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Despite the severe impact of uncommon yeast fungal infections and the pressing need for more research on the topic, there are still few studies available on the identification, epidemiology, and susceptibility profile of those pathogens. The aims of the current study were to define the profile of uncommon yeast species at Fattouma Bourguiba University Hospital using phenotypic, molecular, and proteomic methods and to study their antifungal susceptibility profile. Pre-identified uncommon yeast species were collected from 2018 to 2021. These isolates were further identified using phenotypic methods (ID32C® system and Vitek2® YST), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and sequencing. The antifungal susceptibility profile was studied using the reference CLSI broth microdilution method. In total, 30 strains were collected during the study period. Referring to the sequencing, the most isolated uncommon species were Saprochaete capitata, Candida lusitaniae, Candida kefyr, Candida inconspicua, and Candida guilliermondii. A total of 90% of isolates were correctly identified by MALDI-TOF MS compared to 76.7% and 63.3% by ID32® C and VITEK® 2 YST, respectively. The isolated species showed variable responses to antifungals. Candida guilliermondii showed increased azole minimum inhibitory concentrations. Misidentification of uncommon yeast species was common using commercial phenotypic methods. The high percentage of concordance of MALDI-TOF results with sequencing highlights its high performance and usefulness as a routine diagnosis tool., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Although Candida species are the most common cause of fungemia, non-Candida rare yeasts (NCY) have been increasingly reported worldwide. Although the importance of these yeast infections is recognized, current epidemiological information about these pathogens is limited, and they have variable antifungal susceptibility profiles. In this study, we aimed to evaluate the clinical characteristics for fungemia caused by NCY by comparing with candidemia. The episodes of NCY fungemia between January 2011 and August 2023 were retrospectively evaluated in terms of clinical characteristics, predisposing factor, and outcome. In addition, a candidemia group, including patients in the same period was conducted for comparison. Antifungal susceptibility tests were performed according to the reference method. A total of 85 patients with fungemia episodes were included: 25 with NCY fungemia and 60 with candidemia. Fluconazole had high minimal inhibitory concentration (MIC) values against almost all NCY isolates. The MIC values for voriconazole, posaconazole, and amphotericin B were ≤ 2 µg/ml, and for caspofungin and anidulafungin were ≥ 1 µg/ml against most of isolates. Hematological malignancies, immunosuppressive therapy, neutropenia and prolonged neutropenia, polymicrobial bacteremia/fungemia, preexposure to antifungal drugs, and breakthrough fungemia were associated with NCY fungemia, whereas intensive care unit admission, diabetes mellitus, urinary catheters, and total parenteral nutrition were associated with candidemia. In conclusion, the majority of fungemia due to NCY species was the problem, particularly in hematology units and patients with hematological malignancy. Preexposure to antifungal drugs likely causes a change in the epidemiology of fungemia in favor of non-albicans Candida and/or NCY., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Formaldehyde and acetaldehyde are reactive small molecules produced endogenously in cells as well as being environmental contaminants. Both of these small aldehydes are classified as human carcinogens, since they are known to damage DNA and exposure is linked to cancer incidence. However, the mutagenic properties of formaldehyde and acetaldehyde remain incompletely understood, at least in part because they are relatively weak mutagens. Here, we use a highly sensitive yeast genetic reporter system featuring controlled generation of long single-stranded DNA regions to show that both small aldehydes induced mutational patterns characterized by predominantly C/G → A/T, C/G → T/A, and T/A → C/G substitutions, each in similar proportions. We observed an excess of C/G → A/T transversions when compared to mock-treated controls. Many of these C/G → A/T transversions occurred at TC/GA motifs. Interestingly, the formaldehyde mutational pattern resembles single base substitution signature 40 from the Catalog of Somatic Mutations in Cancer. Single base substitution signature 40 is a mutational signature of unknown etiology. We also noted that acetaldehyde treatment caused an excess of deletion events longer than 4 bases while formaldehyde did not. This latter result could be another distinguishing feature between the mutational patterns of these simple aldehydes. These findings shed new light on the characteristics of 2 important, commonly occurring mutagens., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Heavy metals act as cofactors for several microbial enzymes and are required in low concentrations for the proper biological functioning of yeasts. Because concentrations beyond the permitted threshold can damage a cell's functionality and viability, metal tolerance in yeasts towards such heavy metals is therefore desirable during fermentation. Tyrosol, a quorum-sensing molecule in yeasts, protects yeasts from oxidative stress induced by various factors, but the performance of the molecule under heavy metal-induced stress is not known. In this investigation, the metal tolerance of four species of endemic yeasts from northeast India, Wickerhamomyces anomalus, Candida tropicalis, Saccharomyces cerevisiae and Candida glabrata, isolated from traditional starter culture cakes, was tested towards zinc (Zn+2), manganese (Mn+2), cobalt (Co+2) and copper (Cu+2) in the presence and absence of tyrosols retrieved from these isolates. The decreasing order of the tolerance of isolates was found to be Mn+2 > Zn+2 > Co+2 > Cu+2. Under the influence of tyrosols, isolates showed enhanced growth at their upper metal tolerance limit. Candida tropicalis showed enhanced growth (2-48-fold, P < 0.0001) in all the tested metal consisting medium (2 mM Zn+2, 5 mM Mn+2, 2 mM Co+2 and 1 mM Cu+2), while W. anomalus, C. glabrata and S. cerevisiae showed increased growth (3-17-fold, P < 0.0001) in Zn+2 (2 mM), Mn+2 (5 mM) and Cu+2 (1 mM) augmented medium. The overall result suggests that tyrosol exerts a protective effect under heavy metal-induced stress, which could be useful in enhancing the quality of fermented products., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

In this study, it was aimed to develop a novel disinfectant from various essential oils containing active components with antimicrobial activity. The mixture of oregano, cinnamon and clove oils (1 : 1 : 1) with 10% oil concentration (SOM) was used as potential disinfectant on various areas and showed the highest antimicrobial activity among oil combinations tested. SOM reduced the numbers of total mesophilic aerobic bacteria (TMAB; 2·27 log CFU per 25 cm 2 ) and Escherichia coli (4·60 log CFU per 25 cm 2 ) under the detection limits. Application of SOM (1, 2, 3, 4 and 6%) into incubators reduced TMAB and mould-yeast counts of incubator air by 82·9 and 100% respectively. SOM application (3%) into ambient air also reduced its TMAB and mould-yeast counts by 92 and 84·6% respectively. While ethanol is commonly used for the disinfection of environments, equipment and surfaces, SOM is an important alternative that may also be used for the disinfection of various surfaces as well as air., (© 2020 The Society for Applied Microbiology.)

Aims: The objective of this study was to evaluate the potential of hexanoic acid (Hex) as a silage additive., Methods and Results: The effect of Hex, Lactobacillus plantarum (Lp) and their combination (Hex + Lp) on the aerobic stability of napier grass silage was investigated. Napier grass was ensiled without additives (C) or with Lp, Hex or Hex + Lp for 60 days followed by 7 days of aerobic exposure. After 60 days of ensiling, the Lp silage had the lowest pH and the highest lactic acid (LA) concentration among all silage, whereas the highest water-soluble carbohydrates (WSC) content was observed in Hex + Lp silage, followed by Hex silage. After 60 days of ensiling, the population of yeasts in C and Lp silages was > 3·0 log 10 CFU per g FW, while that of Hex and Hex + Lp was <2·0 log 10 CFU per g FW. During aerobic exposure, the pH in the Lp and C silages increased (P < 0·05) above 7·0 on day 5. The pH of Hex silage was the lowest among all silages on day 3 and 5, followed by a significant (P < 0·05) increase until 7 days of aerobic exposure. There were no significant changes in pH and AA of Hex + Lp silage over the duration of aerobic exposure. The concentrations of LA in C, Lp and Hex silages decreased while that of Hex + Lp silage remained stable after 3 days of aerobic exposure. The Hex delayed the decline of WSC contents and the increase in yeasts over the aerobic exposure period., Conclusion: Addition of Hex (97 h) and Hex + Lp (>168 h) improved aerobic stability (P < 0·05) as compared to the control (83 h)., Significance and Impact of the Study: Hex or in combination with L. plantarum inhibited the proliferation of yeasts during aerobic exposure of napier grass. Thus, Hex is an alternative antifungal additive to improve aerobic stability., (© 2020 The Society for Applied Microbiology.)

Aim: ChloraPrep™ (CHP) is a clear solution of 2% (w/v) chlorhexidine (CHG) in 70% (v/v) isopropyl alcohol (IPA) administered with a specially designed sterile single-use applicator in which a tinting agent can be added to the CHP solution upon activation of applicator immediately prior to patient skin preparation (CHP+T). This study investigated whether the immediate and residual efficacy of CHP vs CHP+T and a stock solution of 2% CHG in 70% IPA varied, and whether CHP was compromised by the addition of the dye., Methods and Results: We compared the immediate and residual activity (in 1 min) of 70% IPA with that of 2% CHG in 70% IPA stock solution prepared in the laboratory against CHP+T and CHP, against 22 micro-organisms (5 ATCC and 18 clinical isolates) on germ-carriers. CHP and CHP+T demonstrated superior immediate and residual efficacy compared to the 70% IPA plus 2% CHG in 70% IPA stock solutions. Each antiseptic tested showed greater efficacy against the Gram-positive bacteria than against the Gram-negative bacteria. However, their antimicrobial effect on yeasts was even lower., Conclusions: CHP and CHP+T have superior immediate and residual efficacy compared to stock 70% IPA and 2% CHG in 70% IPA solutions, and CHP+T is not affected by the tinting agent., Significance and Impact of the Study: ChloraPrep is a product which can be stained just before use. We have demonstrated that the immediate and residual efficacy of the antimicrobial solution is not compromised by the dye. The efficacy of CHP is greater against bacteria than against yeasts obtained from ICU patients. Interestingly, CHP is more effective against bacteria than a formula made in the laboratory with the same basic components (2% chlorhexidine and 70% IPA). The intermittent heat sterilization process of the commercial preparation might hypothetically have improved the residual activity of the CHP solutions., (© 2019 The Society for Applied Microbiology.)

Alkylation is one of the most ubiquitous forms of DNA lesions. However, the motif preferences and substrates for the activity of the major types of alkylating agents defined by their nucleophilic substitution reactions (SN1 and SN2) are still unclear. Utilizing yeast strains engineered for large-scale production of single-stranded DNA (ssDNA), we probed the substrate specificity, mutation spectra and signatures associated with DNA alkylating agents. We determined that SN1-type agents preferably mutagenize double-stranded DNA (dsDNA), and the mutation signature characteristic of the activity of SN1-type agents was conserved across yeast, mice and human cancers. Conversely, SN2-type agents preferably mutagenize ssDNA in yeast. Moreover, the spectra and signatures derived from yeast were detectable in lung cancers, head and neck cancers and tumors from patients exposed to SN2-type alkylating chemicals. The estimates of mutation loads associated with the SN2-type alkylation signature were higher in lung tumors from smokers than never-smokers, pointing toward the mutagenic activity of the SN2-type alkylating carcinogens in cigarettes. In summary, our analysis of mutations in yeast strains treated with alkylating agents, as well as in whole-exome and whole-genome-sequenced tumors identified signatures highly specific to alkylation mutagenesis and indicate the pervasive nature of alkylation-induced mutagenesis in cancers., (Published by Oxford University Press on behalf of Nucleic Acids Research 2020.)

The incidence of opportunistic yeast infections in humans has been increasing over recent years. These infections are difficult to treat and diagnose, in part due to the large number and broad diversity of species that can underlie the infection. In addition, resistance to one or several antifungal drugs in infecting strains is increasingly being reported, severely limiting therapeutic options and showcasing the need for rapid detection of the infecting agent and its drug susceptibility profile. Current methods for species and resistance identification lack satisfactory sensitivity and specificity, and often require prior culturing of the infecting agent, which delays diagnosis. Recently developed high-throughput technologies such as next generation sequencing or proteomics are opening completely new avenues for more sensitive, accurate and fast diagnosis of yeast pathogens. These approaches are the focus of intensive research, but translation into the clinics requires overcoming important challenges. In this review, we provide an overview of existing and recently emerged approaches that can be used in the identification of yeast pathogens and their drug resistance profiles. Throughout the text we highlight the advantages and disadvantages of each methodology and discuss the most promising developments in their path from bench to bedside., (© FEMS 2019.)

The current study aims to compare between the types of yeasts that cause vulvovaginal candidiasis (VVC) in women using corticosteroid medication compared to nonusers and estimate their sensitivity to available commercial antifungal agents. In a descriptive analytical cross-sectional study, we recruited 41 chronic corticosteroid users diagnosed clinically to have VVC from Women's Health Hospital, Assiut University, Egypt. Forty-seven age-matched women with VVC were recruited as a control group. Full history and clinical examination were performed. Vaginal sterile swab obtained from the vagina of each participant was subjected to direct Gram-stained smear examination as well as a culture on Sabouraud's glucose agar and HiCrome Candida agar. Further identification of the isolates was done using traditional methods. Fifty out of 88 samples (56.8%) were positive in culture including 25 samples (61%) from corticosteroid users group and 25 (53.2%) from noncorticosteroid users with no statistically significant difference (P = .302). The chronic corticosteroid users had more incidence of recurrent VVC as compared to nonusers (65.9% vs 40.4%, respectively) (P = .015). There was a significantly higher rate of non-Candida albicans (NCA) infections in corticosteroid users compared with nonusers (48% vs 20%, respectively) (P = .036). A higher significant difference in resistance of the isolates against clotrimazole (P = .003) and ketoconazole (P = .017) was demonstrated in corticosteroid users compared to nonusers. Thus, chronic corticosteroid use causes frequent attacks of VVC and increases the frequency of infection by NCA strains. Also, it increases resistance to common antifungal agents especially azole group., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The objectives of this study were to identify yeasts in 40 feces samples of Agapornis birds and determine their antifungal susceptibility profile by two methodologies. Results indicated 92.5% of positivity (46 isolates). Six genera were identified (14 species): Cryptococcus (39.13%), four species; Candida (36.96%), four species; Rhodotorula (13.05%), two species; Trichosporon (6.52%), two species; Kodamaea ohmeri (2.17%); and Sporobolomyces salmonicolor (2.17%). Regarding the susceptibility profiles, 41.3% of the isolates showed low sensibility to fluconazole and 47.8% to itraconazole. Thus, Agapornis sp. disseminate potentially pathogenic and resistant yeasts. Precautionary measures should be taken regarding breeding and keeping them as pets., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The marine yeast strain Metschnikowia saccharicola DD21-2, isolated from sediments in the Yalu River, produces a killer toxin with a lethal effect on Metschnikowia bicuspidate strain WCY, a pathogenic yeast strain that infects crabs. In this study, the killer toxin was purified and characterized. After sequential purification, the purity of the killer toxin was increased 72.2-fold over the purity of the yeast cell culture supernatant. The molecular weight of the purified killer toxin was 47.0 kDa. The optimal pH and temperature for killing activity were 5.5°C and 16°C, respectively. The killing activity was stable over a pH range of 4.0-6.5 and temperature range of 0°C-40°C. The purified killer toxin was only effective against toxin-sensitive integral cells and had no killing effect on the protoplasts of toxin-sensitive cells. When exerting the killing effect, the toxin bind to a cell wall receptor of the treated strain, disrupted cell wall integrity and eventually caused death. The amino acid sequence identified by mass spectroscopy indicated that the purified killer toxin might be a protein kinase, but did not show β-1,3-glucanase activity, consistent with the laminarin hydrolysis results. These findings provide a basis for disease prevention and control in marine aquaculture.

Aims: The aim of this study was to examine the antimicrobial properties of novel aqueous natural rapeseed oil/saline emulsions containing different soluble components of spruce resin., Methods and Results: The composition of aqueous resin emulsions was analysed by GC-MS and their antimicrobial properties were studied with challenge tests and with turbidometric assays. The emulsions were strongly antimicrobial against common Gram-positive and Gram-negative bacteria (including MRSA) as well as common yeasts. Furthermore, they inhibited the biofilm formation and eradicated the microbial biofilms on tested microbes. Characteristic for the emulsions was the presence of oxidized resin acids. Other main components present in emulsions, such as lignans and coumaric acids, were not antimicrobial, when tested separately., Conclusions: The results indicated that the oxidized resin acids were the antimicrobial components in the emulsions. Also, there appears to be a stoichiometric relationship between the number of resin acid molecules and the number microbe cells in the antimicrobial action., Significance and Impact of the Study: The fact that these solutions do not contain abietic acid, which is the main allergenic compound in resins, suggests that these solutions would be suitable, well-tolerated antimicrobials for various medical applications. The aqueous formulation will also allow the expansion of the use of these emulsions in from medical applications to the food preservatives and disinfectants., (© 2017 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of the Society for Applied Microbiology.)

Aims: This work aimed to evaluate the antibacterial and antifungal activities of two types of pyroligneous acid (PA) obtained from slow pyrolysis of wood of Mimosa tenuiflora and of a hybrid of Eucalyptus urophylla × Eucalyptus grandis., Methods and Results: Wood wedges were carbonized on a heating rate of 1·25°C min -1 until 450°C. Pyrolysis smoke was trapped and condensed to yield liquid products. Crude pyrolysis liquids were bidistilled under 5 mmHg vacuum yielding purified PA. Multi-antibiotic-resistant strains of Escherichia coli, Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 25923) had their sensitivity to PA evaluated using agar diffusion test. Two yeasts were evaluated as well, Candida albicans (ATCC 10231) and Cryptococcus neoformans. GC-MS analysis of both PAs was carried out to obtain their chemical composition. Regression analysis was performed, and models were adjusted, with diameter of inhibition halos and PA concentration (100, 50 and 20%) as parameters. Identity of regression models and equality of parameters in polynomial orthogonal equations were verified. Inhibition halos were observed in the range 15-25 mm of diameter., Conclusions: All micro-organisms were inhibited by both types of PA even in the lowest concentration of 20%., Significance and Impact of the Study: The feasibility of the usage of PAs produced with wood species planted in large scale in Brazil was evident and the real potential as a basis to produce natural antibacterial and antifungal agents, with real possibility to be used in veterinary and zootechnical applications., (© 2017 The Society for Applied Microbiology.)

Aims: The objective of this study was to show whether the edible part of broccoli has antibacterial and antifungal activity against micro-organism of importance in human health and vegetable spoilage, and to test if this effect was partially due to antimicrobial peptides (AMPs)., Methods and Results: Crude extracts were obtained from florets and stems of broccoli cultivar Avenger and the inhibitory effect was demonstrated against pathogenic bacteria (Bacillus cereus, Staphylococcus xylosus, Staphylococcus aureus, Shigella flexneri, Shigella sonnei, Proteus vulgaris), phytopathogenic fungi (Colletotrichum gloeosporioides, Asperigillus niger) and yeasts (Candida albicans and Rhodotorula sp.). It was shown that samples treated with proteolytic enzymes had a reduction of approximately 60% in antibacterial activity against Staph. xylosus, suggesting that proteinaceous compounds might play a role in the inhibitory effect. Antimicrobial components in crude extracts were thermoresistant and the highest activity was observed under acidic conditions. It was shown that antifungal activity of broccoli's crude extracts might not be attributed to chitinases., Conclusions: Organic broccoli cultivar Avenger has antimicrobial activity against pathogenic bacteria, yeast and phytophatogenic fungi. Data suggest that this effect is partially due to AMPs., Significance and Impact of the Study: Broccoli's crude extracts have activity not only against pathogenic bacteria but also against phytophatogenic fungi of importance in agriculture. We suggest for first time that the inhibitory effect is probably due to AMPs., (© 2017 The Society for Applied Microbiology.)

Ribavirin is an antiviral drug that is used to treat a wide range of human viral infections. However, the side effects are reported, and the mechanisms on eukaryotic cells are still largely unknown. Here we report our observation of accumulation followed by reduction of small nuclear (sn)RNAs and small nucleolar (sno)RNAs in Saccharomyces cerevisiae exposed to ribavirin. The three strains reported to contain dsRNA virus-like particle(s) were exposed to 100 μM of ribavirin, and snRNAs and snoRNAs from a total of 31 snR genes were differentially detected between the samples exposed to ribavirin and the respective negative controls by mRNA-Seq. Our results suggest that polyadenylated snRNAs and snoRNAs accumulated at 1 h but reduced to the subbasal levels at 4 h of ribavirin exposure. The tendency was reproducible across the three tested strains. Our study showed ribavirin affected snRNAs and snoRNAs in yeast. There may be a need to scrutinize the relationships between the side effects and such non-coding RNAs in humans who are treated with ribavirin., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study aimed to identify yeasts from the gastrointestinal tract of scarlet ibises (Eudocimus ruber) and from plant material collected from the environment where they live. Then, the isolates phenotypically identified as Candida famata were submitted to molecular identification of their closely related species and evaluated for their antifungal susceptibility and possible resistance mechanisms to antifungal drugs. Cloacal swabs from 20 scarlet ibises kept in captivity at Mangal das Garças Park (Brazil), pooled stool samples (n = 20) and samples of trunks and hollow of trees (n = 20) obtained from their enclosures were collected. The samples were seeded on Sabouraud agar supplemented with chloramphenicol. The 48 recovered isolates were phenotypically identified as 15 Candida famata, 13 Candida catenulata, 2 Candida intermedia, 1 Candida lusitaniae, 2 Candida guilliermondii, 1 Candida kefyr, 1 Candida amapae, 1 Candida krusei, 8 Trichosporon spp., and 4 Rhodotorula spp. The C. famata isolates were further identified as 3 C. famata, 8 Debaryomyces nepalensis, and 4 C. palmioleophila. All C. famata and C. palmioleophila were susceptible to caspofungin and itraconazole, while one D. nepalensis was resistant to fluconazole and voriconazole. This same isolate and another D. nepalensis had lower amphotericin B susceptibility. The azole resistant strain had an increased efflux of rhodamine 6G and an alteration in the membrane sterol content, demonstrating multifactorial resistance mechanism. Finally, this research shows that scarlet ibises and their environment harbor C. famata and closely related species, including antifungal resistant isolates, emphasizing the need of monitoring the antifungal susceptibility of these yeast species., (© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In cases of fungal infection of the bloodstream, rapid species identification is crucial to provide adapted therapy and thereby ameliorate patient outcome. Currently, the commercial Sepsityper kit and the sodium-dodecyl sulfate (SDS) method coupled with MALDI-TOF mass spectrometry are the most commonly reported lysis protocols for direct identification of fungi from positive blood culture vials. However, the performance of these two protocols has never been compared on clinical samples. Accordingly, we performed a two-step survey on two distinct panels of clinical positive blood culture vials to identify the most efficient protocol, establish an appropriate log score (LS) cut-off, and validate the best method. We first compared the performance of the Sepsityper and the SDS protocols on 71 clinical samples. For 69 monomicrobial samples, mass spectrometry LS values were significantly higher with the SDS protocol than with the Sepsityper method (P < .0001), especially when the best score of four deposited spots was considered. Next, we established the LS cut-off for accurate identification at 1.7, based on specimen DNA sequence data. Using this LS cut-off, 66 (95.6%) and 46 (66.6%) isolates were correctly identified at the species level with the SDS and the Sepsityper protocols, respectively. In the second arm of the survey, we validated the SDS protocol on an additional panel of 94 clinical samples. Ninety-two (98.9%) of 93 monomicrobial samples were correctly identified at the species level (median LS = 2.061). Overall, our data suggest that the SDS method yields more accurate species identification of yeasts, than the Sepsityper protocol., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fungemia due to rare yeasts constitutes an emerging but poorly investigated condition. Data on risk factors, clinical features, therapy, and outcome of episodes of fungemia due to rare (non-Candida, non-Cryptococcus) yeasts were analyzed in a population-based surveillance program conducted in 29 Spanish hospitals between May 2010 and April 2011. Species identification (DNA sequencing) and antifungal susceptibility testing (EUCAST and CLSI methods) were centrally performed. Fourteen out of 767 episodes of fungemia (1.8%) were due to rare yeasts: Trichosporon asahii, Magnusiomyces capitatus (three cases each), Rhodotorula mucilaginosa, Wickerhamomyces anomalus (two cases each), and Pichia kudriavzevii, Cyberlindnera fabianii, Kodamaea ohmeri, and Lodderomyces elongisporus (one case each). Misidentification by local laboratories was observed in two isolates. Breakthrough fungemia occurred in two episodes due to M. capitatus MIC values for echinocandins were generally high (particularly for M. capitatus, T. asahii, and R. mucilaginosa isolates [≥2 mg/l]), whereas T. asahii isolates showed MICs ≥1 mg/l to amphotericin B. Patients with fungemia due to rare yeasts were more likely to have hematological malignancies (28.6% vs. 7.8%; P-value = .021), chronic lung disease (50.0% vs. 22.3%; P-value = .023), and prior immunosuppression (57.1% vs. 22.2%; P-value = .005) compared to those with candidemia. The rate of clinical failure (persistent fungemia and/or 30-day mortality) was 46.2% and did not significantly differ from that observed in episodes of candidemia. In conclusion, non-Candida, non-Cryptococcus yeasts are uncommon causes of fungemia, with immunosuppression and chronic lung disease as predisposing factors. Outcome does not appear to be worse than that of candidemia., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Expansion of polyglutamine stretches in several proteins causes neurodegenerative amyloidoses, including Huntington disease. In yeast, mutant huntingtin (mHtt) with a stretch of 103 glutamine residues (HttQ103) forms toxic aggregates. A range of yeast strains have been used to elucidate the mechanisms of mHtt toxicity, and have revealed perturbations of various unrelated processes. HttQ103 aggregates can induce aggregation of cellular proteins, many of which contain glutamine/asparagine-rich regions, including Sup35 and Def1. In the strain 74-D694 HttQ103, toxicity is related to aggregation-mediated depletion of soluble Sup35 and its interacting partner Sup45. Def1 was also implicated in mHtt toxicity, since its lack detoxified HttQ103 in another yeast strain, BY4741. Here we show that in BY4742, deletion of DEF1 lowers HttQ103 toxicity and decreases the amount of its polymers, but does not affect copolymerization of Sup35. Furthermore, in contrast to 74-D694, increasing the levels of soluble Sup35 and Sup45 does not alleviate toxicity of HttQ103 in BY4742. These data demonstrate a difference in the mechanisms underlying mHtt toxicity in different yeast strains and suggest that in humans with Huntington disease, neurons of different brain compartments and cells in other tissues can also be damaged by different mechanisms., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aim: To determine uncovered antifungal activity of lichen-derived compound, vulpinic acid, by using chemical-genetic analyses., Methods and Results: Haploinsufficiency and homozygous-profiling assays were performed, revealing that strains lacking GLC7, MET4, RFC2, YAE1 and PRP18 were sensitive to three concentrations (12·5, 25 and 50% of inhibitory concentration) of vulpinic acid and independently validated. To verify inhibition of those genes, cell cycle analysis using flow cytometry was performed and relative expressions were measured. Under vulpinic acid-treated condition, cell cycle was arrested in S and G2/M phases and sensitive strains' relative expressions were significantly lower than the wild type yeast., Conclusions: Vulpinic acid mainly affects cell cycle, glycogen metabolism, transcription and translation to fungi., Significance and Impact of the Study: Although lichen-derived compounds are commercially valuable, few studies have determined their modes of action. This study used a chemogenomic approach to gain insight into the mechanisms of one of well-known lichen-derived compound, vulpinic acid., (© 2016 The Society for Applied Microbiology.)

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK) pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS), and barcode analysis by sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating., (Copyright © 2016 Sliva et al.)

During screening for potentially antimicrobial actinobacteria, a highly antagonistic strain, designated WAB9, was isolated from a Saharan soil of Algeria. A polyphasic approach characterized the strain taxonomically as a member of the genus Streptomyces. The strain WAB9 exhibited a broad spectrum of antimicrobial activity toward various multidrug-resistant micro-organisms. A PCR-based assay of genomic potential for producing bioactive metabolites revealed the presence of PKS-II gene. After 6 days of strain fermentation, one bioactive compound was extracted from the remaining aqueous phase and then purified by HPLC. The chemical structure of the compound was determined by spectroscopic (UV-visible, and (1)H and (13)C NMR) and spectrometric analysis. The compound was identified to be 2-amino-N-(2-amino-3-phenylpropanoyl)-N-hydroxy-3-phenylpropanamide, a novel hydroxamic acid-containing molecule. The pure molecule showed appreciable minimum inhibitory concentration values against a selection of drug-resistant bacteria, filamentous fungi and yeasts. Significance and impact of the study: This study presents the isolation of a Streptomyces strain, named WAB9, from a Saharan soil in Algeria. This strain was found to produce a new hydroxamic acid-containing molecule with interesting antimicrobial activities towards various multidrug-resistant micro-organisms. Although hydroxamic acid-containing molecules are known to exhibit low toxicities in general, only real evaluations of the toxicity levels could decide on the applications for which this new molecule is potentially most appropriate. Thus, this article provides a new framework of research., (© 2015 The Society for Applied Microbiology.)

Aims: To evaluate the antimicrobial properties of the main Ginjo-flavour components of sake, volatile isoamyl acetate and isoamyl alcohol., Methods and Results: Volatile isoamyl acetate and isoamyl alcohol both inhibited growth of the five yeast and 10 bacterial test strains. The minimum inhibitory dose and minimum bactericidal (fungicidal) dose of isoamyl acetate were higher than those of isoamyl alcohol. Escherichia coli and Acetobacter aceti were markedly sensitive to isoamyl acetate and isoamyl alcohol. In E. coli exposed to isoamyl acetate for 5 h, changes in expression were noted in proteins involved in sugar metabolism (MalE, MglB, TalB and PtsI), tricarboxylic acid cycle (AceA, Pfl and AcnB) and protein synthesis (EF-Tu, EF-G, and GlyS). Expression of acid and alcohol stress-response proteins was altered in E. coli exposed to isoamyl acetate. Esterase activity was detected in E. coli, suggesting that isoamyl acetate was hydrolyzed to acetic acid and isoamyl alcohol. Acetic acid and isoamyl alcohol damaged E. coli cell membranes and inactivated membrane proteins, impairing respiration., Conclusions: Volatile isoamyl acetate and isoamyl alcohol were effective in inactivating various micro-organisms, and antimicrobial mechanism of volatile isoamyl acetate against E. coli was clarified based on proteome analysis., Significance and Impact of the Study: To the best of our knowledge, this is the first report to examine the antimicrobial mechanism of volatile organic compound using proteome analysis combining two-dimensional difference gel electrophoresis with peptide mass fingerprinting., (© 2015 The Society for Applied Microbiology.)

Unlabelled: A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on the reduction in a tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) with 2-methyl-1,4-napthoquinone as the electron mediator was developed. The proposed method was useful to measure the proliferation of 18 kinds of moulds and seven kinds of yeasts, including representative pathogens such as Aspergillus spp., Candida spp. and Cryptococcus spp. Linear relationships between the absorbance and viable fungal cell density were obtained for all fungi, suggesting that the absorbance change reflected the fungal proliferation. In addition, the minimum inhibitory concentrations (MICs) against a variety of different pathogenic moulds and yeasts for amphotericin B, itraconazole and 5-flucytosine were determined by susceptibility testing using the proposed method and compared with those obtained using the conventional broth microdilution method. There was an excellent agreement between the results obtained using the WST-8 colorimetric method and those obtained using the conventional Clinical and Laboratory Standard Institute method. The WST-8 colorimetric assay is a useful method for rapid determination of accurate MICs for a variety of different fungi., Significance and Impact of the Study: A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on reduction in a tetrazolium salt (WST-8) was developed. The WST-8 colorimetric method was useful to measure the proliferation of a variety of different fungi. In the antifungal susceptibility testing, there was a good agreement between the MICs determined after 24 h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The proposed method was superior to conventional methods in terms of its rapidity towards a variety of different fungi., (© 2014 The Society for Applied Microbiology.)

Conversion of lignocellulosic hydrolysates to lipids using oleaginous (high lipid) yeasts requires alignment of the hydrolysate composition with the characteristics of the yeast strain, including ability to utilize certain nutrients, ability to grow independently of costly nutrients such as vitamins, and ability to tolerate inhibitors. Some combination of these characteristics may be present in wild strains. In this study, 48 oleaginous yeast strains belonging to 45 species were tested for ability to utilize carbon sources associated with lignocellulosic hydrolysates, tolerate inhibitors, and grow in medium without supplemented vitamins. Some well-studied oleaginous yeast species, as well as some that have not been frequently utilized in research or industrial production, emerged as promising candidates for industrial use due to ability to utilize many carbon sources, including Cryptococcus aureus, Cryptococcus laurentii, Hannaella aff. zeae, Tremella encephala, and Trichosporon coremiiforme. Other species excelled in inhibitor tolerance, including Candida aff. tropicalis, Cyberlindnera jadinii, Metschnikowia pulcherrima, Schwanniomyces occidentalis and Wickerhamomyces ciferrii. No yeast tested could utilize all carbon sources and tolerate all inhibitors tested. These results indicate that yeast strains should be selected based on characteristics compatible with the composition of the targeted hydrolysate. Other factors to consider include the production of valuable co-products such as carotenoids, availability of genetic tools, biosafety level, and flocculation of the yeast strain. The data generated in this study will aid in aligning yeasts with compatible hydrolysates for conversion of carbohydrates to lipids to be used for biofuels and other oleochemicals.

Inhibitors released by the breakdown of plant cell walls prevent efficient conversion of sugar into ethanol. The aim of this study was to develop a fast and reliable inhibitor sensitivity assay for ethanologenic yeast strains. The assay comprised bespoke 96-well plates containing inhibitors in isolation or combination in a format that was compatible with the Phenotypic Microarray Omnilog reader (Biolog, hayward, CA, USA). A redox reporter within the assay permits analysis of inhibitor sensitivity in aerobic and/or anaerobic conditions. Results from the assay were verified using growth on spot plates and tolerance assays in which maintenance of viability was assessed. The assay allows for individual and synergistic effects of inhibitors to be determined. It was observed that the presence of both acetic and formic acid significantly inhibited the yeast strains assessed, although this impact could be partially mitigated by buffering to neutral pH. Scheffersomyces stipitis, Candida spp., and Pichia guilliermondii demonstrated increased sensitivity to short chain weak acids at concentrations typically present in lignocellulosic hydrolysates. S. cerevisiae exhibited robustness to short chain weak acids at these concentrations. However, S. stipitis, Candida spp., and P. guilliermondii displayed increased tolerance to HMF when compared to that observed for S. cerevisiae. The results demonstrate that the phenotypic microarray assay developed in the current study is a valuable tool that can be used to identify yeast strains with desirable resistance to inhibitory compounds found in lignocellulosic hydrolysates.

Aims: In the present study, it was investigated the antagonistic behaviour of Metschnikowia pulcherrima, as biocontrol agent, against the main wine yeast species involved in the winemaking process., Methods and Results: Seven strains of M. pulcherrima were evaluated for the antimicrobial activity against 114 yeast strains belonging to Pichia, Candida, Hanseniaspora, Kluyveromyces, Saccharomycodes, Torulaspora, Brettanomyces and Saccharomyces genera. Results showed both different inter-generic and intra-generic responses to the antimicrobial action of M. pulcherrima strains. Interestingly, the antimicrobial activity of M. pulcherrima did not have any influence on the growth of Saccharomyces cerevisiae. Instead, M. pulcherrima displayed a broad and effective antimicrobial action on undesired wild spoilage yeasts, such as Brettanomyces/Dekkera, Hanseniaspora and Pichia genera. Fermentation trials carried out in synthetic grape must confirmed the antimicrobial activity of M. pulcherrima, determining the early death of the non-Saccharomyces co-inoculated cultures., Conclusions: The antimicrobial activity of M. pulcherrima does not seem due to proteinaceous compounds such as killer phenomenon, but to the pulcherriminic acid (the precursor of pulcherrimin pigment) that depletes iron present in the medium, making it not available to the other yeasts., Significance and Impact of the Study: These data agree with and further support the potential use of selected M. pulcherrima strains in controlled multistarter fermentations with S. cerevisiae starter cultures., (© 2014 The Society for Applied Microbiology.)

Aims: To investigate the efficacy and mode of action of the fatty acid-based product Foodcoat(®) (FC) against Botrytis cinerea., Methods and Results: In vitro, in vivo and field experiments were carried out to investigate the effect of different concentrations of FC on B. cinerea germination and infection of grape leaves and berries, using three selected isolates and comparing results with those achieved by the commercialized product Protector(HML) (PRT). Furthermore, the effect of field applications of FC on the grape berry microbiota was investigated. FC reduced B. cinerea germination and grape berry severity by up to 54 and 96%, respectively, compared with the untreated controls., Conclusions: Foodcoat demonstrated efficacy that was equal or greater than the registered product, PRT. A multiple mode of action was hypothesized for FC suppression of B. cinerea, including: inhibition of germination and germ tube alteration, protection of host green tissues and enhancement of the natural yeast populations on the berry surface., Significance and Impact of the Study: The efficacy of both products has been quantified and their modes of action described, suggesting them for field applications against B. cinerea, alone or in combined strategies. This is also the first report of a fatty acid-based product stimulating natural yeast populations on grape berries., (© 2014 The Society for Applied Microbiology.)

Aims: The aim of this study was to investigate the effect of clary sage, juniper, lemon and marjoram essential oils (EOs) and their major components on the formation of bacterial and yeast biofilms and on the inhibition of AHL-mediated quorum sensing (QS)., Methods and Results: Biofilm formation was measured by crystal violet and resazurin staining, and QS inhibition was detected by paper disc diffusion assay. Marjoram EO inhibited Bacillus cereus, Pichia anomala, Pseudomonas putida and mixed-culture biofilm formation of Ps. putida and Escherichia coli and showed the best QS inhibitor effect on Chromobacterium violaceum. For B. cereus, all components showed better antibiofilm capacity than the parent EOs. Lemon EO inhibited E. coli and mixed-culture biofilms, and cinnamon was effective against the mixed forms. Scanning electron microscopy showed the loss of three-dimensional structures of biofilms., Conclusions: The EOs and components used seem to be good candidates for prevention of biofilm formation and inhibition of the AHL-mediated QS mechanism., Significance and Impact of the Study: Biofilm formation on foods and food industrial equipment is a serious problem causing food spoilage and emergence of foodborne diseases. This article highlights the importance of studying EOs as potential disinfectants and food preservatives., (© 2013 The Society for Applied Microbiology.)

Fungal infections with multiple resistance to conventional antifungals are increasingly becoming a medical problem, and there is an urgent need for new antifungal compounds with novel mechanisms of action. Here, we show that aurein 2.5, a naturally occurring peptide antibiotic, displays activity against the fungal strains: Rhodotorula rubra and Schizosaccharomyces pombe (MICs < 130 μM). The peptide adopted high levels of membrane-interactive α-helical structure (> 65%) in the presence of lipid membranes derived from these organisms and showed strong propensities to penetrate (π ≥ 13 mN m(-1) ) and lyse them (> 70%). Based on these data, we suggest that aurein 2.5 kills yeasts via membranolytic mechanisms and may act as a template for the development of therapeutically useful antifungal agents., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Aims: The objective of this study was to evaluate fungicidal efficacy of hydrogen peroxide administered in combination with 17 mineral and organic acids authorized for use in the food industry., Methods and Results: The assays were performed on a 96-well microplate using a microdilution technique based on the checkerboard titration method. The six selected strains (one yeast and five fungi) were reference strains and strains representative of contaminating fungi found in the food industry. Each synergistic hydrogen peroxide/acid combination found after fifteen minutes contact time at 20 °C in distilled water was then tested in conditions simulating four different use conditions. Twelve combinations were synergistic in distilled water, eleven of these remained synergistic with one or more of the four mineral and organic interfering substances selected. Hydrogen peroxide/formic acid combination remained effective against four strains and was never antagonistic against the other two fungi. Combinations with propionic acid and acetic acid stayed synergistic against two strains. Those with oxalic acid and lactic acid kept their synergism only against Candida albicans. No synergism was detected against Penicillium cyclopium., Conclusions: Synergistic combinations of disinfectants were revealed, among them the promising hydrogen peroxide/formic acid combination., Significance and Impact of the Study: A rapid screening method developed in our laboratory for bacteria was adapted to fungi and used to reveal the synergistic potential of disinfectants and/or sanitizers combinations., (© 2012 The Society for Applied Microbiology.)

Aims: The aim of the study was to describe the microbiological findings of cardiac implantable electronic devices (CIEDs) infection in the 2000-2011 period at the Cardiology Unit of New Santa Chiara Hospital in Pisa (Italy)., Methods and Results: Removed CIED leads and pocket material were seeded on solid media and isolates tested for antimicrobial susceptibility with the Kirby Bauer method. Electrodes from 1204 patients were analysed and 854 (70.9%) tested positive. In 663 (77.6%) cases only one species was isolated, in 175 (20.5%) two species, and in 14 (1.8%) >2 species. In 116 cases material from the pocket was also cultured. The result was consistent with that from the electrodes in 69 (59%) cases. In 359 cases a blood sample was also obtained for culture. The result was consistent with that from the leads in 124 (35%) cases. A total of 1068 strains were isolated from electrodes. Gram-positive organisms were most frequently isolated (92.5% of isolates); particularly, coagulase-negative staphylococci (CoNS), mainly Staphylococcus epidermidis, in 69% of cases and Staphylococcus aureus in 13.8%, Gram-negative rods in 6.1%, yeasts in 1% and molds in 0.4%. Overall, Oxacillin resistance was 30%, in particular 33% among CoNS and 13% among S. aureus. Oxacillin resistance and quinolones resistance have increased in the period 2006-2011 with respect to the 5 years before. Seventeen percent of Enterobacteriaceae strains had a phenotype compatible with extended spectrum beta-lactamase expression., Conclusions: Culture of the leads offers the possibility of an aetiological diagnosis in the majority of cases. When material from the pocket can be obtained, the microbiological result is often consistent with that from the electrodes, while species isolated from blood cultures are often different and more likely to be the result of contamination. Cardiac implantable electronic device infection is more often monomicrobial, CoNS are most frequently isolated and S. epidermidis is largely the main single agent. Very early infections were associated with S. aureus infection. The pattern of susceptibility to antimicrobials is in general that of community-acquired infections, although oxacillin resistance and quinolones resistance has increased in the last 5 years.

Yeast flocculation is an important property for the brewing industry as well as for ethanol fermentation to facilitate biomass recovery by sedimentation from the fermentation broth, which is cost-effective. In this study, a new flocculating gene FLO10 (spsc) of 4,221 bp homologous to FLO10 was identified in the industrial flocculating yeast SPSC01. Sequence analysis indicated that the N- and C-terminus of the deduced protein of this new FLO gene are 99 % identical to that of FLO10, but more intragenic repeats are included. The study on the function of FLO10 (spsc) by its integrative expression in the non-flocculating industrial yeast indicated severe inhibition in the flocculation of the transformant by mannose and maltose, moderate inhibition by sucrose and glucose and no inhibition by xylose and galactose, and thus the NewFlo type was established. Meanwhile, the flocculation of the transformant was stable when the temperature was below 50 °C and the pH was in the range of 4.0-6.0. Furthermore, the medium containing 250 g/l glucose was completely fermented within 48 h by the transformant, with about 110 g/l ethanol and 5.5 g(DCW)/l biomass produced, and no significant difference in ethanol fermentation performance was observed compared to its wide-type strain. Therefore, the FLO gene and corresponding transformation strategy provide a platform for engineering yeast strains with the flocculation phenotype to facilitate biomass recovery.

Rapid and accurate identification of clinically important yeasts is essential given their inherent differences in antifungal susceptibility. We implemented nucleic acid sequencing for those species that could not be identified by phenotypic methods. Internal Transcribed Spacer region 1 and 2 (ITS1 and ITS2) sequences were investigated using SmartGene IDNS software, an rDNA sequence database and analysis program for microbial identification (ID). Over a 2.5-year period, 2,938 specimens were evaluated. Most (94%) isolates were fully identified by conventional methods, with Candida species accounting for the majority of them. Of the 169 organisms that required molecular analysis, 79% were identified to species level, 19% to genus and 2% remained unresolved. Sequenced isolates encompassed 33 unique species of which approximately half (52%) were common pathogens with atypical biochemical profiles and the remainder were rarer yeast species. A significant proportion (33%) of sequenced organisms displayed elevated MICs to fluconazole. Our experience supports the use of molecular techniques as an adjunct to conventional methods for the identification of medically important yeasts. Susceptibility testing alone may provide valuable treatment information in situations where phenotypic assessments are inconclusive and molecular or proteomic testing is not readily available.

Aims: The in vitro antimicrobial activity of three essential oil samples of frankincense (Boswellia rivae, Boswellia neglecta and Boswellia papyrifera) and two essential oil samples of myrrh and sweet myrrh (Commiphora guidotti and Commiphora myrrha), collected from different regions of Ethiopia, was investigated independently and in combination to determine their anti-infective properties., Methods and Results: The microdilution minimum inhibitory concentration (MIC) assay was performed, whereby it was noted that generally Cryptococcus neoformans (MIC values in the range of 0·8-1·4 mg ml(-1)) and Pseudomonas aeruginosa (MIC values in the range of 0·5-1·3 mg ml(-1)) often appeared to be the most susceptible micro-organisms against oils of both Boswellia and Commiphora spp. When assayed in various combinations, the frankincense and myrrh oils displayed synergistic, additive and noninteractive properties, with no antagonism noted. When investigating different ratio combinations against Bacillus cereus, the most favourable combination was between B. papyrifera and C. myrrha. The composition of the oils was determined by gas chromatography coupled to mass spectrometry (GC-MS) to document the specific chemotypes used in the study, and the chemical profiles were found to be congruent with previously reported data., Conclusions: The majority of interactions identified synergistic and additive effects, with strong synergism noted between B. papyrifera and C. myrrha., Significance and Impact of the Study: Frankincense and myrrh essential oils have been used in combination since 1500 bc; however, no antimicrobial investigations have been undertaken to confirm their effect in combination. This study validates the enhanced efficacy when used in combination against a selection of pathogens., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

Nickel has long been known to be an important human toxicant, including having the ability to form carcinomas, but until recently nickel was believed to be an issue only to microorganisms living in nickel-rich serpentine soils or areas contaminated by industrial pollution. This assumption was overturned by the discovery of a nickel defense system (RcnR/RcnA) found in microorganisms that live in a wide range of environmental niches, suggesting that nickel homeostasis is a general biological concern. To date, the mechanisms of nickel toxicity in microorganisms and higher eukaryotes are poorly understood. In this review, we summarize nickel homeostasis processes used by microorganisms and highlight in vivo and in vitro effects of exposure to elevated concentrations of nickel. On the basis of this evidence we propose four mechanisms of nickel toxicity: (1) nickel replaces the essential metal of metalloproteins, (2) nickel binds to catalytic residues of non-metalloenzymes; (3) nickel binds outside the catalytic site of an enzyme to inhibit allosterically and (4) nickel indirectly causes oxidative stress.

Aims: The objectives of this research were to (i) optimize the concentration of cycloheximide for use in WL media used in the wine industry and (ii) evaluate Dekkera/Brettanomyces differential medium (DBDM) as a means to detect Dekkera., Methods and Results: Dekkera bruxellensis and other yeasts were transferred into WL broths containing 0, 10, 50 or 100 mg l(-1) of cycloheximide. While several grew in 10 mg l(-1) , only Hanseniaspora uvarum, Pichia guillermondii, Schizosaccharomyces pombe and D. bruxellensis tolerated ≥50 mg l(-1) of the antibiotic. On solidified WL media after 8-days incubation, colony sizes of two strains of D. bruxellensis (B1b and ATCC 52905) decreased with increased concentrations of cycloheximide, while others (F3 and P2) were unaffected. Although D. bruxellensis B1b did not grow well on another selective medium, DBDM, colony development was improved by the addition of sterilized red wine., Conclusions: Of the concentrations tested, 50 mg l(-1) cycloheximide inhibited many grape/wine yeasts yet generally yielded countable colonies of Dekkera (1-2.5 mm diameter). Several strains of Dekkera did not grow well on DBDM, probably due to the lack of an unidentified nutrient(s)., Significance and Impact of the Study: Better media formulations will improve the detection of Dekkera, thereby increasing microbiological control during winemaking., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Objectives: The aim of this study was to investigate the flavonoid composition of Scutellaria immaculata and S. ramosissima (Lamiaceae) and the in-vitro biological activity of their extracts and flavonoids., Methods: The flavonoid composition of S. immaculata (Si) and S. ramosissima (Sr) were analysed using LC-MS. Antimicrobial activity was studied in vitro against a range of bacteria and fungi using diffusion and microdilution methods. Anti-trypanosomal and cell proliferation inhibitory activity of the extracts and flavonoids was assessed using MTT. The antioxidant activity of the flavonoids and extracts were evaluated using DPPH* test., Key Findings: LC-MS investigation of Si and Sr plants allowed the identification, for the first time, of an additional 9 and 16 flavonoids, respectively. The methanol, chloroform and water extracts from these plants and six flavonoids (scutellarin, chrysin, apigenin, apigenin-7-O-glucoside, cynaroside and pinocembrine) exhibited significant inhibition of cell growth against HeLa, HepG-2 and MCF-7 cells. The chloroform extract of Sr showed potent cytotoxic effects with IC50 (drug concentration which resulted in a 50% reduction in cell viability) values of 9.25 ± 1.07 µg/ml, 12.83 ± 1.49 µg/ml and 17.29 ± 1.27 µg/ml, respectively. The highest anti-trypanosomal effect against T. b. brucei was shown by the chloroform extract of Sr with an IC50 (drug concentration which resulted in a 50% inhibition of the biological activity) of 61 µg/ml. The pure flavonoids showed an IC50 range between 3 and 29 µm, with cynaroside as the most active compound with an IC50 value of 3.961 ± 0.133 µm. The chloroform extract of Sr has potent antimicrobial activity against Streptococcus pyogenes (minimum inhibitory concentration, MIC = 0.03 mg/ml). Pinocembrine exhibited a strong activity against the all bacteria except Escherichia coli and yeasts. Water extracts of Sr and Si exhibited potent antioxidant activity with IC50 values of 5.62 ± 0.51 µg/ml and 3.48 ± 0.02 µg/ml, respectively. Scutellarin exerted stronger antioxidant activity than other flavonoids., Conclusions: This is the first study reporting an in-vitro biological investigation for Si and Sr. Especially the chloroform extract of Sr showed potent anticancer and antimicrobial activity. Cynaroside had a highly selective and strong cytotoxicity against T. b. brucei while showing only mild effects against cancer cells., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

[URE3] is a prion (infectious protein) of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. We show that wild S. paradoxus can be infected with a [URE3] prion, supporting the use of S. cerevisiae as a prion test bed. We find that the Ure2p of Candida albicans and C. glabrata also regulate nitrogen catabolism. Conservation of amino acid sequence within the prion domain of Ure2p has been proposed as evidence that the [URE3] prion helps its host. We show that the C. albicans Ure2p, which does not conserve this sequence, can nonetheless form a [URE3] prion in S. cerevisiae, but the C. glabrata Ure2p, which does have the conserved sequence, cannot form [URE3] as judged by its performance in S. cerevisiae. These results suggest that the sequence is not conserved to preserve prion forming ability.

Aims: This study is focused on the possibility to control microbial contamination of strawberries by chlorophyllin (Na-Chl)-based photosensitization. Moreover, photosensitization-induced effects on key quality attributes of treated strawberries was evaluated., Methods and Results:   Strawberries were inoculated with Listeria monocytogenes ATC(L3) C 7644, soaked in 1mmol l(-1) Na-Chl for 5 min and illuminated for 30 min with visible light (λ = 400 nm, energy density 12 mW cm(-2) ). Results indicated that the decontamination of strawberries using photosensitization was 98% compared to control sample. Naturally occurring yeasts/microfungi and mesophiles were inhibited by 86 and 97%, respectively. The shelf life of treated strawberries was extended by 2 days. The total antioxidant activity of treated strawberries increased by 19%. No impact on the amount of phenols, anthocyanins or surface colour was detected., Conclusions:   Photosensitization may be an effective, nonthermal and environmentally friendly microbial decontamination technique which expands the shelf life of strawberries without any negative impact on antioxidant activity, and phenols, anthocyanins or colour formation., Significance and Impact of the Study: Experimental data support the idea that Na-Chl-based photosensitization can be a useful tool for the future development of nonthermal food preservation technology., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Motivation: With the growing availability of high-throughput protein-protein interaction (PPI) data, it has become possible to consider how a protein's local or global network characteristics predict its function., Results: We introduce a graph-theoretic approach that identifies key regulatory proteins in an organism by analyzing proteins' local PPI network structure. We apply the method to the yeast genome and describe several properties of the resulting set of regulatory hubs. Finally, we demonstrate how the identified hubs and putative target gene sets can be used to identify causative, functional regulators of differential gene expression linked to human disease., Availability: Code is available at http://bcb.cs.tufts.edu/hubcomps., Contact: fox.andrew.d@gmail.com; slonim@cs.tufts.edu, Supplementary Information: Supplementary data are available at Bioinformatics online.

A large number of non-Saccharomyces yeasts were isolated from grapes of Bobal variety and identified according to their physiological and molecular characteristics. The yeasts were tested to determine the presence of β-glucosidase, β-xylosidase, α-arabinosidase, and α-rhamnosidase activities and five isolates were selected. All enzymatic activities were induced by the presence of glycosides as the only carbon source in the medium, which seems to be a characteristic of the yeast isolate, and were characterized according to different parameters of enological interest.

Squalamine and other aminosterols have demonstrated interesting antimicrobial activities against clinical bacterial isolates and a limited number of reference yeast strains. We aimed to test whether squalamine and a synthetic aminosterol derivative (ASD) display any in vitro activity comparable to currently available systemic antifungals, an acceptable safety index, as well as to provide insights into their mechanism of action. The minimum inhibitory concentrations (MICs) of squalamine, ASD and available antifungals were determined against 21 yeast isolates that were recovered from cases of fungemia. Remarkably, homogeneous MICs ranging from 8-16 mg/L and from 1-2 mg/L were noted for squalamine and ASD, respectively, as opposes the heterogeneous in vitro activity of available systemic antifungals. Aminosterols induced haemolysis, a surrogate for toxic effects to mammalian cells, at concentrations high above their MICs. In time-kill studies, killing was as fast with ASD as with amphotericin B. Both aminosterols induced a time-dependent disruption of yeast membrane, as evidenced by gradual increase of ATP efflux. In conclusion, our preliminary data indicate that aminosterols have the potential to be further developed as antifungals. Additional work is warranted to assess their toxicity and activity in experimental models.

Aims: Investigating the effect of perturbations, with relatively low Cu concentrations, on yeast community composition in low-nutrient virgin soil., Methods and Results: Culturable soil yeast populations were monitored at an experimental site treated with the fungicide copper oxychloride (10 mg Cu per kg soil). Yeast numbers were unaffected by additional Cu; however, a shift in yeast community composition from Hymenomycetes to Urediniomycetes species occurred. Subsequent growth experiments conducted with a synthetic liquid medium revealed that hymenomycetous and urediniomycetous yeasts were affected differently by 1 and 10 mg l(-1) Cu. Soil microcosm experiments then indicated that additional 10 mg kg(-1) Cu may improve the competitive ability of urediniomycetous yeasts in the presence of hymenomycetous yeasts., Conclusions: The shift from hymenomycetous to urediniomycetous yeasts, as a result of slightly increased soil Cu levels, may be because of hymenomycetous yeasts being more sensitive to elevated Cu levels and urediniomycetous yeasts having an improved competitive ability in the presence of elevated Cu levels., Significance and Impact of the Study: Yeast community composition of pristine low-nutrient soils may change as a result of perturbations with relatively low concentrations of Cu. Urediniomycetous yeasts should be studied as potential bio-indicators of Cu perturbations., (© 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.)

The stress sensitivity of different wild-type strains was evaluated, as well as the response of cells arrested at different cell cycle positions to high hydrostatic pressure (HPP). HHP was chosen both for its importance in food decontamination and assessment of its suitability as a model for stress in general and understanding the yeast stress response. Studies were conducted with four industrial strains and four laboratory wild-type yeast strains (two haploid and two diploid) that differed in their backgrounds. Fundamental differences were found between the laboratory and industrial populations. Industrial strains were clearly more sensitive to hydrostatic pressure and ethanol stresses than the laboratory strains. However, ethanol production was higher in industrial strains than laboratory strains. Furthermore, no correlation was observed between ploidy and stress resistance. Yeast cells arrested in the G1 phase led to an enhancement in pressure tolerance compared to unarrested, G2 arrested, and S arrested cells. Moreover, cells arrested in the S phase were more sensitive to hydrostatic pressure than cells arrested in the G2 phase. Again, industrial strains were more sensitive than laboratory strains. HHP responses of industrial yeasts correlated well with both ethanol concentration and temperature stress, which suggests that it would be a useful model stress.

Aims: In this study, the potential of calcium chloride (CaCl(2)) application to improve the efficacy of the marine antagonist Rhodosporidium paludigenum in controlling postharvest diseases of cherry tomatoes was assessed., Methods and Results: CaCl(2) alone was found not to have any direct influence on the population growth of R. paludigenum in NYDB cultures or in cherry tomato wounds. However, the combined treatments with 1 x 10(8) cells ml(-1)R. paludigenum and CaCl(2) at the concentration from 0.5 to 2% showed high activities to reduce black rot caused by Alternaria alternata in cherry tomato wounds, significantly higher than those of R. paludigenum or CaCl(2) alone. Meanwhile, 0.5% CaCl(2) in combination with 1 x 10(8) cells ml(-1)R. paludigenum greatly inhibited the natural decay of cherry tomatoes in 21 days' storage at 25 degrees C., Conclusions: The combination of R. paludigenum and CaCl(2) enhances the inhibition of black rot and natural decay of postharvest cherry tomatoes. The results from this study provide a new way to improve the efficiency of R. paludigenum in maintaining the quality of postharvest fruits and vegetables., Significance and Impact of the Study: The marine yeast R. paludigenum combined with CaCl(2) has greatly potential use as an alternative to chemical fungicides in inhibiting postharvest decay on cherry tomatoes.