Da Vinci's yeast: Blastobotrys davincii f.a., sp. nov.

A new species of the yeast genus Blastobotrys was discovered during a worldwide survey of culturable xerophilic fungi in house dust. Several culture-dependent and independent studies from around the world detected the same species from a wide range of substrates including indoor air, cave wall paintings, bats, mummies, and the iconic self-portrait of Leonardo da Vinci from ca 1512. However, none of these studies identified their strains, clones, or OTUs as Blastobotrys. We introduce the new species as Blastobotrys davincii f.a., sp. nov. (holotype CBS H-24879) and delineate it from other species using morphological, phylogenetic, and physiological characters. The new species of asexually (anamorphic) budding yeast is classified in Trichomonascaceae and forms a clade along with its associated sexual state genus Trichomonascus. Despite the decade-old requirement to use a single generic name for fungi, both names are still used. Selection of the preferred name awaits a formal nomenclatural proposal. We present arguments for adopting Blastobotrys over Trichomonascus and introduce four new combinations as Blastobotrys allociferrii (≡ Candida allociferrii), B. fungorum (≡ Sporothrix fungorum), B. mucifer (≡ Candida mucifera), and Blastobotrys vanleenenianus (≡ Trichomonascus vanleenenianus). We provide a nomenclatural review and an accepted species list for the 37 accepted species in the Blastobotrys/Trichomonascus clade. Finally, we discuss the identity of the DNA clones detected on the da Vinci portrait, and the importance of using appropriate media to isolate xerophilic or halophilic fungi.

Mould spoilage of foods and beverages: Using the right methodology.

Fungal spoilage of products manufactured by the food and beverage industry imposes significant annual global revenue losses. Mould spoilage can also be a food safety issue due to the production of mycotoxins by these moulds. To prevent mould spoilage, it is essential that the associated mycobiota be adequately isolated and accurately identified. The main fungal groups associated with spoilage are the xerophilic, heat-resistant, preservative-resistant, anaerobic and psychrophilic fungi. To assess mould spoilage, the appropriate methodology and media must be used. While classic mycological detection methods can detect a broad range of fungi using well validated protocols, they are time consuming and results can take days or even weeks. New molecular detection methods are faster but require good DNA isolation techniques, expensive equipment and may detect viable and non-viable fungi that probably will not spoil a specific product. Although there is no complete and easy method for the detection of fungi in food it is important to be aware of the limitation of the methodology. More research is needed on the development of methods of detection and identification that are both faster and highly sensitive.

Identification of Immunoglobulin E-Binding Proteins of the Xerophilic Fungus Aspergillus penicillioides Crude Mycelial Mat Extract and Serological Reactivity Assessment in Subjects with Different Allergen Reactivity Profiles.

BACKGROUND: Aspergillus penicillioides is a very common indoor xerophilic fungus and potential causative agent of respiratory conditions. Although people are constantly exposed to A. penicillioides, no proteins with allergenic potential have been described. Therefore, we aim to confirm allergic sensitization to A. penicillioides through reactivity in serological assays and detect immunoglobulin E (IgE)-binding proteins. METHODS: In an indirect ELISA, we compared the serological reactivity to A. penicillioides between subjects with specific IgE (sIgE) (group 1, n = 54) and no sIgE reactivity (group 2, n = 15) against commercial allergens. Correlations and principal component analysis were performed to identify associations between reactivity to commercial allergens and A. penicillioides. IgE-binding proteins in A. penicillioides were visualized using Western blotting (WB) in group 1. The IgE-binding proteins with the highest reactivity were analyzed by mass spectrometry and confirmed by transcript matching. RESULTS: There was no statistical significance (p = 0.1656) between the study groups in serological reactivity. Correlations between reactivity to A. penicillioides, dog epithelia, Aspergillus fumigatus, and Penicillium chrysogenum were observed. WB experiments showed 6 IgE-binding proteins with molecular weights ranging from 45 to 145 kDa. Proteins of 108, 83, and 56 kDa showed higher reactivity. Mass spectrometry analysis of these 3 proteins led to the putative identification of NADP-specific glutamate dehydrogenase and catalase B. This was confirmed with transcriptome analysis. CONCLUSIONS: These results provide evidence of the presence of potential allergenic components in A. penicillioides. Further analysis of the putatively identified proteins should reveal their allergenic potential.

Phylogeny of xerophilic aspergilli (subgenus Aspergillus) and taxonomic revision of section Restricti.

Aspergillus section Restricti together with sister section Aspergillus (formerly Eurotium) comprises xerophilic species, that are able to grow on substrates with low water activity and in extreme environments. We adressed the monophyly of both sections within subgenus Aspergillus and applied a multidisciplinary approach for definition of species boundaries in sect. Restricti. The monophyly of sections Aspergillus and Restricti was tested on a set of 102 isolates comprising all currently accepted species and was strongly supported by Maximum likelihood (ML) and Bayesian inferrence (BI) analysis based on ß-tubulin (benA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) loci. More than 300 strains belonging to sect. Restricti from various isolation sources and four continents were characterized by DNA sequencing, and 193 isolates were selected for phylogenetic analyses and phenotypic studies. Species delimitation methods based on multispecies coalescent model were employed on DNA sequences from four loci, i.e., ID region of rDNA (ITS + 28S), CaM, benA and RPB2, and supported recognition of 21 species, including 14 new. All these species were also strongly supported in ML and BI analyses. All recognised species can be reliably identified by all four examined genetic loci. Phenotype analysis was performed to support the delimitation of new species and includes colony characteristics on seven cultivation media incubated at several temperatures, growth on an osmotic gradient (six media with NaCl concentration from 0 to 25 %) and analysis of morphology including scanning electron microscopy. The micromorphology of conidial heads, vesicle dimensions, temperature profiles and growth parameters in osmotic gradient were useful criteria for species identification. The vast majority of species in sect. Restricti produce asperglaucide, asperphenamate or both in contrast to species in sect. Aspergillus. Mycophenolic acid was detected for the first time in at least six members of the section. The ascomata of A. halophilicus do not contain auroglaucin, epiheveadride or flavoglaucin which are common in sect. Aspergillus, but shares the echinulins with sect. Aspergillus.

Detection and identification of xerophilic fungi in Belgian chocolate confectionery factories.

Chocolate confectionery fillings are generally regarded as microbiologically stable. The stability of these fillings is largely due to the general practice of adding either alcohol or preservatives. Consumer demands are now stimulating producers to move away from adding alcohol or other preservatives to their confectionery fillings and instead to search for innovative formulations. Such changes in composition can influence the shelf life of the product and may lead to spoilage by xerophilic fungi. The aim of this study was to test whether the production environment of Belgian chocolate confectionery factories and common ingredients of chocolate confectioneries could be potential sources of contamination with xerophilic fungal species. In the factory environment, the general and strictly xerophilic fungal spore load was determined using an RCS Air Sampler device in combination with DG18 and MY50G medium, respectively. Four basic ingredients of chocolate confectionery fillings were also examined for fungal spore levels using a direct plating technique. Detected fungi were identified to species level by a combination of morphological characterization and sequence analysis. Results indicated a general fungal spore load in the range of 50-250 colony forming units per cubic meter of air (CFU/m(3) air) and a more strict xerophilic spore load below 50 CFU/m(3) air. These results indicate rather low levels of fungal spores present in the factory environment. The most prevalent fungi in the factory environment were identified as Penicillium spp., particularly Penicillium brevicompactum. Examination of the basic ingredients of confectionery fillings revealed nuts to be the most likely potential source of direct contamination. In nuts, the most prevalent fungal species identified were Eurotium, particularly Eurotium repens.

Synergistic antifungal effects of the preservative ammonium propionate and medium chain fatty acids against dormant and germinating conidia, germ tubes and hyphae of Aspergillus chevalieri, a feed spoilage fungus.

In feed, propionic acid is the weak organic acid of choice to prevent growth of spoilage fungi. For safe and easy industrial handling this antifungal agent is applied in the presence of neutralizing ammonium, which however has the disadvantage to negatively affect the efficacy of fungus-inhibiting properties of the formulation. In the present study we investigated the impact of medium chain fatty acids (MCFA) on the antifungal efficacy of an ammonium propionate formulation on dormant- and germinating conidia as well as germ tubes and hyphae of Aspergillus chevalieri, a xerophilic fungus predominant on moulded feed. Dormant conidia were not affected by 32 mM of ammonium propionate after a 28 h-treatment in demi water. Similar results were obtained with solely 0.52 mM MCFA. However, the combination of both components nearly eradicated formation of colonies from these conidia and was accompanied by distortion of the cellular structure as was visible with light- and transmission electron microscopy. Germination of conidia, characterised by swelling and germ tube formation, was significantly decreased in the presence of 16 mM ammonium propionate and 0.26 mM MCFA, while the latter component itself did not significantly decrease germination. We conclude that a combination of ammonium propionate and MCFA had a synergistic antifungal effect on dormant and germinating conidia. When the combination of ammonium propionate and MCFA was tested on hyphae for 30 min, we observed that cell death was significantly increased in comparison to components alone. Treatment of the hyphae with 16 mM of ammonium propionate caused aberrant mitochondria, as evidenced by irregularly shaped and enlarged mitochondria that contained electron-dense inclusions as observed by transmission electron microscopy. When the combination of ammonium propionate and MCFA was applied against the hyphae, more severe cell damage was observed, with signs of autophagy. Summarised, our results demonstrate synergistic antifungal effects of ammonium propionate and medium chain fatty acids on fungal survival structures, during their germination and after a short (sudden) treatment of growing cells. This is of potential importance for several areas of feed and food storage and shelf-life.

Big Sound and Extreme Fungi-Xerophilic, Halotolerant Aspergilli and Penicillia with Low Optimal Temperature as Invaders of Historic Pipe Organs.

Recent investigations have shown that xerophilic fungi may pose a biodeterioration risk by threatening objects of cultural heritage including many types of materials, including wood, paint layers, organic glues or leather and even metal. Historic—and also new built—pipe organs combine all those materials. In this study, halotolerant aspergilli and penicillia with low optimal temperatures were shown to be the most frequent invaders of pipe organs. The fungi form white mycelia on the organic components of the organs with a clear preference for the bolus paint of the wooden pipes, the leather-made hinges of the stop actions and all parts fixed by organic glue. Physiological tests showed that the strains isolated from the instruments all show a halotolerant behavior, although none was halophilic. The optimum growth temperature is below 20 °C, thus the fungi are perfectly adapted to the cool and relatively dry conditions in the churches and organs respectively. The de-novo genome sequences analyses of the strains are currently ongoing and will reveal the genomic basis for the halotolerant behavior of the fungi.

Efeito da adição de dicloran ao diluente, para enumeração de fungos em alimentos desidratados utilizando-se o sistema petrifilm para bolores e leveduras / Effect of the addition of dicloran to the diluent, for enumeration of fungi in sustenances dehydrated utilizing itself the system petrifilm for fungi and yeasts

A contagem de bolores e leveduras nos alimentos é indicativa de falhas higiênicas ao longo do processamento ou matérias-primas de má qualidade. Nos alimentos desidratados, os fungos xerofílicos representam os principais microrganismos responsáveis de multiplicação. Diversas técnicas e meios de cultura tem sido utilizados para a sua enumeração para alimentos com baixa atividade de água. Dez amostras de diferentes alimentos foram divididas em 5 subamostras cada, totalizando 50 amostras. Os seguintes tratamentos foram avaliados: Petrifilm (controle), Petrifilm (com dicloran) e DG-18 (controle). Os resultados da análise estatística permitem concluir que não há diferença significativa, ao nível de 5 por cento, entre os tratamentos avaliados. A ocorrência de colônias espalhadas parece não estar associada a um tipo de alimento, mas às amostras de um determinado alimento. Ao se usar altas diluições, as colônias espalhadas não estiveram comumente presentes, não afetando, portanto, a contagem no Petrifilm Bolores e Leveduras.

The yeast and mold enumeration of foods is indicative of poor hygienical conditions in the processing or raw materials of bad quality. In dehydrated foods, the xerophilic fungi are the major responsible of deterioration. Several techniques and culture media have been used for its enumeration, being agar DG-18 recommended for foods with low water activity. Ten differents food samples were divided into five subsamples, totalizing 50 samples. The following treatments were evaluaied: PetrifiIm (control), PetrifiIm (with dicloran) and DG-18 (control). The results of the statistical analysis allow to conclude that it does not have significant difference to the 5% level between the evaluated treatments. The occurrence of spread colonies, seems not to be associated to a type of food, but with samples of one determined food. When high dilutions were used, spread colonies weren't present, not affecting, therefore, the Petrifilm Yeast and Mold counts.