Construction of the advanced flavin mononucleotide producers in the flavinogenic yeast Candida famata.

Flavin mononucleotide (FMN, riboflavin-5'-phosphate) is flavin coenzyme synthesized in all living organisms from riboflavin (vitamin B2 ) after phosphorylation in the reaction catalyzed by riboflavin kinase. FMN has several applications mostly as yellow colorant in food industry due to 200 times better water solubility as compared to riboflavin. Currently, FMN is produced by chemical phosphorylation of riboflavin, however, final product contains up to 25% of flavin impurities. Microbial overproducers of FMN are known, however, they accumulate this coenzyme in glucose medium. Current work shows that the recombinant strains of the flavinogenic yeast Candida famata with overexpressed FMN1 gene coding for riboflavin kinase in the recently isolated by us advanced riboflavin producers due to overexpression of the structural and regulatory genes of riboflavin synthesis and of the putative exporter of riboflavin from the cell, synthesized elevated amounts of FMN in the media not only with glucose but also in lactose and cheese whey. Activation of FMN accumulation on lactose and cheese whey was especially strong in the strains which expressed the gene of transcription activator SEF1 under control of the lactose-induced LAC4 promoter. The accumulation of this coenzyme by the washed cells of the best recombinant strain achieved 540 mg/L in the cheese whey supplemented only with ammonium sulfate during 48 h in shake flask experiments.

Cheese whey supports high riboflavin synthesis by the engineered strains of the flavinogenic yeast Candida famata.

BACKGROUND: Riboflavin is a precursor of FMN and FAD which act as coenzymes of numerous enzymes. Riboflavin is an important biotechnological commodity with annual market sales exceeding nine billion US dollars. It is used primarily as a component of feed premixes, a food colorant, a component of multivitamin mixtures and medicines. Currently, industrial riboflavin production uses the bacterium, Bacillus subtilis, and the filamentous fungus, Ashbya gossypii, and utilizes glucose and/or oils as carbon substrates. RESULTS: We studied riboflavin biosynthesis in the flavinogenic yeast Candida famata that is a genetically stable riboflavin overproducer. Here it was found that the wild type C. famata is characterized by robust growth on lactose and cheese whey and the engineered strains also overproduce riboflavin on whey. The riboflavin synthesis on whey was close to that obtained on glucose. To further enhance riboflavin production on whey, the gene of the transcription activator SEF1 was expressed under control of the lactose-induced promoter of the native ß-galactosidase gene LAC4. These transformants produced elevated amounts of riboflavin on lactose and especially on whey. The strain with additional overexpression of gene RIB6 involved in conversion of ribulose-5-phosphate to riboflavin precursor had the highest titer of accumulated riboflavin in flasks during cultivation on whey. Activation of riboflavin synthesis was also obtained after overexpression of the GND1 gene that is involved in the synthesis of the riboflavin precursor ribulose-5-phosphate. The best engineered strains accumulated 2.5 g of riboflavin/L on whey supplemented only with (NH4)2SO4 during batch cultivation in bioreactor with high yield (more than 300 mg/g dry cell weight). The use of concentrated whey inhibited growth of wild-type and engineered strains of C. famata, so the mutants tolerant to concentrated whey were isolated. CONCLUSIONS: Our data show that the waste of dairy industry is a promising substrate for riboflavin production by C. famata. Possibilities for using the engineered strains of C. famata to produce high-value commodity (riboflavin) from whey are discussed.

Jerveratrum-Type Steroidal Alkaloids Inhibit ß-1,6-Glucan Biosynthesis in Fungal Cell Walls.

The limited number of available effective agents necessitates the development of new antifungals. We report that jervine, a jerveratrum-type steroidal alkaloid isolated from Veratrum californicum, has antifungal activity. Phenotypic comparisons of cell wall mutants, K1 killer toxin susceptibility testing, and quantification of cell wall components revealed that ß-1,6-glucan biosynthesis was significantly inhibited by jervine. Temperature-sensitive mutants defective in essential genes involved in ß-1,6-glucan biosynthesis, including BIG1, KEG1, KRE5, KRE9, and ROT1, were hypersensitive to jervine. In contrast, point mutations in KRE6 or its paralog SKN1 produced jervine resistance, suggesting that jervine targets Kre6 and Skn1. Jervine exhibited broad-spectrum antifungal activity and was effective against human-pathogenic fungi, including Candida parapsilosis and Candida krusei. It was also effective against phytopathogenic fungi, including Botrytis cinerea and Puccinia recondita. Jervine exerted a synergistic effect with fluconazole. Therefore, jervine, a jerveratrum-type steroidal alkaloid used in pharmaceutical products, represents a new class of antifungals active against mycoses and plant-pathogenic fungi. IMPORTANCE Non-Candida albicans Candida species (NCAC) are on the rise as a cause of mycosis. Many antifungal drugs are less effective against NCAC, limiting the available therapeutic agents. Here, we report that jervine, a jerveratrum-type steroidal alkaloid, is effective against NCAC and phytopathogenic fungi. Jervine acts on Kre6 and Skn1, which are involved in ß-1,6-glucan biosynthesis. The skeleton of jerveratrum-type steroidal alkaloids has been well studied, and more recently, their anticancer properties have been investigated. Therefore, jerveratrum-type alkaloids could potentially be applied as treatments for fungal infections and cancer.

Virulence Factors as Promoters of Chronic Vulvovaginal Candidosis: A Review.

INTRODUCTION: The vast majority of the species of the genus Candida spp. is commensal in humans; however, some are opportunistic pathogens that can cause infection, called candidosis. Among the different types of candidosis, we highlight the vulvovaginal (VVC) which can occur in two main clinical variants: chronic (cVVC) and episodic or sporadic. The incidence of cVVC has been worrying the scientific community, promoting the research on genotypic and phenotypic causes of its occurrence. We summarize important findings on factors that favor chronic vulvovaginal candidosis with respect to molecular epidemiology and the expression of various virulence factors, while clarifying the terminology involving these infections. AIM AND METHODOLOGY: The aim of this review was to gather research that linked virulence factors to VVC and its persistence and recurrence, using two databases (Pubmed and Google Scholar). Predisposing factors in women for the occurrence of cVVC and some studies that refer new preventive and alternative therapies were also included, where appropriate. RESULTS AND DISCUSSION: Several studies have been shedding light on the increasing number of persistence and recurrences of VVC. The expression of virulence factors has been related to both chronic forms of VVC and antifungal resistance. Other studies report mutations occurring in the genome of Candida spp. during the infection phase which may be important indications for new therapies. The introduction of preventive therapies and new therapies has revealed great importance and is also highlighted here.

Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility to antifungal agent, and reduces infection.

Candida is an opportunistic pathogen and a common cause of fungal infections worldwide. Anti-fungal use against Candida infections has resulted in the appearance of resistant strains. The limited choice of anti-fungal therapy means alternative strategies are needed to control fungal infectious diseases. The aim of this study was to evaluate the inhibition of Candida biofilm formation by Hedera rhombea (Korean name: songak) extract. Biofilm formation was assessed using the crystal violet assay which showed a dose dependent reduction in the presence of extract with the biofilm formation inhibitory concentration of C. albicans (IC50 = 12.5µg/ml), C. tropicalis var. tropicalis (IC50 = 25µg/ml), C. parapsilosis var. parapsilosis (IC50 = 6.25µg/ml), C. glabrata (IC50 = 6.25µg/ml), C. tropicalis (IC50 = 12.5µg/ml), and C. parapsilosis (IC50 = 12.5µg/ml) without directly reducing Candida growth. Treatment with 6.25µg/mL of extract increased the antifungal susceptibility to miconazole from 32% decreasing of fungal growth to 98.8% of that based on the fungal growth assay. Treatment of extract dose-dependently reduced the dimorphic transition of Candida based on the dimorphic transition assay and treatment of 3.125µg/mL of extract completely blocked the adherence of Candida to the HaCaT cells. To know the molecular mechanisms of biofilm formation inhibition by extract, qRT-PCR analysis was done, and the extract was found to dose dependently reduce the expression of hyphal-associated genes (ALS3, ECE1, HWP1, PGA50, and PBR1), extracellular matrix genes (GSC1, ZAP1, ADH5, and CSH1), Ras1-cAMP-PKA pathway genes (CYR1, EFG1, and RAS1), Cph2-Tec1 pathway gene (TEC1) and MAP kinases pathway gene (HST7). In this study, Hedera rhombea extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is good screen for developing the antifungal adjuvant and Hedera rhombea extract should be a good candidate against biofilm-related fungal infection.

Spectrum and Antifungal Resistance of Candidemia in Neonates With Early- and Late-Onset Sepsis in Pakistan.

BACKGROUND: Neonatal candidemia leads to high morbidity and mortality in developing countries. We studied the trends, spectrum and antifungal resistance in neonatal candidemia isolates from the year 2014 to 2019. METHODS: This was a cross-sectional study conducted at the Aga Khan University, Pakistan. Neonates with positive blood cultures with Candida species were retrospectively identified from the laboratory database (2014-2018) and prospectively in 2019 where clinical information was also collected as part of routine laboratory reporting. RESULTS: We identified 669 neonates with Candida species in blood cultures. Three hundred forty-six neonates had early-onset disease (EOD age ≤7 days) and 323 had late-onset disease (LOD age >7 days). Non-albicans Candida species (86.7%) were predominant versus C. albicans (13.3%; P-value 0.024) with Candida tropicalis being most common in both EOD and LOD. Candida pelliculosa and Candida guilliermondii were associated with EOD and C. albicans with LOD. Isolation of fluconazole nonsusceptible non-albicans Candida species was significantly higher in early-onset (5.9%) versus late-onset (2%) neonatal candidemia (P-value 0.005; crude odds ratio [COR] 2.73, 95% CI: 1.34-5.53). LOD in neonates was more likely associated with the use of vancomycin (COR 3.89, 95% CI: 1.39-10.89). EOD was more likely seen in patients with vaginal delivery (COR 4.16, 95% CI: 1.42-12.23) and in neonates with respiratory distress leading to intensive care unit admission (COR 3.31, 95% CI: 1.05-10.42). CONCLUSIONS: Non-albicans Candida species were increasingly isolated from neonates with candidemia during recent years from Pakistan. Amphotericin remains first-line option for neonatal candidemia in our setting as fluconazole nonsusceptible Candida species are commonly isolated.

Evaluation of the antifungal activity in vitro of midazolam against fluconazole-resistant Candida spp. isolates.

Aim: The purpose of this study was to evaluate the antifungal activity of midazolam, alone and in association with azoles, against isolates of clinical Candida spp. in planktonic and biofilm form. Materials & methods: The antifungal activity was observed using the broth microdilution technique. Flow cytometry tests were performed to investigate the probable mechanism of action and the comet test and cytotoxicity test were applied to evaluate DNA damage. Results: Midazolam (MIDAZ) showed antifungal activity against planktonic cells (125-250 µg/ml) and reduced the viability of Candida spp. biofilms (125 a 2500 µg/ml). The interaction of MIDAZ against Candida spp. biofilms was observed through scanning electron microscopy, causing alteration of their appearance. Therefore, MIDAZ has antifungal potential against Candida spp.

Oligonucleotide-capped nanoporous anodic alumina biosensor as diagnostic tool for rapid and accurate detection of Candida auris in clinical samples.

Candida auris has arisen as an important multidrug-resistant fungus because of several nosocomial outbreaks and elevated rates of mortality. Accurate and rapid diagnosis of C. auris is highly desired; nevertheless, current methods often present severe limitations and produce misidentification. Herein a sensitive, selective, and time-competitive biosensor based on oligonucleotide-gated nanomaterials for effective detection of C. auris is presented. In the proposed design, a nanoporous anodic alumina scaffold is filled with the fluorescent indicator rhodamine B and the pores blocked with different oligonucleotides capable of specifically recognize C. auris genomic DNA. Gate opening modulation and cargo delivery is controlled by successful DNA recognition. C. auris is detected at a concentration as low as 6 CFU/mL allowing obtaining a diagnostic result in clinical samples in one hour with no prior DNA extraction or amplification steps.

Candida metrosideri pro tempore sp. nov. and Candida ohialehuae pro tempore sp. nov., two antifungal-resistant yeasts associated with Metrosideros polymorpha flowers in Hawaii.

Flowers produce an array of nutrient-rich exudates in which microbes can thrive, making them hotspots for microbial abundance and diversity. During a diversity study of yeasts inhabiting the flowers of Metrosideros polymorpha (Myrtaceae) in the Hawai'i Volcanoes National Park (HI, USA), five isolates were found to represent two novel species. Morphological and physiological characterization, and sequence analysis of the small subunit ribosomal RNA (rRNA) genes, the D1/D2 domains of the large subunit rRNA genes, the internal transcribed spacer (ITS) regions, and the genes encoding the largest and second largest subunits of the RNA polymerase II (RPB1 and RPB2, respectively), classified both species in the family Metschnikowiaceae, and we propose the names Candida metrosideri pro tempore sp. nov. (JK22T = CBS 16091 = MUCL 57821) and Candida ohialehuae pro tempore sp. nov. (JK58.2T = CBS 16092 = MUCL 57822) for such new taxa. Both novel Candida species form a well-supported subclade in the Metschnikowiaceae containing species associated with insects, flowers, and a few species of clinical importance. The ascosporic state of the novel species was not observed. The two novel yeast species showed elevated minimum inhibitory concentrations to the antifungal drug amphotericin B (>4 µg/mL). The ecology and phylogenetic relationships of C. metrosideri and C. ohialehuae are also discussed.

Ultrastructural investigation of acidocalcisomes and ATPase activity in yeast Candida guilliermondii NP-4 as 'complementary' stress-targets.

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25-150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.

Mutations in TAC1B: a Novel Genetic Determinant of Clinical Fluconazole Resistance in Candida auris.

Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro Comparative analyses of comprehensive sterol profiles, [3H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and relative expression levels of C. aurisERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into fluconazole-susceptible isolate AR0387 was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance.IMPORTANCECandida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.

Effect of initial antifungal therapy on mortality among patients with bloodstream infections with different Candida species and resistance to antifungal agents: A multicentre observational study by the Turkish Fungal Infections Study Group.

This study aimed to describe the effect of initial antifungal therapy on patient mortality and to detail the current distribution and resistance patterns of Candida spp. among patients with candidaemia. A prospective observational study was performed among consecutive patients with candidaemia from 10 Turkish medical centres between January 2015 and November 2018. The primary outcome was 10-day mortality. Species were identified using MALDI-TOF/MS. A total of 342 patients with candidaemia were included, of which 175 (51.2%) were male and 68 (19.9%) were aged <18 years. The most common species were Candida albicans (47.4%), Candida parapsilosis (26.6%), Candida tropicalis (9.6%) and Candida glabrata (7.6%). Among all Candida spp., the 10-day case fatality rate (CFR) was 32.2%. The CFR was highest in patients with C. albicans (57.3%) and lowest in patients with C. parapsilosis (21.8%). The resistance rate to fluconazole was 13% in C. parapsilosis, with no significant effect on mortality. No resistance to echinocandins was detected. In the multivariate analysis, being in the ICU [OR = 2.1 (95% CI 1.32-3.57); P = 0.002], renal failure [OR = 2.4 (1.41-3.97); P = 0.001], total parenteral nutrition [OR = 2 (1.22-3.47); P = 0.006], C. albicans infection [OR = 1.7 (1.06-2.82); P = 0.027] and echinocandin as primary agent [OR = 0.6 (0.36-0.99); P = 0.047] were significantly associated with mortality. Candidaemia is a deadly infection. Fluconazole resistance is emerging, although it was not significantly related to mortality. Using an echinocandin as the primary agent could be life-saving.

Characterizing fungal communities in medicinal and edible Cassiae Semen using high-throughput sequencing.

Cassiae Semen (CS) has been widely used as roasted tea and traditional Chinese medicine for decades. However, CS is easily contaminated by fungi and mycotoxins during pre-harvest and post-harvest process, thus posing a potential threat to consumer health. In this study, we used the Illumina MiSeq PE300 platform and targeted the internal transcribed spacer 2 sequences to survey the occurrence of fungi in raw and roasted CS samples. Results showed the fungal contamination in all 12 test samples. Ascomycota was the prevailing fungus at the phylum level, with the relative abundance of 66.50%-99.42%. At the genus level, Aspergillus, Cladosporium, and Penicillium were the most dominant genera, accounting for 0.66%-85.51%, 0.20%-29.11%, and 0.11%-32.92% of the fungal reads, respectively. A total of 68 species were identified, among which six potential toxigenic fungi belonging to Aspergillus, Penicillium, Candida, and Schizophyllum genera were detected. Moreover, differences in fungal communities were observed in raw and roasted CS samples. In conclusion, amplicon sequencing is feasible for analyzing fungal communities in CS samples, which provides a new approach to investigate the fungal contamination in edible-medicinal herb, thereby ensuring food safety and drug efficacy.

Diagnosis, management and prevention of Candida auris in hospitals: position statement of the Australasian Society for Infectious Diseases.

Candida auris is an emerging drug-resistant yeast responsible for hospital outbreaks. This statement reviews the evidence regarding diagnosis, treatment and prevention of this organism and provides consensus recommendations for clinicians and microbiologists in Australia and New Zealand. C. auris has been isolated in over 30 countries (including Australia). Bloodstream infections are the most frequently reported infections. Infections have crude mortality of 30-60%. Acquisition is generally healthcare-associated and risks include underlying chronic disease, immunocompromise and presence of indwelling medical devices. C. auris may be misidentified by conventional phenotypic methods. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry or sequencing of the internal transcribed spacer regions and/or the D1/D2 regions of the 28S ribosomal DNA are therefore required for definitive laboratory identification. Antifungal drug resistance, particularly to fluconazole, is common, with variable resistance to amphotericin B and echinocandins. Echinocandins are currently recommended as first-line therapy for infection in adults and children ≥2 months of age. For neonates and infants <2 months of age, amphotericin B deoxycholate is recommended. Healthcare facilities with C. auris should implement a multimodal control response. Colonised or infected patients should be isolated in single rooms with Standard and Contact Precautions. Close contacts, patients transferred from facilities with endemic C. auris or admitted following stay in overseas healthcare institutions should be pre-emptively isolated and screened for colonisation. Composite swabs of the axilla and groin should be collected. Routine screening of healthcare workers and the environment is not recommended. Detergents and sporicidal disinfectants should be used for environmental decontamination.

Candida auris in Germany and Previous Exposure to Foreign Healthcare.

The emerging yeast Candida auris has disseminated worldwide. We report on 7 cases identified in Germany during 2015-2017. In 6 of these cases, C. auris was isolated from patients previously hospitalized abroad. Whole-genome sequencing and epidemiologic analyses revealed that all patients in Germany were infected with different strains.

Enzyme cocktail containing NADH regeneration system for efficient bioremediation of oil sludge contamination.

Oil sludge is one kind of toxic and persistent contamination to ecology system from petroleum industry. In order to recycle contaminated sands and reduce environmental impacts at a lower operating cost, enzyme cocktail 21/CbFDH including NADH regeneration system for oily sludge bioremediation was constructed for the first time. The intracellular enzymes of oil-degrading strain Acinetobacter calcoaceticus 21 were prepared and the formate dehydrogenase gene Cbfdh from Candida boidinii was cloned and functionally expressed in E.coli BL21 induced by lactose. The activity and stability of CbFDH was enhanced through self-induction medium optimization using Box-Behnken design. The CbFDH activity was 12.2 times increased and was only decreased 3.9% upon storage at 30 °C for 5 d. The CbFDH increased the degradation rate of oil in high concentration. For the sludge with 10% oil (w/w), the degradation rate achieved 35.6% after 12 h using enzyme 21/CbFDH with the protein ratio of 1:4. The results will provide novel perspectives for creation and operation of petroleum-degrading enzymes involving formate dehydrogenase with higher efficiency and lower cost comparing to current microbial strains or consortium.

Multilocus sequence typing analysis of Candida africana from vulvovaginal candidiasis.

BACKGROUND: Candida africana is distributed worldwide and colonized in human genitalia and cause mainly vulvovaginal candidiasis (VVC). We report the multilocus sequence typing (MLST) analysis of C. africana from VVC. METHODS: MLST analysis of 43 strains of C. africana, which were isolated from vaginal specimens of patients with VVC, was performed. The enzymatic activity of phospholipase, esterase and haemolysis enzyme production was evaluated.The level of virulent genes and resistant genes mRNA expression was determined by using real-time PCR. Antifungal susceptibilities of the isolates were assayed by using the broth microdilution method. The statistical of the results was determined by the T test and Pearson chi-squared test. RESULTS: The MLST analysis revealed a substantial degree of genetic homogeneity. The DST782 and DST182 were the main MLST genotypes in C. africana. All the patients were symptomatic and with a high mycological cure rate when treated with commonly used antifungal agents.There were statistically significant differences in biofilm formation and phospholipase activity between C. africana and C.albicans. The level of virulent genes and resistant genes mRNA expression was higher in fluconazole-resistant strains. All C. africana isolates were susceptible to fluconazole, itraconazole, voriconazole, caspofungin, and micafungin. These isolates also exhibited low MICs to amphotericin B, flucytosine, and posaconazole. CONCLUSIONS: Candida africana appear to be with a low level of sequence variation in MLST loci. Candida africana, a lower virulence candida, is susceptible to commonly used antifungal agents. This paper was presented at the conference of 8th Trend in Medical Mycology (6-9 October 2017, Belgrade, Serbia) and was published on conference abstract.

Effect of different starter cultures on chemical and microbial parameters of buckwheat honey fermentation.

The aim of this study was to analyze the microbiology of buckwheat honey fermentation inoculated with different starter cultures by culturing and PCR-DGGE, taking as a model for comparison a spontaneously fermented batch. The inoculants tested were (i) cider lees (from a cider factory), (ii) sourdough (from a bakery), and (iii) a commercial Saccharomyces cerevisiae strain. The results of the culturing and culture-independent techniques agreed well and detected the same dominant species along the fermentations. Our results suggest that S. cerevisiae strains, which constituted a majority population in all batches including the uninoculated one, carried out the fermentations. The highest microbial diversity was found at the beginning of the fermentation in the uninoculated batch; this contained in addition to S. cerevisiae bacteria (Paracoccus sp., Staphylococcus sp., and Bacillus sp.) and yeast (Candida sp.) species. Candida sp. was also common in batches inoculated with sourdough and cider lees cultures. Lactobacillus species were found throughout the fermentation of the sourdough-inoculated batch. Basic chemical analysis and testing trials demonstrated that the overall sensory acceptance of the four meads were highly similar. Yeast and bacteria isolated in this study could serve as a source of technologically relevant microorganisms for mead production.

Molecular mechanisms of azole resistance in Candida bloodstream isolates.

BACKGROUND: Antifungal resistance rates are increasing. We investigated the mechanisms of azole resistance of Candida spp. bloodstream isolates obtained from a surveillance study conducted between 2012 and 2015. METHODS: Twenty-six azole non-susceptible Candida spp. clinical isolates were investigated. Antifungal susceptibilities were determined using the Sensititre YeastOne® YO10 panel. The ERG11 gene was amplified and sequenced to identify amino acid polymorphisms, while real-time PCR was utilised to investigate the expression levels of ERG11, CDR1, CDR2 and MDR1. RESULTS: Azole cross-resistance was detected in all except two isolates. Amino acid substitutions (A114S, Y257H, E266D, and V488I) were observed in all four C. albicans tested. Of the 17 C. tropicalis isolates, eight (47%) had ERG11 substitutions, of which concurrent observation of Y132F and S154F was the most common. A novel substitution (I166S) was detected in two of the five C. glabrata isolates. Expression levels of the various genes differed between the species but CDR1 and CDR2 overexpression appeared to be more prominent in C. glabrata. CONCLUSIONS: There was interplay of various different mechanisms, including mechanisms which were not studied here, responsible for azole resistance in Candida spp in our study.