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Nondestructive detection and discrimination of fungal pathogens is essential for rapid and precise treatment, which further effectively prevents antifungal resistance from overused drugs. In this work, fluorescent gold nanoclusters served as the basis for discriminating Candida species. Varied on surface ligands, these gold nanoclusters demonstrated different optical properties as a result of the perturbation effects of ligands. The biointerface interaction between the surface ligands of gold nanoclusters and the cell walls of Candida species can be constructed, and their restriction on ligands perturbation effect produced enhanced fluorescence signals. Owing to the variation of the cell wall composition, cells of different Candida species demonstrated different degrees of association with the gold nanoclusters, leading to discriminable amounts of fluorescence enhancements. The reverse signal response from these gold nanoclusters gives rise to a synergistic and effective assay that allows identification of Candida species.

Developing mathematical models to accurately predict microbial growth dynamics remains a key challenge in ecology, evolution, biotechnology, and public health. To reproduce and grow, microbes need to take up essential nutrients from the environment, and mathematical models classically assume that the nutrient uptake rate is a saturating function of the nutrient concentration. In nature, microbes experience different levels of nutrient availability at all environmental scales, yet parameters shaping the nutrient uptake function are commonly estimated for a single initial nutrient concentration. This hampers the models from accurately capturing microbial dynamics when the environmental conditions change. To address this problem, we conduct growth experiments for a range of micro-organisms, including human fungal pathogens, baker's yeast, and common coliform bacteria, and uncover the following patterns. We observed that the maximal nutrient uptake rate and biomass yield were both decreasing functions of initial nutrient concentration. While a functional form for the relationship between biomass yield and initial nutrient concentration has been previously derived from first metabolic principles, here we also derive the form of the relationship between maximal nutrient uptake rate and initial nutrient concentration. Incorporating these two functions into a model of microbial growth allows for variable growth parameters and enables us to substantially improve predictions for microbial dynamics in a range of initial nutrient concentrations, compared to keeping growth parameters fixed., Competing Interests: The authors have declared that no competing interests exist.

Objective: Candida kefyr causes invasive candidiasis in immunocompromised patients, particularly among those with oncohematological diseases. This study determined the prevalence of C. kefyr among yeast isolates collected during 2011-2018 in Kuwait. Antifungal susceptibility testing (AST) and genotypic heterogeneity among C. kefyr was also studied., Methods: Clinical C. kefyr isolates recovered from bloodstream and other specimens during 2011 to 2018 were retrospectively analyzed. All C. kefyr isolates were identified by CHROMagar Candida, Vitek2 and PCR amplification of rDNA. AST was performed by Etest. Molecular basis of resistance to fluconazole and echinocandins was studied by PCR-sequencing of ERG11 and FKS1, respectively. Genotypic heterogeneity was determined with microsatellite-/minisatellite-based primers and for 27 selected isolates by PCR-sequencing of IGS1 region of rDNA., Results: Among 8257 yeast strains, 69 C. kefyr (including four bloodstream) isolates were detected by phenotypic and molecular methods. Isolation from urine and respiratory samples from female and male patients was significantly different (P = 0.001). Four isolates showed reduced susceptibility to amphotericin B and one isolate to all (amphotericin B, fluconazole, voriconazole and caspofungin/micafungin) antifungals tested. Fluconazole-resistant isolate contained only synonymous mutations in ERG11. Echinocandin-resistant isolate contained wild-type hotspot-1 and hotspot-2 of FKS1. Fingerprinting with microsatellite-/minisatellite-based primers identified only three types. IGS1 sequencing identified seven haplotypes among 27 selected isolates., Conclusions: The overall prevalence of C. kefyr among clinical yeast isolates and among candidemia cases was recorded as 0.83% and 0.32%, respectively. The frequency of isolation of C. kefyr from bloodstream and other invasive samples was stable during the study period. The C. kefyr isolates grown from invasive (bloodstream, bronchoalveolar lavage, abdominal drain fluid, peritonial fluid and gastric fluid) samples and amphotericin B-resistant isolates were genotypically heterogeneous strains., Competing Interests: The authors have declared that no competing interests exist.

Candida hispaniensis is an oleaginous yeast with a great potential for production of single cell oil according to its naturally high lipid accumulation capacity. Its unusual small genome size trait is also attractive for fundamental research on genome evolution. Our physiological study suggests a great potential for lipid production, reaching 224 mg/g of cell dry weight in glucose minimum medium. C. hispaniensis is also able to secrete up to 34.6 mg/L of riboflavin promising further riboflavin production improvements by cultivation optimization and genetic engineering. However, while its genome sequence has been released very recently, no genetic tools have been described up to now for this yeast limiting its use for fundamental research and for exploitation in an industrial biotechnology. We report here the first genetic modification of C. hispaniensis by introducing a heterologous invertase allowing the growth on sucrose using a biolistic transformation approach using a dedicated vector. The first genetic tool and transformation method developed here appear as a proof of concept, and while it would benefit from further optimization, heterogeneous expression of invertase allows for metabolism of an additional sugar and shows heterologous enzyme production capacity., (© 2020 John Wiley & Sons, Ltd.)

Fungal infections are a major contributor to infectious disease-related deaths worldwide. Recently, global emergence of the fungal pathogen Candida auris has caused considerable concern because most C. auris isolates are resistant to fluconazole, the most commonly administered antifungal, and some isolates are resistant to drugs from all three major antifungal classes. To identify novel agents with bioactivity against C. auris , we screened 2,454 compounds from a diversity-oriented synthesis collection. Of the five hits identified, most shared a common rocaglate core structure and displayed fungicidal activity against C. auris These rocaglate hits inhibited translation in C. auris but not in its pathogenic relative Candida albicans Species specificity was contingent on variation at a single amino acid residue in Tif1, a fungal member of the eukaryotic initiation factor 4A (eIF4A) family of translation initiation factors known to be targeted by rocaglates. Rocaglate-mediated inhibition of translation in C. auris activated a cell death program characterized by loss of mitochondrial membrane potential, increased caspase-like activity, and disrupted vacuolar homeostasis. In a rocaglate-sensitized C. albicans mutant engineered to express translation initiation factor 1 (Tif1) with the variant amino acid that we had identified in C. auris , translation was inhibited but no programmed cell death phenotypes were observed. This surprising finding suggests divergence between these related fungal pathogens in their pathways of cellular responses to translation inhibition. From a therapeutic perspective, the chemical biology that we have uncovered reveals species-specific vulnerability in C. auris and identifies a promising target for development of new, mechanistically distinct antifungals in the battle against this emerging pathogen. IMPORTANCE Emergence of the fungal pathogen Candida auris has ignited intrigue and alarm within the medical community and the public at large. This pathogen is unusually resistant to antifungals, threatening to overwhelm current management options. By screening a library of structurally diverse molecules, we found that C. auris is surprisingly sensitive to translation inhibition by a class of compounds known as rocaglates (also known as flavaglines). Despite the high level of conservation across fungi in their protein synthesis machinery, these compounds inhibited translation initiation and activated a cell death program in C. auris but not in its relative Candida albicans Our findings highlight a surprising divergence across the cell death programs operating in Candida species and underscore the need to understand the specific biology of a pathogen in attempting to develop more-effective treatments against it., (Copyright © 2020 Iyer et al.)

Although infrequent in our laboratory, growth of bacterial colonies has been observed on top of the purified cultures of yeasts. In this study, the likelihood of bacterial excision from yeast under aging and starvation stresses was assessed using 10 gastric and 10 food-borne yeasts. Yeasts were identified as members of Candida or Saccharomyces genus by amplification and sequencing of D1/D2 region of 26S rDNA. For aging stress, yeasts were cultured on brain heart infusion agar supplemented with sheep blood and incubated at 30 °C for 3-4 weeks. For starvation stress, yeasts were inoculated into distilled water and incubated similarly. After seven days, starved yeasts were cultured on yeast extract glucose agar, incubated similarly and examined daily for appearance of bacterial colonies on top of the yeast's growth. Outgrowth of excised bacteria was observed on top of the cultures of 4 yeasts (Y1, Y3, Y13 and Y18) after 3-7 days. The excised bacteria (B1, B3, B13 and B18) were isolated and identified at the genus level according to their biochemical characteristics as well as amplification and sequencing of 16S rDNA. B1 (Arthrobacter) were excised from Y1 (Candida albicans) upon aging and B3 (Staphylococcus), B13 (Cellulomonas) and B18 (Staphylococcus) were excised from their respective yeasts; Y3 (Candida tropicalis), Y13 (Saccharomyces cerevisiae) and Y18 (Candida glabrata) upon starvation. DNA from yeasts was used for detection of 16S rDNA of their intracellular bacteria and sequencing. Amplified products from yeasts showed sequence similarity to those of excised bacteria. Under normal conditions, yeast exerts tight control on multiplication of its intracellular bacteria. However, upon aging and starvation the control is no longer effective and bacterial outgrowth occurs. Unlimited multiplication of excised bacteria might provide yeast with plenty of food in close vicinity. This could be an evolutionary dialogue between yeast and bacteria that ensures the survival of both partners., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Methylene blue viability staining has been traditionally used to assess viability of Saccharomyces cerevisiae in brewing and wine making. Here, this method was tested and validated with the emerging fungal pathogen Candida auris to determine if this species would also deferentially stain, which could provide utility in assessing microbial control and disinfectant efficacy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antifungal drug resistance exhibits a major clinical challenge for treating nosocomial fungal infections. To find a possible solution, we synthesized and studied the antifungal activities of three different arginolipids (N α -acyl-arginine ethyl ester) against clinical drug-resistant isolates of Candida. The most active arginolipid, oleoyl arginine ethyl ester (OAEE) consisting of a long unsaturated hydrophobic chain, was tested for its mode of action, which revealed that it altered ergosterol biosynthesis and compromised the fungal cell membrane. Also, OAEE was found to exhibit synergistic interactions with fluconazole (FLU) or amphotericin B (AmB) against planktonic Candida cells, wherein it reduced the inhibitory concentrations of these drugs to their in vitro susceptible range. Studies conducted against the C. tropicalis biofilm revealed that the OAEE+AmB combination synergistically reduced the metabolic activity and hyphal density in biofilms, whereas OAEE+FLU was found to be additive against most cases. Finally, the evaluated selective toxicity of OAEE toward fungal cells over mammalian cells could establish it as an alternative treatment for combating drug-resistant Candida infections., (© 2019 Deutsche Pharmazeutische Gesellschaft.)

The existence of the multi-drug resistant (MDR) pathogenic fungus, Candida auris came to light in 2009. This particular organism is capable of causing nosocomial infections in immunecompromised persons. This pathogen is associated with consistent candidemia with high mortality rate and presents a serious global health threat. Whole genome sequence (WGS) investigation detected powerful phylogeographic Candida auris genotypes which are specialized to particular geological areas indicating dissemination of particular genotype among provinces. Furthermore, this organism frequently exhibits multidrug-resistance and displays an unusual sensitivity profile. Identification techniques that are commercialized to test Candida auris often show inconsistent results and this misidentification leads to treatment failure which complicates the management of candidiasis. Till date, Candida auris has been progressively recorded from several countries and therefore its preventive control measures are paramount to interrupt its transmission. In this review, we discussed prevalence, biology, drug-resistance phenomena, virulence factors and management of Candida auris infections., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Azole antifungals inhibit the biosynthesis of ergosterol, the fungal equivalent of cholesterol in mammalian cells. Here we report an investigation of the activity of coumarin-substituted azole antifungals. Screening against a panel of Candida pathogens, including a mutant lacking CYP51, the target of antifungal azoles, revealed that this enzyme is inhibited by triazole-based antifungals, whereas imidazole-based derivatives have more than one mode of action. The imidazole-bearing antifungals more effectively reduced trailing growth associated with persistence and/or recurrence of fungal infections than triazole-based derivatives. The imidazole derivatives were more toxic to mammalian cells and more potently inhibited the activity of CYP3A4, which is one of the main causes of azole toxicity. Using live cell imaging, we showed that regardless of the type of azole ring fluorescent 7-diethylaminocoumarin-based azoles localized to the endoplasmic reticulum, the organelle that harbors CYP51. This study suggests that the coumarin is a promising scaffold for development of novel azole-based antifungals that effectively localize to the fungal cell endoplasmic reticulum., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Candida auris is an emerging pathogenic yeast responsible for nosocomial infections with high mortality, on a global scale. A 65-year-old woman with hypovolemic shock and severe metabolic acidosis was intubated and admitted to the intensive care unit (ICU). Shortly after admission, she developed ventilator-associated pneumonia caused by multidrug-resistant Acinetobacter baumannii , which necessitated treatment with high-dose ampicillin-sulbactam. Two weeks later, a yeast was cultured from her blood. It formed pale pink colonies on CHROMagar Candida medium and produced predominantly oval budding yeast cells with the occasional rudimentary pseudohyphae on cornmeal agar. ID 32 C identified the yeast as Candida sake However, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and sequencing of the D1/D2 region of the 28S rRNA gene identified the yeast as C. auris ., (© 2019 by the Association of Clinical Scientists, Inc.)

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Selective manipulation of single cells is an important step in sample preparation for biological analysis. A highly specific and automated device is desired for such an operation. An ideal device would be able to selectively pick several single cells in parallel from a heterogeneous population and transfer those to designated sites for further analysis without human intervention. The robotic manipulator developed here provides the basis for development of such a device. The device in this work is designed to selectively pick cells based on their inherent properties using dielectrophoresis (DEP) and automatically transfer and release those at a transfer site. Here we provide proof of concept of such a device and study the effect of different parameters on its operation. Successful experiments were conducted to separate Candida cells from a mixture with 10 μm latex particles and a viability assay was performed for separation of viable rat adipose stem cells (RASCs) from non-viable ones. The robotic DEP device was further used to pick and transfer single RASCs. This work also discusses the advantages and disadvantages of our current setup and illustrates the future steps required to improve the performance of this robotic DEP technology.

Fungal cell surface carbohydrates and proteins are useful antigens for the development of antifungal vaccines. In this study, glycopeptides consisting of the β-1,2-mannan and N-terminal peptide epitopes of Candida albicans (C. albicans) cell wall phosphomannan complex and Als1p (rAls1p-N) protein, respectively, were synthesized and covalently conjugated with keyhole limpet hemocyanin (KLH) and human serum albumin (HSA) through homobifunctional disuccinimidyl glutarate. The resultant KLH-conjugates were immunologically evaluated using Balb/c mice to reveal that they induced high levels of IgG antibodies. Furthermore, these conjugates showed self-adjuvanting property, as they could promote robust antibody responses without the presence of an external adjuvant. More significantly, the obtained antisera could effectively recognize both the carbohydrate and the Als1 peptide epitopes and immunofluorescence and flow cytometry assays also demonstrated that the elicited antibodies could react with the cell surface of a number of fungi, including C. albicans, C. tropicalis, C. lustaniae and C. glabrata. These results suggested the great potential of these conjugates as antifungal vaccines., (Copyright © 2019. Published by Elsevier Masson SAS.)

Purpose: The pathogenicity in Candida spp was attributed by several virulence factors such as production of tissue damaging extracellular enzymes, germ tube formation, hyphal morphogenesis and establishment of drug resistant biofilm. The objective of present study was to investigate the effects of silver nanoparticles (AgNPs) on growth, cell morphology and key virulence attributes of Candida species. Methods: AgNPs were synthesized by the using seed extract of Syzygium cumini (Sc), and were characterized by UV-Vis spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and transmission electron microscopy (TEM). ScAgNPs were used to evaluate their antifungal and antibacterial activity as well as their potent inhibitory effects on germ tube and biofilm formation and extracellular enzymes viz. phospholipases, proteinases, lipases and hemolysin secreted by Candida spp. Results: The MICs values of ScAgNPs were ranged from 0.125-0.250 mg/ml, whereas the MBCs and MFCs were 0.250 and 0.500 mg/ml, respectively. ScAgNPs significantly inhibit the production of phospholipases by 82.2, 75.7, 78.7, 62.5, and 65.8%; proteinases by 82.0, 72.0, 77.5, 67.0, and 83.7%; lipase by 69.4, 58.8, 60.0, 42.9, and 65.0%; and hemolysin by 62.8, 69.7, 67.2, 73.1, and 70.2% in C. albicans , C. tropicalis , C. dubliniensis , C. parapsilosis and C. krusei , respectively, at 500 μg/ml. ScAgNPs inhibit germ tube formation in C. albicans up to 97.1% at 0.25 mg/ml. LIVE/DEAD staining results showed that ScAgNPs almost completely inhibit biofilm formation in C. albicans. TEM analysis shows that ScAgNPs not only anchored onto the cell surface but also penetrated and accumulated in the cytoplasm that causes severe damage to the cell wall and cytoplasmic membrane. Conclusion: To summarize, the biosynthesized ScAgNPs strongly suppressed the multiplication, germ tube and biofilm formation and most importantly secretion of hydrolytic enzymes (viz. phospholipases, proteinases, lipases and hemolysin) by Candia spp. The present research work open several avenues of further study, such as to explore the molecular mechanism of inhibition of germ tubes and biofilm formation and suppression of production of various hydrolytic enzymes by Candida spp., Competing Interests: The authors declare that they have no conflicts of interest in this work.

Aim: To evaluate the efficacy of photodynamic inactivation (PDI) mediated by hypericin encapsulated in P-123 copolymeric micelles (P123-Hyp) alone and in combination with fluconazole (FLU) against planktonic cells and biofilm formation of Candida species Materials & methods: PDI was performed using P123-Hyp and an LED device with irradiance of 3.0 mW/cm 2  . Results: Most of isolates (70%) were completely inhibited with concentrations up to 2.0 μmol/l of HYP and light fluence of 16.2 J/cm 2 . FLU-resistant strains had synergic effect with P123-HYP-PDI and FLU. The biofilm formation was inhibited in all species, in additional the changes in Candida morphology observed by scanning electron microscopy. Conclusion: P123-Hyp-PDI is a promising option to treat fungal infections and medical devices to prevent biofilm formation and fungal spread.

Background: Plant defensins have a hallmark γ-core motif (GXCX 3-9 C) that is related to their antimicrobial properties. The aim of this work was to design synthetic peptides based on the region corresponding to the Pv D 1 defensin γ-core that are the smallest amino acid sequences that bear the strongest biological activity., Methods: We made rational substitutions of negatively charged amino acid residues with positively charged ones, and the reduction in length in the selected Pv D 1 γ-core sequence to verify whether the increased net positive charges and shortened length are related to the increase in antifungal activity. Herein, we opted to evaluate the action mechanism of γ 33-41 Pv D 1 ++ peptide due to its significant inhibitory effect on tested yeasts. In addition, it is the smallest construct comprising only nine amino acid residues, giving it a better possibility to be a prototype for designing a new antifungal drug, with lower costs to the pharmaceutical industry while still maintaining the strongest antimicrobial properties., Results: The γ 33-41 Pv D 1 ++ peptide caused the most toxic effects in the yeast Candida buinensis , leading to membrane permeabilization, viability loss, endogenous reactive oxygen species increase, the activation of metacaspase, and the loss of mitochondrial functionality, suggesting that this peptide triggers cell death via apoptosis., Conclusion: We observed that the antifungal activity of Pv D 1 is not strictly localized in the structural domain, which comprises the γ-core region and that the increase in the net positive charge is directly related to the increase in antifungal activity., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

High rate of reported cases of infections in humans caused by fungal pathogens pose serious concern. Potentially these commensal fungi remain harmless to the healthy individuals but can cause severe systemic infection in patients with compromised immune system. Effective drug remedies against these infections are rather limited. Moreover, frequently encountered multidrug resistance poses an additional challenge to search for alternate and novel targets. Notably, imbalances in lipid homeostasis which impact drug susceptibility of Candida albicans cells do provide clues of novel therapeutic strategies. Sphingolipids (SPHs) are unique components of Candida cells, hence are actively exploited as potential drug targets. In addition, recent research has uncovered that several SPH intermediates and of other lipids as well, govern cell signaling and virulence of C. albicans. In this chapter, we highlight the role of lipids in the physiology of Candida, particularly focusing on their roles in the development of drug resistance. Considering the importance of lipids, the article also highlights recent high-throughput analytical tools and methodologies, which are being employed in our understanding of structures, biosynthesis, and roles of lipids in fungal pathogens.

Dendritic cells (DCs) abundantly express diverse receptors to recognize mannans in the outer surface of Candida cell wall, and these interactions dictate the host immune responses that determine disease outcomes. C. krusei prevalence in candidiasis worldwide has increased since this pathogen has developed multidrug resistance. However, little is known how the immune system responds to C. krusei. Particularly, the molecular mechanisms of the interplay between C. krusei mannan and DCs remain to be elucidated. We investigated how C. krusei mannan affected DC responses in comparison to C. albicans, C. tropicalis and C. glabrata mannan. Our results showed that only C. krusei mannan induced massive cytokine responses in DCs, and led to apoptosis. Although C. krusei mannan-activated DCs underwent apoptosis, they were still capable of initiating Th17 response. C. krusei mannan-mediated DC apoptosis was obligated to the TLR2 and MyD88 pathway. These pathways also controlled Th1/Th17 switching possibly by virtue of the production of the polarizing cytokines IL-12 and IL-6 by the C. krusei mannan activated-DCs. Our study suggests that TLR2 and MyD88 pathway in DCs are dominant for C. krusei mannan recognition, which differs from the previous reports showing a crucial role of C-type lectin receptors in Candida mannan sensing.

Scientific advances have not been sufficient to accompany the growing resistance to antimicrobial medicines. High mortality rates due to opportunistic infections have threated human health. The development of new drugs, such as those obtained from plant sources, is a world priority. Herein, we report the purification of a trypsin inhibitor from Enterolobium timbouva seeds (EtTI) with regard to its homology, physico-chemical and inhibitory properties. Furthermore, we evaluated its activity against Candida strains, opportunistic pathogens regularly found in hospital infections. EtTI belongs to the Kunitz family and inhibits two trypsin molecules simultaneously; a feature shared among double-headed Kunitz inhibitors. A high inhibitory activity against trypsin was determined (Ki = 0.5 nM), and refractory to digestion by pepsin. EtTI was candidicidal against C. albicans, C. buinensis and C. tropicalis, triggering disturbances on integrity of the plasma membrane and morphological alterations, presumably mediated via apoptosis. The presence of two reactive sites is an unusual feature detected in EtTI. Numerous diseases and pathologies involve changes in peptidase activities, encouraging studies with multifunctional inhibitors. Accordingly, the further exploration of EtTI could provide new insights into the Kunitz inhibitors and their applications in disease control., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Spondias tuberosa is a plant that produces a fruit crop with high economic relevance at Brazilian Caatinga. Its roots and leaves are used in folk medicine., Methods: Chemical composition of a hexane extract from S. tuberosa leaves was evaluated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and 1 H nuclear magnetic resonance (NMR). Antioxidant potential was investigated by DPPH and ABTS assays. Antifungal action on Candida species was evaluated determining the minimal inhibitory concentration (MIC 50 ) and putative mechanisms were determined by flow cytometry analysis. In addition, hemolytic activity on human erythrocytes was assessed and the concentration required to promote 50% hemolysis (EC 50 ) was determined., Results: Phytochemical analysis by TLC showed the presence of flavonoids, hydrolysable tannins, saponins and terpenes. The HPLC profile of the extract suggested the presence of gallic acid (0.28 ± 0.01 g%) and hyperoside (1.27 ± 0.01 g%). The representative 1 H NMR spectrum showed saturated and unsaturated fatty acids among the main components. The extract showed weak and moderate antioxidant activity in DPPH (IC 50 : 234.00 μg/mL) and ABTS (IC 50 : 123.33 μg/mL) assays, respectively. It was able to inhibit the growth of C. albicans and C. glabrata with MIC 50 of 2.0 and 0.078 mg/mL, respectively. The treatment of C. glabrata cells with the extract increased levels of mitochondrial superoxide anion, caused hyperpolarization of mitochondrial membrane, and compromised the lysosomal membrane. Weak hemolytic activity (EC 50 : 740.8 μg/mL) was detected., Conclusion: The results demonstrate the pharmacological potential of the extract as antioxidant and antifungal agent, aggregating biotechnological value to this plant and stimulating its conservation.

Candida auris has recently emerged as a multi-drug resistant fungal pathogen that poses a serious global health threat, especially for patients in hospital intensive care units (ICUs). C. auris can colonize human skin and can spread by physical contact or contaminated surfaces and equipment. Here, we show that the mycoparasitic yeast Saccharomycopsis schoenii efficiently kills both sensitive and multi-drug resistant isolates of C. auris belonging to the same clade, as well as clinical isolates of other pathogenic species of the Candida genus suggesting novel approaches for biocontrol.

Yeasts are the most important microorganisms in the fermentation of Chinese Baijiu and the interaction of these yeasts could impact the quality of Baijiu. In this study, we initially characterized the Baijiu yeasts and evaluated their fermentation potential. Wickerhamomyces anomalus GZ3 and Saccharomyces cerevisiae G20 were found to generate high yields of ethyl acetate (2.76 g/L) and 2-phenylethanol, respectively. Results also indicated that the use of W. anomalus along with S. cerevisiae increased volatile compounds production, the maximum ethyl acetate production was observed in S. cerevisiae and W. anomalus at 10 6 :10 6 ratio and have increased by 33 % compared with single culture of W. anomalus. Besides, there was a significant increase of 2-phenylethanol (3.29 g/L) production in single culture of S. cerevisiae with the addition of l-phenylalanine. However, the conversion of l-phenylalanine in mixed culture had significant impact on the yeasts interaction and end flavor of Baijiu. Thus, the present study provided new insights into yeasts interactions in Baijiu fermentation and the effect of some primary metabolites on the end flavor and Baijiu quality., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC 50 of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.

Soft X-ray microscopy has excellent characteristics for imaging cells and subcellular structures. In this paper, the yeast strain, Candida utilis, was imaged by soft X-ray microscopy and three-dimensional volumes were reconstructed with the SART-TV method. We performed segmentation on the reconstruction in three dimensions and identified several types of subcellular architecture within the specimen cells based on their linear absorption coefficient (LAC) values. Organelles can be identified by the correlation between the soft X-ray LAC values and the subcellular architectures. Quantitative analyses of the volume ratio of organelles to whole cell in different phases were also carried out according to the three-dimensional datasets. With such excellent features, soft X-ray imaging has a great influence in the field of biological cellular and subcellular research., (© 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.)

Recently, the mortality of life-threatening fungal infections increased dramatically. However, there are few antifungals existed. Antimicrobial peptides (AMPs) as promising antifungal candidates have attracted much attention. Here, we present a small antimicrobial peptide Jelleine-I that had potent in vitro and in vivo antifungal activity. Negligible hemolytic activity and in vivo toxicity were observed. Selectivity index (SI) of Jelleine-I is at least 4.6 times higher than amphotericin B. Jelleine-I could increase the production of cellular ROS and bind with genome DNA. This may contribute to its antifungal activity. Furthermore, drug resistance is not induced when the fungal cells were repeatedly treated by Jelleine-I. In conclusion, our results suggest that Jelleine-I may have the potential to be developed as a novel antifungal agent.

The pathogenicity of Candida species in human is dependent on a variety of virulence factor such as adhesion factors, germ tube and hyphal formation, secretion of hydrolytic phospholipases and proteinases and drug resistance biofilm. ZnO NPs have been synthesized by using leaf extract of Crinum latifolium and were characterized by UV-Vis spectrophotometer, FTIR, SEM, EDX and TEM. In this study for the first time, potent inhibitory effects of ZnO NPs on principal virulence factors of Candida albicans and non-albicans such as germ tube formation, secretion of hydrolytic phospholipases and proteinases and biofilm formation has been investigated. ZnO NPs remarkably reduced the germ tube formation of C. albicans at 1 (86.4%), 0.5 (75.0%), 0.25 (61.4%), 0.125 (34.1%) and 0.062 mg/ml (11.4%). ZnO NPs significantly lowered the phospholipase and proteinase secretion by 58.8 and 95.2% at 0.25 mg/ml, respectively. CSLM results showed that ZnO NPs suppressed biofilm formation up to 85% at 0.25 mg/ml. SEM and TEM micrograph showed that ZnO NPs penetrated inside the cell and causes extensive damaged in cell wall and cell membrane. Inhibition of Candida growth and various virulent factors by ZnO NPs provides an insight towards their therapeutic application for the treatment of Candida-associated infections.

The incidence of oral candidosis has increased in recent years due to the escalation in HIV-infection, cancer treatments, organ transplantation, and diabetes. In addition, corticosteroid use, dentures, and broad-spectrum antibiotic use have also contributed to the problem. Treatment of oral candidosis has continued to be problematic because of the potential toxicity of antifungals in clinical use, and, above all, development of drug resistance among patients. In this study, the antifungal effect of magnolol was investigated against 64 strains of Candida spp. (four standard and 60 oral isolates) through minimum inhibitory concentration (MIC) and growth curve assays. Insight into the mechanisms of the antifungal action has been gained through ultrastructural studies using confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Molecular docking was done for predicting the interactions of magnolol with ergosterol at supramolecular level. The toxicity of magnolol on human erythrocytes was measured by in vitro hemolytic assay. MIC values of magnolol ranged from 16-64 μg/ml, respectively. All tested isolates showed a marked sensitivity towards magnolol in growth curve assays. Biofilm results suggested that magnolol showed strong anti-biofilm activity. The results obtained for four different Candida spp. demonstrated that MBIC values of magnolol showed the average biofilm inhibition by 69.5%, respectively. CLSM experiments showed that cells exposed to magnolol (MIC) exhibited cell membrane disruption. SEM analysis of magnolol treated cells resulted in deformed cells. TEM micrographs showed rupturing of the cell wall and plasma membrane, releasing the intracellular content, and swelling of the cell wall. Hemolytic activity of magnolol is 11.9% at its highest MIC compared to an activity level of 25.4% shown by amphotericin B (Amp B) at 1 μg/ml. Lipinski's parameters calculated for magnolol suggested its good oral bioavailability. Docking studies indicated that magnolol might be interacting with ergosterol in the fungal cell membranes. Together, the present study provides enough evidence for further work on magnolol so that better strategies could be employed to treat oral candidosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Candida albicans is a diploid fungus that is a frequent cause of mucosal and systemic infections in humans. This species exhibits an unusual parasexual cycle in which mating produces tetraploid cells that undergo a nonmeiotic program of concerted chromosome loss to return to a diploid or aneuploid state. In this work, we used a multipronged approach to examine the capacity of parasex to generate diversity in C. albicans First, we compared the phenotypic properties of 32 genotyped progeny and observed wide-ranging differences in fitness, filamentation, biofilm formation, and virulence. Strikingly, one parasexual isolate displayed increased virulence relative to parental strains using a Galleria mellonella model of infection, establishing that parasex has the potential to enhance pathogenic traits. Next, we examined parasexual progeny derived from homothallic, same-sex mating events, and reveal that parasex can generate diversity de novo from identical parental strains. Finally, we generated pools of parasexual progeny and examined resistance of these pools to environmental stresses. Parasexual progeny were generally less fit than control strains across most test conditions, but showed an increased ability to grow in the presence of the antifungal drug fluconazole (FL). FL-resistant progeny were aneuploid isolates, often being diploid strains trisomic for both Chr3 and Chr6. Passaging of these aneuploid strains frequently led to loss of the supernumerary chromosomes and a concomitant decrease in drug resistance. These experiments establish that parasex generates extensive phenotypic diversity de novo , and that this process has important consequences for both virulence and drug resistance in C. albicans populations., (Copyright © 2017 by the Genetics Society of America.)

Xylose is the second major fermentable sugar present in hard woods and herbs (after d-glucose). Therefore, efficient conversion of xylose to ethanol is essential for the commercialization of lignocellulosic ethanol, which may provide an ideal alternative to fossil fuels in the future. ZLYRHZ7 is a fusant produced by protoplast fusion between two different yeast species, Saccharomyces cerevisiae W5 and Candida shehatae 20335, which is able to utilize xylose to produce ethanol. To improve ethanol production and to quantitatively analyze metabolic pathway in ZLYRHZ7, we used high performance liquid chromatography (HPLC) to assess the utilization rates of xylose, xylitol, and xylulose, and to measure ethanol yields using xylose, xylitol, and xylulose as sole carbon sources. The ethanol yields reached 0.549±0.003, 0.567±0.003 and 0.544±0.005 g/g in 72 h, which indicated that the metabolic pathways from xylose to xylitol, xylitol to xylulose, and xylulose to ethanol, respectively, were functional. In addition, enzyme activity and qRT-PCR analyses showed that the xylose metabolism-related enzymes xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XK) and their respective genes were expressed at significantly higher levels in ZLYRHZ7 than in both S. cerevisiae W5 and C. shehatae 20335 at 24, 48, and 72 h of fermentation. These results clearly show that the fusant ZLYRHZ7, obtained by protoplast fusion of two different yeast species, has the ability to ferment xylose to produce ethanol., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

This study provides a new insight into our understanding of yeast response to starvation conditions (sole acetate as carbon source) and applied polarization and offers important information about the role of the glyoxylate cycle in the carbohydrate synthesis and extracellular charge transfer processes in biofuel cells. The biosynthetic capabilities of yeast C. melibiosica 2491 and the up/down-regulation of the glyoxylate cycle are evaluated by modifying the cellular metabolism by feedback inhibition or carbohydrate presence and establishing the malate dehydrogenase activity and carbohydrate content together with the electric charge passed through bioelectrochemical system. 10mM malate leads to a decrease of the produced quantity of electricity with ca. 55%. At the same time, 24-times lower intracellular malate dehydrogenase activity is established. At polarization conditions the glyoxylate pathway is up-regulated and huge amount of malate is intra-converted into oxaloacetate. The yeasts are able to synthesize carbohydrates from acetate and a part of them is used for the electricity generation. It is recognized that the enhanced charge transfer in acetate fed yeast-based biofuel cell is implemented by secreted endogenous mediator and changes in the cellular surface redox activity depending on the addition of carbohydrate in the medium., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aims: This study aimed to determine how electric field strength, pulse width and shape, and specific energy input relate to the effect of pulsed electric fields (PEF) on viability and membrane permeabilization in Candida humilis and Saccharomyces cerevisiae suspended in potassium phosphate buffer., Methods and Results: Cells were treated with a micro-scale system with parallel plate electrodes. Propidium iodide was added before or after treatments to differentiate between reversible and irreversible membrane permeabilization. Treatments of C. humilis with 71kV/cm and 48kJ/kg reduced cell counts by 3.9±0.6 log (cfu/mL). Pulse shape or width had only a small influence on the treatment lethality. Variation of electric field strength (17-71kV/cm), pulse width (0.086-4μs), and specific energy input (8-46kJ/kg) demonstrated that specific energy input correlated to the membrane permeabilization (r 2 =0.84), while other parameters were uncorrelated. A minimum energy input of 3 and 12kJ/kg was required to achieve reversible membrane permeabilization and a reduction of cell counts, respectively, of C. humilis., Conclusions: Energy input was the parameter that best described the inactivation efficiency of PEF., Significance and Impact of Study: This study is an important step to identify key process parameters and to facilitate process design for improved cost-effectiveness of commercial PEF treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recent research has shown broad antifungal activity of the classic antidepressants selective serotonin reuptake inhibitors (SSRIs). This fact, combined with the increased cross-resistance frequency of the genre Candida regarding the main treatment today, fluconazole, requires the development of novel therapeutic strategies. In that context, this study aimed to assess the antifungal potential of fluoxetine, sertraline, and paroxetine against fluconazole-resistant Candida spp. planktonic cells, as well as to assess the mechanism of action and the viability of biofilms treated with fluoxetine. After 24 h, the fluconazole-resistant Candida spp. strains showed minimum inhibitory concentration (MIC) in the ranges of 20-160 μg/mL for fluoxetine, 10-20 μg/mL for sertraline, and 10-100.8 μg/mL for paroxetine by the broth microdilution method (M27-A3). According to our data by flow cytometry, each of the SSRIs cause fungal death after damaging the plasma and mitochondrial membrane, which activates apoptotic signaling pathways and leads to dose-dependant cell viability loss. Regarding biofilm-forming isolates, the fluoxetine reduce mature biofilm of all the species tested. Therefore, it is concluded that SSRIs are capable of inhibit the growth in vitro of Candida spp., both in planktonic form, as biofilm, inducing cellular death by apoptosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Three yeast strains designated as S44, XF1 and XF2, respectively, were isolated from Scolytus scheryrewi Semenov of apricot tree in Shule County, Xinjiang, China, and were demonstrated to be a new member of the genus Candida by sequence comparisons of 26S rRNA gene D1/D2 domain and internal transcribed spacer (ITS) region. BLASTn alignments on NCBI showed that the similarity of 26S rRNA gene sequences of S44 (type strain) to all sequences of other Candida yeasts was very low (≦93 %). The phylogenetic tree based on the 26S rRNA gene D1/D2 domain and ITS region sequences revealed that the strain S44 is closely related to C. blattae, C. dosseyi, C. pruni, C. asparagi, C. fructus and C. musae. However, the strain S44 is distinguished from these Candida species by the physiological characteristics. Moreover, the strain S44 formed typical pseudohyphae when grown on cornmeal agar at 25 °C for 7 days, but did not form ascospores in sporulation medium for 3-4 weeks. Therefore, the name Candida xinjiangensis is proposed for the novel species, with S44 (=KCTC T 27747) as the type strain.

Background: Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear., Methods: The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model., Results: Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2μg/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load., Conclusion: Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent., General Significance: The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development., Competing Interests: None to declare., (Copyright © 2016 Elsevier B.V. All rights reserved.)

NAD + -dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10C fdh ) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10C fdh , which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances., Importance: FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application., (Copyright © 2016 Zheng et al.)

The extracellular matrix of biofilm is unique to the biofilm lifestyle, and it has key roles in community survival. A complete understanding of the biochemical nature of the matrix is integral to the understanding of the roles of matrix components. This knowledge is a first step toward the development of novel therapeutics and diagnostics to address persistent biofilm infections. Many of the assay methods needed for refined matrix composition analysis require milligram amounts of material that is separated from the cellular components of these complex communities. The protocol described here explains the large-scale production and isolation of the Candida biofilm extracellular matrix. To our knowledge, the proposed procedure is the only currently available approach in the field that yields milligram amounts of biofilm matrix. This procedure first requires biofilms to be seeded in large-surface-area roller bottles, followed by cell adhesion and biofilm maturation during continuous movement of the medium across the surface of the rotating bottle. The formed matrix is then separated from the entire biomass using sonication, which efficiently removes the matrix without perturbing the fungal cell wall. Subsequent filtration, dialysis and lyophilization steps result in a purified matrix product sufficient for biochemical, structural and functional assays. The overall protocol takes ∼11 d to complete. This protocol has been used for Candida species, but, using the troubleshooting guide provided, it could be adapted for other fungi or bacteria.

Cells of Candida guilliermondii permeabilized with Triton X-100 were able to efficiently produce xylitol from a medium composed only by D-xylose and MgCl 2 ·6H 2 O in potassium phosphate buffer, at 35 °C and pH 6.5. Under these conditions, the results were similar to those obtained when cofactor and co-substrate or nutrients were added to the medium (about 95 % D-xylose was assimilated producing 42 g/L of xylitol, corresponding to 0.80 g/g yield and 2.65 g/L h volumetric productivity). Furthermore, the permeabilized cells kept the D-xylose assimilation in about 90 % and the xylitol production in approx. 40 g/L during three bioconversion cycles of 16 h each. These values are highly relevant when compared to others reported in the literature using enzyme technology and fermentative process, thereby demonstrating the effectiveness of the proposed method. The present study reveals that the use of permeabilized cells is an interesting alternative to obtain high xylitol productivity using low cost medium formulation. This approach may allow the future development of xylitol production from xylose present in lignocellulosic biomass, with additional potential for implementation in biorefinery strategies.

The study evaluated the efficiency of eugenyl acetate (EA), a phytochemical in clove essential oil, against clinical isolates of Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida glabrata. Minimum inhibitory concentrations (MIC) of EA against Candida isolates were in the range between 0.1% and 0.4% (v/v). Spot assay further confirmed the susceptibility of Candida isolates to the compound upon treatment with respective 1 × MIC. Growth profile measured in time kill study evidence that the compound at 1 × MIC and 1/2 × MIC retarded the growth of Candida cells, divulging the fungicidal activity. Light microscopic observation demonstrated that upon treated with EA, rough cell morphology, cell damage, and fragmented patterns were observed in C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata. Furthermore, unusual morphological changes of the organism were observed in scanning electron microscopic study. Therefore, it is validated that the compound could cause cell damage resulting in the cell death of Candida clinical isolates. Eventually, the compound at sub-MIC (0.0125% v/v) significantly inhibited serum-induced germ tube formation by C. albicans. Eugenyl acetate inhibited biofilm forming ability of the organisms as well as reduced the adherence of Candida cells to HaCaT keratinocytes cells. In addition, upon treatment with EA, the phagocytic activity of macrophages was increased significantly against C. albicans (P < 0.05). The results demonstrated the potential of EA as a valuable phytochemical to fight against emerging Candida infections., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This review highlights the importance of the biocatalyst, Candida parapsilosis for oxidation and reduction reactions of organic compounds and establishes its versatility to generate a variety of chiral synthons. Appropriately designed reactions using C. parapsilosis effect efficient catalysis of organic transformations such as deracemization, enantioselective reduction of prochiral ketones, imines, and kinetic resolution of racemic alcohols via selective oxidation. This review includes the details of these biotransformations, catalyzed by whole cells (wild type and recombinant strains), purified enzymes (oxidoreductases) and immobilized whole cells of C. parapsilosis. The review presents a bioorganic perspective as it discusses the chemo, regio and stereoselectivity of the biocatalyst along with the structure of the substrates and optical purity of the products. Fermentation scale biocatalysis using whole cells of C. parapsilosis for several biotransformations to synthesize important chiral synthons/industrial chemicals is included. A comparison of C. parapsilosis with other whole cell biocatalysts for biocatalytic deracemization and asymmetric reduction of carbonyl and imine groups in the synthesis of a variety of enantiopure products is presented which will provide a basis for the choice of a biocatalyst for a desired organic transformation. Thus, a wholesome perspective on the present status of C. parapsilosis mediated organic transformations and design of new reactions which can be considered for large scale operations is provided. Taken together, C. parapsilosis can now be considered a 'reagent' for the organic transformations discussed here., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although most cases of candidiasis have been attributed to Candida albicans, non-C. albicans Candida species have been isolated in increasing numbers in patients. In this study, we determined the inhibition of carboxymethyl chitosan (CM-chitosan) on single and mixed species biofilm of non-albicans Candida species, including Candida tropicalis, Candida parapsilosis, Candida krusei and Candida glabrata. Biofilm by all tested species in microtiter plates were inhibited nearly 70%. CM-chitosan inhibited mixed species biofilm in microtiter plates and also on medical materials surfaces. To investigate the mechanism, the effect of CM-chitosan on cell viability and biofilm growth was employed. CM-chitosan inhibited Candida planktonic growth as well as adhesion. Further biofilm formation was inhibited with CM-chitosan added at 90min, 12h or 24h after biofilm initiation. CM-chitosan was not only able to inhibit the metabolic activity of Candida cells, but was also active upon the establishment and the development of biofilms., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Olive leaf extract is characterized by a high content of polyphenols (oleuropein, hydroxytyrosol and their derivatives), which is associated with its therapeutic properties. The objective of the present research was to evaluate the antifungal activity of olive leaf extract against Candida albicans ATCC 10231 and C. dubliniensis CBS 7987 strains. Minimum inhibitory concentrations (MIC) of the extract were determined by several in vitro assays. The extract showed a concentration depended effect on the viability of C. albicans with MIC value of 46.875 mg mL-1 and C. dubliniensis with MIC value 62.5 mg mL-1. Most sensitive methods for testing the antifungal effect of the extracts were the trypan blue exclusion method and fluorescent dye exclusion method while MIC could not be determined by the method according to the EUCAST recommendation suggesting that herbal preparations contain compounds that may interfere with this susceptibility testing. The fluorescent dye exclusion method was also used for the assessment of morphological changes in the nuclei of treated cells. According to the obtained results, olive leaf extract is less effective against the tested strains than hydroxytyrosol, an olive plant constituent tested in our previous study.

Adhesion to buccal epithelial cells (BEC) and denture acrylic surfaces (DAS), germ tube (GT) formation and cell surface hydrophobicity (CSH) are all virulence traits involved in the pathogenicity of Candida. Post-antifungal effect (PAFE) also have a bearing on pathogenicity and virulence of Candida. Candida dubliniensis is associated with oral and systemic candidosis, which can be managed with caspofungin. There is no published information on caspofungin-induced PAFE and its impact on adhesion traits of C. dubliniensis isolates. Thus, the purpose of this investigation was to determine the in vitro duration of PAFE on 20 C. dubliniensis isolates following transient exposure to caspofungin. Furthermore the impacts of caspofungin-induced PAFE on adhesion to BEC and DAS, GT formation and CSH of these isolates were also determined. After establishing the minimum inhibitory concentration (MIC) of caspofungin, C. dubliniensis isolates were exposed to sub-lethal concentrations (×3 MIC) of caspofungin for 1 hr. Thereafter the duration of PAFE, adhesion to BEC and DAS, GT formation and CSH were determined by previously described in-vitro assays. MIC (μg/mL) of C. dubliniensis isolates to caspofungin ranged from 0.004 to 0.19. Caspofungin-induced mean PAFE on C. dubliniensis isolates was 2.17 hr. Exposure to caspofungin suppressed the ability of C. dubliniensis isolates to adhere to BEC and DAS, form GT and CSH by 69.97%, 71.95%, 90.06% and 32.29% (P < 0.001 for all), respectively. Thus, transient exposure of C. dubliniensis isolates to caspofungin produces an antifungal effect not only by suppressing its growth but also by altering its adhesion traits., (© 2016 The Societies and John Wiley & Sons Australia, Ltd.)

Candida parapsilosis is a common cause of invasive candidiasis, especially in premature infants, even surpassing Candida albicans as the most frequently identified Candida species in some newborn intensive care units. Whereas many molecular tools are available to facilitate the study of C. albicans, relatively few have been developed for C. parapsilosis. In this study, we show that plasmids harbouring green, yellow and mCherry fluorescent protein sequences, previously developed for expression in C. albicans, can be used to construct fluorescent fusion proteins in C. parapsilosis by PCR-mediated gene modification. Further, the strategy can be used in clinical isolates of C. parapsilosis, which are typically prototrophic, because the plasmids include NAT1, a dominant selectable trait that confers resistance to the antibiotic nourseothricin. Overall, these tools will be useful to yeast researchers who require the ability to visualize C. parapsilosis directly, e.g. in in vitro and in vivo infection models. In addition, this strategy can be used to generate fluorescence in other C. parapsilosis clinical isolates and to tag sequences of interest for protein localization studies. Lastly, the ability to express up to three different fluorescent proteins will allow researchers to visualize and differentiate C. parapsilosis and/or C. albicans clinical isolates from each other in mixed infection models., (Copyright © 2015 John Wiley & Sons, Ltd.)

An original high-pressure microscopy chamber has been designed for real-time visualization of biological cell growth during high isostatic (gas or liquid) pressure treatments up to 200 MPa. This new system is highly flexible allowing cell visualization under a wide range of pressure levels as the thickness and the material of the observation window can be easily adapted. Moreover, the design of the observation area allows different microscope objectives to be used as close as possible to the observation window. This chamber can also be temperature controlled. In this study, the resistance and optical properties of this new high-pressure chamber have been tested and characterized. The use of this new chamber was illustrated by a real-time study of the growth of two different yeast strains - Saccharomyces cerevisiae and Candida viswanathii - under high isostatic gas pressure (30 or 20 MPa, respectively). Using image analysis software, we determined the evolution of the area of colonies as a function of time, and thus calculated colony expansion rates.

As an eco-friendly management method, biological control of postharvest diseases, utilizing antagonistic yeasts, is a research topic receiving considerable attention. Detailed knowledge on the biology of yeast antagonists is crucial when considering their potential application and development as biocontrol products. Changes in the growth form, such as single-cell to pseudohyphae, have been associated with the mode of action in postharvest biocontrol yeasts. In this study, the antagonistic yeast, Candida diversa, reversibly shifted from a single-cell morphology on yeast peptone dextrose (YPD) medium with 2 % agar to a pseudohyphal morphology on YPD with 0.3 % agar. The tolerance of the pseudohyphal form to heat and oxidative stresses, as well as the biocontrol efficacy against Botrytis cinerea on apple and kiwifruit stored at 25 and 4 °C, was significantly higher as compared to the single-cell form. This study provides new information on the ability of C. diversa to change its morphology and the impact of the morphology shift on stress tolerance and biocontrol performance.

A series of novel 3-substituted isocoumarins was prepared via Pd-catalysed coupling processes and screened in vitro for antifungal activity against Candida species. The study revealed antifungal potential of isocoumarins possessing the azole substituents, which, in some cases, showed biological properties equal to those of clinically used voriconazole. Selected compounds were also screened against voriconazole resistant Candida krusei 6258 and a clinical isolate Candida parapsilosis CA-27. Although the activity against these targets needs to be improved further, the results emphasise additional potential of this new class of antifungal compounds., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The influence of mitochondrial electron transport chain inhibitors on the electricity outputs of Candida melibiosica yeast-based biofuel cell was investigated. The addition of 30 μM rotenone or antimycin A to the yeast suspension results in a decrease in the current generation, corresponding to 25.7±1.3%, respectively 38.8±1.9% reduction in the electric charge passed through the bioelectrochemical system. The latter percentage coincides with the share of aerobic respiration in the yeast catabolic processes, determined by the decrease of the ethanol production during cultivation in the presence of oxygen compared with that obtained under strict anaerobic conditions. It was established that the presence of both inhibitors leads to almost complete mitochondrial dysfunction, expressed by inactivation of cytochrome c oxidase and NADH:ubiquinone oxidoreductase as well as reduced electrochemical activity of isolated yeast mitochondria. It was also found that methylene blue partially neutralized the rotenone poisoning, probably serving as alternative intracellular electron shuttle for by-passing the complex I blockage. Based on the obtained results, we suppose that electrons generated through the aerobic respiration processes in the mitochondria participate in the extracellular electron transfer from the yeast cells to the biofuel cell anode, which contributes to higher current outputs at aerobic conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO2 induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO2, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)