Kodamaea ohmeri: A rare yeast causing invasive infections in immunocompromised patients.

INTRODUCTION: Kodamaea ohmeri is a rare, recognized pathogen that has previously been isolated from environmental sources. The patients commonly affected by this yeast include immunocompromised as well as immunocompetent patients having several associated risk factors. METHODOLOGY: We report three cases in which K. ohmeri was isolated from blood using Bact T/ALERT. Identification was carried out by MALDI-TOF MS (Vitek-MS, BioMérieux, Marcy-l'Etoile, France) in addition to color characteristics on chromogenic media. The patients had diminished immune response on account of a multitude of comorbidities. RESULTS: K. ohmeri can be misidentified as Candida tropicalis, Candida albicans, or Candida hemolounii by conventional methods; correct and timely identification can be achieved by MALDI-TOF MS. Antifungal susceptibility breakpoints for K. ohmeri are currently not defined. An Echinocandin was added to the treatment regimen of all three of the cases. CONCLUSIONS: Identification of K. ohmeri using conventional methods is difficult and unusual yeasts should be carefully observed, especially upon prolonged incubation.

Effects of a proteasome inhibitor on the riboflavin production in Ashbya gossypii.

AIMS: Effects of a proteasome inhibitor, MG-132, on the riboflavin production in Ashbya gossypii were investigated to elucidate the relationship of the riboflavin production with flavoprotein homeostasis. METHODS AND RESULTS: The addition of MG-132 to the liquid medium reduced the specific riboflavin production by 79% in A. gossypii at 25 µM after 24 h. The addition of the inhibitor also caused the accumulation of reactive oxygen species and ubiquitinated proteins. These results indicated that MG-132 works in A. gossypii without any genetic engineering and reduces riboflavin production. In the presence of 25 µM MG-132, specific NADH dehydrogenase activity was increased by 1.4-fold compared to DMSO, but specific succinate dehydrogenase (SDH) activity was decreased to 52% compared to DMSO. Additionally, the amount of AgSdh1p (ACR052Wp) was also reduced. Specific riboflavin production was reduced to 22% when 20 mM malonate, a SDH inhibitor, was added to the culture medium. The riboflavin production in heterozygous AgSDH1 gene-disrupted mutant (AgSDH1-/+ ) was reduced to 63% compared to that in wild type. CONCLUSIONS: MG-132 suppresses the riboflavin production and SDH activity in A. gossypii. SDH is one of the flavoproteins involved in the riboflavin production in A. gossypii. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that MG-132 has a negative influence on the riboflavin production and SDH activity in A. gossypii and leads to the elucidation of the connection of the riboflavin production with flavoproteins.

Carbon starvation-induced synthesis of GDH2 and PEPCK is essential for the survival of Pichia pastoris.

The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs. GDH2 and PEPCK synthesis is abrogated in Δatg1 indicating a key role for autophagy or an autophagy-related process. Finally, carbon-starved Δgdh2 and Δpepck exhibit poor survival. This study demonstrates a key role for amino acid catabolism during carbon starvation, a phenomenon hitherto unreported in other yeast species.

The Effects of Photosensitizing Dyes Fagopyrin and Hypericin on Planktonic Growth and Multicellular Life in Budding Yeast.

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John's wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.

Two different clones of Candida pelliculosa bloodstream infection in a tertiary neonatal intensive care unit.

INTRODUCTION: Fungemia in preterm infants results in high mortality and morbidity. The genotypes, drug susceptibilities of Candida pelliculosa strains, and clinical features of two outbreaks of neonatal candidemia caused by C. pelliculosa were analyzed, in order to provide evidence for the outbreaks and characteristics of C. pelliculosa neonatal candidemia. METHODOLOGY: The strains were genotyped by pulsed-field gel electrophoresis to investigate their genetic relatedness. The broth microdilution method was used to determine in vitro susceptibility of the isolates to antifungal drugs. Clinical features of the infected patients were collected to analyze the risks for C. pelliculosa infection. RESULTS: Fourteen neonates, hospitalized in the neonatal intensive care unit from November 2012 to October 2013, were infected by C. pelliculosa. All 14 patients were cured after treatment with fluconazole and discharged without any complications. The C. pelliculosa isolates from the 14 patients were clustered into two groups, indicating that the outbreaks were caused by two types of strains. Eight of nine strains isolated from the 2013 outbreak were clustered into the same group, while one isolate was grouped together with five isolates from the 2012 outbreak. In vitro experiments demonstrated high antifungal activity of fluconazole, voriconazole, amphotericin B, and 5-fluorocytosine to C. pelliculosa. The common symptoms of C. pelliculosa candidaemia were fever, cyanosis, polypnea, hypoactivity, and apnea. CONCLUSIONS: The current study revealed high in vitro susceptibility of C. pelliculosa to antifungals. As C. pelliculosa candidaemia cannot be characterized by clinical symptoms and routine blood testing alone, monitoring unusual strains isolated from immunodeficient hosts is very important to prevent possible outbreaks.

Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast.

BACKGROUND: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin. RESULTS: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways. CONCLUSIONS: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.

Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1.

The evolution of pathogens in response to selective pressures present during chronic infections can influence their persistence and virulence and the outcomes of antimicrobial therapy. Because subpopulations within an infection can be spatially separated and the host environment can fluctuate, an appreciation of the pathways under selection may be most easily revealed through the analysis of numerous isolates from single infections. Here, we continued our analysis of a set of clonally derived Clavispora (Candida) lusitaniae isolates from a single chronic lung infection with a striking enrichment in the number of alleles of MRR1 Genetic and genomic analyses found evidence for repeated acquisition of gain-of-function mutations that conferred constitutive Mrr1 activity. In the same population, there were multiple alleles with both gain-of-function mutations and secondary suppressor mutations that either attenuated or abolished the constitutive activity, suggesting the presence of counteracting selective pressures. Our studies demonstrated trade-offs between high Mrr1 activity, which confers resistance to the antifungal fluconazole, host factors, and bacterial products through its regulation of MDR1, and resistance to hydrogen peroxide, a reactive oxygen species produced in the neutrophilic environment associated with this infection. This inverse correlation between high Mrr1 activity and hydrogen peroxide resistance was observed in multiple Candida species and in serially collected populations from this individual over 3 years. These data lead us to propose that dynamic or variable selective pressures can be reflected in population genomics and that these dynamics can complicate the drug resistance profile of the population.IMPORTANCE Understanding microbial evolution within patients is critical for managing chronic infections and understanding host-pathogen interactions. Here, our analysis of multiple MRR1 alleles in isolates from a single Clavispora (Candida) lusitaniae infection revealed the selection for both high and low Mrr1 activity. Our studies reveal trade-offs between high Mrr1 activity, which confers resistance to the commonly used antifungal fluconazole, host antimicrobial peptides, and bacterial products, and resistance to hydrogen peroxide. This work suggests that spatial or temporal differences within chronic infections can support a large amount of dynamic and parallel evolution and that Mrr1 activity is under both positive and negative selective pressure to balance different traits that are important for microbial survival.

Phialemoniopsis limonesiae sp. nov. causing cutaneous phaeohyphomycosis in an immunosuppressed woman.

Rare or opportunistic fungal infections are mostly described in immunosuppressed patients. We present a case of a cutaneous phaeohyphomycosis that developed on the dorsal foot in an immunosuppressed woman suffering from AIDS, caused by a novel Phialemoniopsis species. It clinically presented as an indurated violaceous plaque, surmounted by nodules exuding a sero-purulent discharge. A filamentous fungus was isolated from pus and cutaneous biopsy. ITS and LSU sequences phylogenetically resolved the fungus as an unknown species of Phialemoniopsis, which is an unresolved family within Sordariomycetes. In this study we describe the new species as Phialemoniopsis limonesiae, which clusters on a single branch clearly separated from its closest phylogenetic neighbours. This new strain showed low MIC to itraconazole, voriconazole and posaconazole.

Gelatin expression from an engineered Saccharomyces cerevisiae CUP1 promoter in Pichia pastoris.

The methylotrophic yeast Pichia pastoris (reclassified as Komagataella phaffii) is a versatile protein expression system, yet many commonly used promoters have attributes undesirable for fermentation or its optimization. Hence, the copper-inducible CUP1 gene promoter from the related yeast Saccharomyces cerevisiae was used to express human gelatin. Multimerization of a potential copper response element in the CUP1 promoter, a S. cerevisiae Ace1p binding site, significantly increased gelatin expression. Expression was induced by copper in a dose-dependent fashion and was not dependent on cell density. Gelatin was additionally induced in standard copper-containing fermentation basal salts media. Removal of a S. cerevisiae heat shock factor (Hsf1p) binding site reduced copper-dependent gelatin induction suggesting that a similar protein may regulate this promoter in P. pastoris. This engineered copper inducible promoter expands the yeast recombinant protein production tool kit.

High-performance thin-layer chromatography in combination with a yeast-based multi-effect bioassay to determine endocrine effects in environmental samples.

Effect-directed analysis (EDA) that combines effect-based methods (EBMs) with high-performance thin-layer chromatography (HPTLC) is a useful technique for spatial, temporal, and process-related effect evaluation and may provide a link between effect testing and responsible substance identification. In this study, a yeast multi endocrine-effect screen (YMEES) for the detection of endocrine effects is combined with HPTLC. Simultaneous detection of estrogenic, androgenic, and gestagenic effects on the HPTLC plate is achieved by mixing different genetically modified Arxula adeninivorans yeast strains, which contain either the human estrogen, androgen, or progesterone receptor. Depending on the yeast strain, different fluorescent proteins are formed when an appropriate substance binds to the specific hormone receptor. This allows to measure hormonal effects at different wavelengths. Two yeast cell application approaches, immersion and spraying, are compared. The sensitivity and reproducibility of the method are shown by dose-response investigations for reference compounds. The spraying approach indicated similar sensitivities and higher precisions for the tested hormones compared to immersion. The EC10s for estrone (E1), 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), 5α-dihydrotestosterone (DHT), and progesterone (P4) were 95, 1.4, 10, 7.4, and 15 pg/spot, respectively. Recovery rates of E1, E2, EE2, DHT, and P4 between 88 and 120% show the usability of the general method in combination with sample enrichment by solid phase extraction (SPE). The simultaneous detection of estrogenic, androgenic, and gestagenic effects in wastewater and surface water samples demonstrates the successful application of the YMEES in such matrices. This promising method allows us to identify more than one endocrine effect on the same HPTLC plate, which saves time and material. The method could be used for comparison, evaluation, and monitoring of different river sites and wastewater treatment steps and should be tested in further studies.

Evaluation of the inhibitory activity of essential oils against spoilage yeasts and their potential application in yogurt.

Yeasts are the leading cause of spoilage in yogurt. Considering the high demand from consumers to use natural products as an alternative to additives, essential oils (EOs) could be a promising solution to guarantee high microbiological standards. The present study highlighted the in vitro antifungal potential of cinnamon, ginger, lemongrass, mandarin, orange, lemon and lime EOs against spoilage yeasts isolated from yogurts prepared with pasteurized buffalo milk. A total of 74 isolates represented by 14 different species of Candida, Rhodotorula, Debaryomyces, Kluyveromyces and Yarrowia genera were subjected to a disc diffusion assay, showing lemongrass EO to have the highest antifungal activity (40.97 ± 9.86 mm), followed by cinnamon (38.46 ± 6.59 mm) and orange (12.00 ± 4.52 mm) EOs. Yarrowia lipolytica was less susceptible to lemongrass EO than Candida sake and Yarrowia deformans isolates. Ginger EO exhibited the lowest efficacy. A minimum inhibitory concentration (MIC) assay showed the ability of lemongrass and cinnamon EOs to inhibit the growth of all selected isolates at concentrations between ≤0.31 and 1.25 µL/mL. Therefore, for the first time, the two best-performing EOs (lemongrass and cinnamon) based on in vitro assays were assessed for their potential roles as preservatives in an in vivo yogurt model prepared at the laboratory scale. Since some limitations, such as the inhibition of lactic acid bacteria by cinnamon EO, consequently leading to fermentation failure as well as species-specific antifungal activity of lemongrass EO, were observed, further studies are needed to explore the possibility of using a slightly higher concentration of lemongrass EO and/or combinations of different EOs and/or their components. Finally, since yogurt spoilage could also be prevented by correct sanitation procedures of the production environment, the sanitizers commonly used in the food industry were tested against all isolates, showing the high efficiency of alcohol-based sanitizers and the ineffectiveness of chlorine-based sanitizers.

Inhibition of alternative respiration system of Scheffersomyces stipitis and effect on glucose or xylose fermentation.

Scheffersomyces stipitis is a Crabtree-negative pentose fermenting yeast, which shows a complex respiratory system involving a cytochrome and an alternative salicylhydroxamic acid (SHAM)-sensitive respiration mechanism that is poorly understood. This work aimed to investigate the role of the antimycin A (AA) sensitive respiration and SHAM-sensitive respiration in the metabolism of xylose and glucose by S. stipitis, upon different agitation conditions. Inhibition of the SHAM-sensitive respiration caused a significant (P < 0.05) decrease in glycolytic flux and oxygen consumption when using glucose and xylose under agitation conditions, but without agitation, only a mild reduction was observed. The combination of SHAM and AA abolished respiration, depleting the glycolytic flux using both carbon sources tested, leading to increased ethanol production of 21.05 g/L at 250 rpm for 0.5 M glucose, and 8.3 g/L ethanol using xylose. In contrast, inhibition of only the AA-sensitive respiration, caused increased ethanol production to 30 g/L using 0.5 M glucose at 250 rpm, and 11.3 g/L from 0.5 M xylose without agitation. Results showed that ethanol production can be induced by respiration inhibition, but the active role of SHAM-sensitive respiration should be considered to investigate better conditions to increase and optimize yields.

Kazachstania slooffiae: An unexpected journey to a human pleural sample.

We report a case of a 50-year-old shepherd hospitalized in intensive care unit for hiatal hernia complicated by an occlusive syndrome. In post-surgery, an acute respiratory distress occurs due to mediastinitis with large pleural effusion. At the laboratory, direct examination of the pleural sample revealed the presence of pseudohyphae. Kazachstania slooffiae was identified by Mass Spectrometry and confirmed by DNA sequencing. This uncommon yeast has never been previously described in human infections. Although its pathogenicity is not well known, K. slooffiae should be considered in the case of critically ill patients.

8-Hydroxyquinoline 1,2,3-triazole derivatives with promising and selective antifungal activity.

Fungal infections that affect humans and plants have increased significantly in recent decades. However, these pathogens are still neglected when compared to other infectious agents. Due to the high prevalence of these infections, the need for new molecules with antifungal potential is recognized, as pathogenic species are developing resistance to the main drugs available. This work reports the design and synthesis of 1,2,3-triazole derivatives of 8-hydroxyquinoline, as well as the determination of their activities against a panel of fungal species: Candida spp., Trichosporon asahii, Magnusiomyces capitatus, Microsporum spp., Trichophyton spp. and Fusarium spp. The triazoles 5-(4-phenyl-1H-1,2,3-triazol-1-yl)quinolin-8-ol (12) and 5-(4-(cyclohex-1-en-1-yl)-1H-1,2,3-triazol-1-yl)quinolin-8-ol (16) were more promising, presenting minimum inhibitory concentration (MIC) values between 1-16 µg/ml for yeast and 2-4 µg/ml for dermatophytes. However, no relevant anti-Fusarium spp. activity was observed. In the time-kill assays with Microsporum canis, 12 and 16 presented time-dependent fungicide profile at 96 h and 120 h in all evaluated concentrations, respectively. For Candida guilliermondii, 12 was fungicidal at all concentrations at 6 h and 16 exhibited a predominantly fungistatic profile. Both 12 and 16 presented low leukocyte toxicity at 4 µg/ml and the cell viability was close to 100% after the treatment with 12 at all tested concentrations. The sorbitol assay combined with SEM suggest that damages on the fungal cell wall could be involved in the activity of these derivatives. Given the good results obtained with this series, scaffold 4-(cycloalkenyl or phenyl)-5-triazol-8-hydroxyquinoline appears to be a potential pharmacophore for exploration in the development of new antifungal agents.

Epidemiology of candidemia in Shiraz, southern Iran: A prospective multicenter study (2016-2018).

Systematic candidemia studies, especially in southern Iran, are scarce. In the current prospective study, we investigated candidemia in three major healthcare centers of Shiraz, the largest city in southern Iran. Yeast isolates from blood and other sterile body fluids were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method. Clinical data were retrieved from patients' medical records. In total, 113 yeast isolates were recovered from 109 patients, over 60% of whom received fluconazole. Antifungal drugs were prescribed without considering species identification or AFST. The all-cause mortality rate was 28%. Almost 30% of the patients were from intensive care units (ICUs). Candida albicans (56/113; 49.5%) was the most prevalent species followed by C. glabrata (26/113; 23%), C. parapsilosis (13/113; 11.5%), C. tropicalis (7/113; 6.2%), and C. dubliniensis (5/113; 4.4%). Only five isolates showed antifungal resistance or decreased susceptibility to fluconazole: one C. orthopsilosis isolate from an azole-naïve patient and two C. glabrata, one C. albicans, and one C. dubliniensis isolates from patients treated with azoles, who developed therapeutic failure against azoles later. Our results revealed a low level of antifungal resistance but a notable rate of azole therapeutic failure among patients with candidemia due to non-albicans Candida species, which threaten the efficacy of fluconazole, the most widely used antifungal in southern regions of Iran. Candidemia studies should not be confined to ICUs and treatment should be administered based on species identification and AFST results.

Landscape of candidemia is blurred in Iran, and only two studies from Tehran have extensively explored the epidemiology of candidemia. However, candidemia data from the other regions are notoriously scarce, which precludes from reaching a consensus regarding species distribution, the burden of antifungal resistance, and the clinical features of infected patients. Therefore, we conducted the current prospective candidemia study in Shiraz, one of the largest cities located in the south of Iran, from April 2016 to April 2018. More than 63% of the candidemia infections were treated by fluconazole and species identification and antifungal susceptibility testing were not used for decision making regarding the choice of antifungal treatment. Approximately 70% of the candidemia cases occurred in the wards outside of the ICUs. Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis were the five leading causative agents of candidemia. Antifungal resistance was rare and fluconazole resistance and/or non-wild type phenotypes were noticed in five isolates, only one was C. albicans and the rest were non-albicans Candida (NAC) species, including C. glabrata, C. dubliniensis, and C. orthopsilosis. Except for C. orthopsilosis, which was isolated from an azole-naïve patient, the rest of isolates were recovered from patients treated with azoles and all showed therapeutic failure to azoles. Collectively, our data will complete the candidemia picture in Iran and show that, although the level of resistance was rare, the therapeutic failure was notable among NAC species, which threatens the efficacy of fluconazole, the most widely used antifungal in Southern regions of Iran. Moreover, we showed that candidemia is poorly managed in Iran since species identification tools along with antifungal susceptibility testing were not used to select appropriate antifungal treatment.

Construction of Tight Conditional Mutants Using the Improved Auxin-Inducible Degron (iAID) Method in the Budding Yeast Saccharomyces cerevisiae.

Conditional mutants, such as temperature-sensitive (ts) mutants, are effective tools for the analysis of essential genes. However, such mutants are frequently leaky. To overcome this problem, it is helpful to isolate a "tight" conditional mutant of a gene of interest, e.g., by using ubiquitin-mediated protein degradation to eliminate the gene product. One such strategy is the auxin-inducible degron (AID) system, which is easy to use because the simple addition of auxin can induce the degradation of a target protein. Sometimes, however, elimination of the target protein is not sufficient, and an AID mutant exhibits a "leaky" phenotype. To address this issue, the improved AID (iAID) system was developed. In this approach, transcriptional repression by the "Tet-OFF" promoter is combined with proteolytic elimination of the target protein by the AID system, yielding a much tighter mutant. Because simple addition of tetracycline is sufficient to repress the Tet-OFF promoter, the combination of Tet-OFF and AID maintains the ease of use of the original AID system. In this manuscript, we describe how to construct and use iAID mutants in the budding yeast Saccharomyces cerevisiae.

First Isolation of Candida nivariensis, an Emerging Fungal Pathogen, in Kuwait.

OBJECTIVE: C. nivariensis is a rare Candida species which is phenotypically closely related to Candida glabrata and Candida bracarensis. The 3 species form the C. glabrata sensu lato complex. Here, we describe the first isolation and characterization of a C. nivariensis isolate cultured from the tracheal aspirate obtained from a young man in Kuwait. MATERIALS AND METHODS: The yeast isolate was initially tested by VITEK 2 followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multiplex PCR. The identification was confirmed by sequencing of internal transcribed spacer (ITS) region of rDNA. Antifungal susceptibility testing was performed by Etest, and phylogenetic comparison with other international strains was carried out by using MEGA version 7 software. RESULTS: The C. nivariensis isolate was misidentified by VITEK 2, but correctly identified by MALDI-TOF MS with updated software and multiplex PCR. The identity was confirmed by sequence comparisons of ITS region of rDNA. Antifungal susceptibility testing revealed high minimum inhibitory concentration (MIC) against fluconazole, but low MICs against amphotericin B and echinocandins. Phylogenetically, our isolate was closely related to Indian isolates. CONCLUSIONS: This report extends the geographic distribution of C. nivariensis to the Arabian Peninsula. MALDI-TOF MS with updated software and molecular tests are needed to correctly identify C. nivariensis. Since C. nivariensis may exhibit reduced susceptibility to antifungal agents, accurate identification and antifungal susceptibility testing are essential, particularly for isolates from sterile sites, for optimal patient management.

Tartary buckwheat protein hydrolysates enhance the salt tolerance of the soy sauce fermentation yeast Zygosaccharomyces rouxii.

Supplementation of protein hydrolysate is an important strategy to improve the salt tolerance of soy sauce aroma-producing yeast. In the present study, Tartary buckwheat protein hydrolysates (BPHs) were prepared and separated by ultrafiltration into LM-1 (<1 kDa) and HM-2 (1-300 kDa) fractions. The supplementation of HM-2 fraction could significantly improve cell growth and fermentation of soy sauce aroma-producing yeast Zygosaccharomyces rouxii As2.180 under high salt (12%, w/w) conditions. However, the LM-1 fraction inhibited strain growth and fermentation. The addition of HM-2 promoted yeast cell accumulation of K+, removal of cytosolic Na+ and accumulation of glycerol. Furthermore, the HM-2 fraction improved the cell membrane integrity and mitochondrial membrane and decreased intracellular ROS accumulation of the strain. The above results indicated that the supplementation of BPHs with a molecular weight of 1-300 kDa is a potentially effective and feasible strategy for improving the salt tolerance of soy sauce aroma-producing yeast Z. rouxii.

A case-series of bloodstream infections caused by the Meyerozyma guilliermondii species complex at a reference center of oncology in Brazil.

Bloodstream infections (BSI) caused by Candida species are the fourth cause of healthcare associated infections worldwide. Non-albicans Candida species emerged in the last decades as agents of serious diseases. In this study, clinical and microbiological aspects of six patients with BSI due to the Meyerozyma (Candida) guilliermondii species complex from an oncology reference center in Brazil, were evaluated. To describe demographic and clinical characteristics, medical records of the patients were reviewed. Molecular identification of the isolates was performed by ITS1-5.8S-ITS2 region sequencing. Antifungal susceptibility was evaluated by the EUCAST method and the minimal inhibitory concentrations (MIC) assessed according to the epidemiological cutoff values. Virulence associated phenotypes of the isolates were also studied. Ten isolates from the six patients were evaluated. Five of them were identified as Meyerozyma guilliermondii and the others as Meyerozyma caribbica. One patient was infected with two M. caribbica isolates with different genetic backgrounds. High MICs were observed for fluconazole and echinocandins. Non-wild type isolates to voriconazole appeared in one patient previously treated with this azole. Additionally, two patients survived, despite infected with non-wild type strains for fluconazole and treated with this drug. All isolates produced hemolysin, which was not associated with a poor prognosis, and none produced phospholipases. Aspartic proteases, phytase, and esterase were detected in a few isolates. This study shows the reduced antifungal susceptibility and a variable production of virulence-related enzymes by Meyerozyma spp. In addition, it highlights the poor prognosis of neutropenic patients with BSI caused by this emerging species complex. LAY ABSTRACT: Our manuscript describes demographic, clinical and microbiological characteristics of patients with bloodstream infection by the Meyerozyma guilliermondii species complex at a reference center in oncology in Brazil.

Closely related budding yeast species respond to different ecological signals for spore activation.

Spore activation is one of the most important developmental decisions in fungi as it initiates the transition from dormant and stress-resistant cells to vegetative cells. Because in many species mating follows spore activation and germination, signals that trigger this developmental transition can also contribute to species reproductive barriers. Here, we examine the biochemical signals triggering spore activation in a natural species complex of budding yeast, Saccharomyces paradoxus (lineages SpA, SpB, SpC and SpC*). We first demonstrate that we can quantitatively monitor spore activation in these closely related lineages. Second, we dissect the composition of culture media to identify components necessary and/or sufficient to activate spores in the four lineages. We show that, contrary to expectation, glucose is necessary but not sufficient to trigger spore activation. We also show that two of the North American lineages (SpC and SpC*) diverge from the other North American (SpB) and European (SpA) lineages in terms of germination signal as their spore activation requires inorganic phosphate. Our results show that the way budding yeast interpret environmental conditions during spore activation diverged among closely related and incipient species, which means that it may play a role in their ecological differentiation and reproductive isolation. TAKE AWAY: Sensing of multiple compounds allows spore activation in non-domesticated budding yeast. Spore activation cues differ among Saccharomyces paradoxus lineages. Dextrose and phosphate signal activation in SpC and SpC* spores.