environment. Yeasts associated with grape skin depend on grape variety, vintage and degree of grape maturation. These species from grape surface constituted the predominant microbiota in must and they developed during the first stages of the process. Yeasts colonies were also isolated and identified from the walls of a fermentation vat some days before the harvest. Contray to what was expected, Saccharomyces cerevisiae was not the major species isolated as Candida sorbosa represented 76% of the species isolated. Saccharomyces strains isolated from the fermentation vat had been previously isolated in wine fermentations in this cellar. Therefore, these strains should be considered as constant residents of this winery.

Antifungal Susceptibilities of the Species of the Pseudallescheria boydii Complex

ABSTRACT Eighty-four isolates belonging to eight species that constitute the Pseudallescheria boydii complex were tested against 11 antifungal agents by using the microdilution method. There were significant differences among the species, with Scedosporium aurantiacum being the most resistant. In general, voriconazole was the most active drug, followed by posaconazole.

Phenotypic and Molecular Characterization of Candida nivariensis sp. nov., a Possible New Opportunistic Fungus

ABSTRACT The new species Candida nivariensis, isolated from the clinical samples of three patients in Spain over a 3-year period, is presented here. This species can be easily differentiated from Candida glabrata, the closest genetic species, by different colony color on CHROMagar and by its ability to ferment trehalose. The analyses of the internal transcribed spacer region and the D1-D2 region of the 26S rRNA gene sequences support a new species designation.

Molecular Phylogeny of Sporothrix schenckii

ABSTRACT The pathogenic dimorphic fungus Sporothrix schenckii is the agent responsible for sporotrichosis, an important fungal infection with a worldwide distribution. Little is known about the population structure of S. schenckii, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to determine, by sequence analysis of three protein coding loci (chitin synthase, β-tubulin, and calmodulin), whether this variability is due to species divergence or intraspecific diversity in S. schenckii. We included in the analysis 60 isolates (59 of clinical and 1 of environmental origin) of this species from a wide geographical range. DNA sequence data from the three nuclear regions were used in a phylogenetic analysis. The combined analysis of the three loci revealed the presence of three major clades, one grouping all of the European isolates, another with only Brazilian isolates, and the third with isolates from other South American countries and Africa. A total of 14 100% bootstrap-supported nodes were shown, 6 of them representing putative phylogenetic species. Our data also demonstrated that most of these species prevail in different geographical regions.

In Vitro Antifungal Susceptibilities of Five Species of Sporothrix

ABSTRACT Ninety-two isolates belonging to five species of the Sporothrix schenckii complex were tested in vitro against 12 antifungal agents, using a reference microdilution method. There were significant differences among the species; Sporothrix brasiliensis was the species that showed the best response to antifungals, and S. mexicana had the worst response. In general, terbinafine was the most active drug, followed by ketoconazole and posaconazole.

Molecular and Morphological Identification of Colletotrichum Species of Clinical Interest

ABSTRACT Colletotrichum species have caused human infections in recent years. Because of the difficulties in recognizing them in vitro, we have designed a quick and unambiguous molecular test, based on the amplification of a specific fragment of the internal transcribed spacer 1 region, to distinguish any Colletotrichum isolate from other fungi, including the common pathogenic species. Analysis of the sequences of the ribosomal DNA (rDNA) fragment showed sufficient variability to clearly separate the five species of Colletotrichum that are of clinical interest, i.e., Colletotrichum coccodes, C. crassipes, C. dematium, C. gloeosporioides, and C. graminicola. Sequencing of the D1-D2 region of the large-subunit rDNA gene also supported these results. Additionally, we reviewed the most suitable morphological characteristics for the in vitro identification of these increasingly important opportunistic fungi.