Co-inoculation of yeasts starters: A strategy to improve quality of low altitude Arabica coffee.

The study aimed to improve the quality of dry-processed coffee grown at low altitudes through yeast inoculation, using three species (Saccharomyces cerevisiae CCMA 0543, Torulaspora delbrueckii CCMA 0684, and Candida parapsilosis CCMA 0544) singly and with co-inoculation for fermentation. Important chemical compounds and groups were analyzed by liquid and gas chromatography and Fourier-transform infrared spectroscopy (FTIR). The inoculated coffees with yeast populations around 106 cell/g obtained the highest scores, and the co-inoculation with C. parapsilosis CCMA 0544 and T. delbrueckii CCMA 0684 had the highest score in the sensory analysis (85). Different descriptors were observed in each treatment, and body, flavor, balance, and aftertaste are strongly related to C. parapsilosis CCMA 0544. The fermentation process improved the quality of low-altitude coffees, and the combination of non-Saccharomyces yeasts (C. parapsilosis CCMA 0544 and T. delbrueckii CCMA 0684) was the most indicated as starter cultures.

Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 µg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and ß(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of ß(1→3) glucans and ß(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses.

Yeast identification by sequencing, biochemical kits, MALDI-TOF MS and rep-PCR DNA fingerprinting.

No study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI-TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, using 71 clinically important yeast isolates (excluding Candida albicans) collected from a single centre, we determined the concordance of 28S nrDNA and ITS sequencing and evaluated the performance of two commercial test kits, two MALDI-TOF MS platforms, and rep-PCR DNA fingerprinting. 28S nrDNA and ITS sequencing showed complete agreement on the identities of the 71 isolates. Using sequencing results as the standard, 78.9% and 71.8% isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively; and 90.1% and 80.3% isolates were correctly identified using the Bruker and Vitek MALDI-TOF MS platforms, respectively. Of the 18 strains belonging to the Candida parapsilosis species complex tested by DiversiLab automated rep-PCR DNA fingerprinting, all were identified only as Candida parapsilosis with similarities ≥93.2%, indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, hierarchical cluster analysis of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters, indicating that this technology can potentially differentiate the three species. To achieve higher accuracies of identification, the databases of commercial biochemical test kits, MALDI-TOF MS platforms, and DiversiLab automated rep-PCR DNA fingerprinting needs further enrichment, particularly for uncommonly encountered yeast species.

Impact of ERG3 mutations and expression of ergosterol genes controlled by UPC2 and NDT80 in Candida parapsilosis azole resistance.

OBJECTIVES: Candida parapsilosis is a healthcare-related fungal pathogen particularly common among immunocompromised patients. Our understanding of antifungal resistance mechanisms in C. parapsilosis remains very limited. We previously described an azole-resistant strain of C. parapsilosis (BC014RPSC), obtained following exposure in vitro to posaconazole. Resistance was associated with overexpression of ergosterol biosynthetic genes (ERG genes), together with the transcription factors UPC2 (CPAR2-207280) and NDT80 (CPAR2-213640). The aim of this study was to identify the mechanisms underlying posaconazole resistance of the BC014RPSC strain. METHODS: To identify the causative mutation, we sequenced the genomes of the susceptible (BC014S) and resistant (BC014RPSC) isolates, using Illumina technology. Ergosterol content was assessed in both strains by mass spectrometry. UPC2 and NDT80 genes were deleted in BC014RPSC strain. Mutants were characterized regarding their azole susceptibility profile and ERG gene expression. RESULTS: One homozygous missense mutation (R135I) was found in ERG3 (CPAR2-105550) in the azole-resistant isolate. We show that Erg3 activity is completely impaired, resulting in a build up of sterol intermediates and a failure to generate ergosterol. Deleting UPC2 and NDT80 in BC014RPSC reduces the expression of ERG genes and restores susceptibility to azole drugs. CONCLUSIONS: A missense mutation in the ERG3 gene results in azole resistance and up-regulation of ERG genes expression. We propose that this mutation prevents the formation of toxic intermediates when cells are treated with azoles. Resistance can be reversed by deleting Upc2 and Ndt80 transcription factors. UPC2 plays a stronger role in C. parapsilosis azole resistance than does NDT80.

Structure-based designing of sordarin derivative as potential fungicide with pan-fungal activity.

Fungal infections have become a significant problem for immunosuppressed patients. Sordarin, a promising fungicidal agent, inhibits fungal protein synthesis by impairing elongation factor-2 (eEF2) function. Intriguingly, despite high sequence similarity among eEF2s from different species, sordarin has been shown to inhibit translation specifically in certain fungi while unable to do so in some other fungal species (e.g. Candida parapsilosis and Candida lusitaniae). The sordarin binding site on eEF2 as well as its mechanism of action is known. In a previous study, we have detailed the interactions between sordarin and eEF2 cavities from different fungal species at the molecular level and predicted the probable cause of sordarin sensitivity. Guided by our previous analysis, we aimed for computer-aided designing of sordarin derivatives as potential fungicidal agents that still remain ineffective against human eEF2. We have performed structural knowledge-based designing of several sordarin derivatives and evaluated predicted interactions of those derivatives with the sordarin-binding cavities of different eEF2s, against which sordarin shows no inhibitory action. Our analyses identify an amino-pyrrole derivative as a good template for further designing of promising broad-spectrum antifungal agents. The drug likeness and ADMET prediction on this derivative also supports its suitability as a drug candidate.