Diversity of Saccharomyces cerevisiae yeasts associated to spontaneous and inoculated fermenting grapes from Spanish vineyards.

The use of commercial yeast strains is a common practice in winemaking leading to a predictable quality in wine production, avoiding stuck or sluggish fermentations. However, the use of commercial yeasts leads to a consequent reduction in autochthonous microbial diversity. In this study, 1047 isolates from three Spanish appellations of origin were checked for fingerprinting on interdelta polymorphisms and the strain composition and diversity analysed using an extensible open-source platform for processing and analysis of an in-house polymorphism database developed for this study. Ancient vineyards managed with organic practices showed intermediate to low levels of strains diversity indicating the existence of stable populations of Saccharomyces cerevisiae strains. A drastic reduction in the number of different S. cerevisiae strains was observed in vineyards with cellars using a selected autochthonous S. cerevisiae strain for winemaking. Contrary, the use of allochthonous commercial strains in wineries did not seem to affect the native S. cerevisiae strain composition and diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: The aim of this study was to compare different viticulture and oenological practices to determine their influence on the composition and diversity of Saccharomyces cerevisiae strains in wine fermentations. The study shows that the use of autochthonous strains of S. cerevisiae as starters for wine fermentation could have an important incidence on S. cerevisiae strains diversity in surrounding vineyards. The use of autochthonous strains of S. cerevisiae reduced the detected number of S. cerevisiae strains, a fact that was not observed when allochthonous commercial strains were used. Furthermore, vineyards managed with organic practices showed intermediate to low levels of S. cerevisiae strain diversity, whereas conventional practices showed higher levels.

Precursors consumption preferences and thiol release capacity of the wine yeasts Saccharomyces cerevisiae, Torulaspora delbrueckii, and Lachancea thermotolerans.

The aromatic profile of wine determines its overall final quality, and among the volatile molecules that define it, varietal thiols are responsible for shaping the distinctive character of certain wine varieties. In grape must, these thiols are conjugated to amino acids or small peptides in a non-volatile form. During wine fermentation, yeasts play a principal role in expressing these aromatic compounds as they internalise and cleavage these precursors, releasing the corresponding free and aroma-impacting fraction. Here, we investigate the impact of three wine yeasts (Saccharomyces cerevisiae, Torulaspora delbrueckii and Lachancea thermotolerans) on thiol releasing in synthetic grape must fermentations supplemented with different cysteinylated (Cys-4MSP and Cys-3SH) and glutathionylated (GSH-4MSP and GSH-3SH) precursors. We demonstrate higher consumption levels of cysteinylated precursors, and consequently, higher amounts of thiols are released from them compared to glutathionylated ones. We also report a significant impact of yeast inoculated on the final thiols released. Meanwhile T. delkbrueckii exhibits a great 3SHA releasing capacity, L. thermotolerans stands out because of its high 3SH release. We also highlight the synergic effect of the co-inoculation strategy, especially relevant in the case of S. cerevisiae and L. thermotolerans mixed fermentation, that has an outstanding release of 4MSP thiol. Although our results stem from a specific experimental approach that differs from real winemaking situations, these findings reveal the potential of unravelling the specific role of different yeast species, thiol precursors and their interaction, to improve wine production processes in the context of wine aroma enhancement.

Phenotypic and transcriptional study of the antimicrobial activity of silver and zinc oxide nanoparticles on a wastewater biofilm-forming Pseudomonas aeruginosa strain.

The extensive use of nanoparticles (NPs) in industrial processes makes their potential release into the environment an issue of concern. Ag and ZnO NPs are among the most frequently used NPs, potentially reaching concentrations of 1-4 and 64 mg/kg, respectively, in Wastewater Treatment Plants (WWTPs), with unknown effects over microbial populations. Thus, we examined, in depth, the effect of such NPs on a P. aeruginosa strain isolated from a WWTP. We evaluated the growth, ROS production and biofilm formation, in addition to the transcriptomic response in presence of Ag and ZnO NPs at concentrations potentially found in sewage sludge. The transcriptomic and phenotypic patterns of P. aeruginosa in presence of Ag NPs were, in general, similar to the control treatment, with some specific transcriptional impacts affecting processes involved in biofilm formation and iron homeostasis. The biofilms formed under Ag NPs treatment were, on average, thinner and more homogeneous. ZnO NPs also alters the biofilm formation and iron homeostasis in P. aeruginosa, however, the higher and more toxic concentrations utilized caused an increase in cell death and eDNA release. Thus, the biofilm development was characterized by EPS production, via eDNA release. The number of differentially expressed genes in presence of ZnO NPs was higher compared to Ag NPs treatment. Even though the responses of P. aeruginosa to the presence of the studied metallic NPs was at some extent similar, the higher and more toxic concentrations of ZnO NPs produced greater changes concerning cell viability and ROS production, causing disruption in biofilm development.

The transcriptional response of Saccharomyces cerevisiae to proapoptotic concentrations of Pichia membranifaciens killer toxin.

PMKT (Pichia membranifaciens killer toxin) reportedly has antimicrobial activity against yeasts and filamentous fungi. In previous research we posited that high PMKT concentrations pose a serious challenge for cell survival by disrupting plasma membrane electrochemical gradients, inducing a transcriptional response similar to that of certain stimuli such as hyperosmotic shock. This response was related to the HOG-pathway with Hog1p phosphorylation and a transitional increase in intracellular glycerol accumulation. Such a response was consistent with the notion that the effect induced by high PMKT concentrations lies in an alteration to the ionic homeostasis of the sensitive cell. By contrast, the evidence presented here shows that low PMKT doses lead to a cell death process in Saccharomyces cerevisiae accompanied by cytological and biochemical indicators of apoptotic programmed cell death, namely, the production of reactive oxygen species, DNA strand breaks, metacaspase activation and cytochrome c release. Furthermore, dying cells progressed from an apoptotic state to a secondary necrotic state, and the rate at which this change occurred was proportional to the intensity of the stimulus. We have explored the global gene expression response of S. cerevisiae during that stimulus. The results obtained from DNA microarrays indicate that genes related with an oxidative stress response were induced in response to proapoptotic concentrations of PMKT, showing that the coordinated transcriptional response is not coincident with that obtained when ionophoric concentrations of PMKT are used. By contrast, cwp2Δ mutants showed no signs of apoptosis, indicating that the initial steps of the killer mechanism coincide when proapoptotic (low) or ionophoric (high) PMKT concentrations are used. Additionally, low dosages of PMKT promoted Hog1p phosphorylation and glycerol accumulation.

Acylase enzymes disrupting quorum sensing alter the transcriptome and phenotype of Pseudomonas aeruginosa, and the composition of bacterial biofilms from wastewater treatment plants.

Biofilms represent an essential way of life and colonization of new environments for microorganisms. This feature is regulated by quorum sensing (QS), a microbial communication system based on autoinducer molecules, such as N-acyl-homoserine lactones (AHLs) in Gram negative bacteria. In artificial ecosystems, like Wastewater Treatment Plants (WWTPs), biofilm attachment in filtration membranes produces biofouling. In this environment, the microbial communities are mostly composed of Gram-negative phyla. Thus, we used two AHLs-degrading enzymes, obtained from Actinoplanes utahensis (namely AuAAC and AuAHLA) to determine the effects of degradation of QS signals in the biofilm formation, among other virulence factors, of a Pseudomonas aeruginosa strain isolated from a WWTP, assessing molecular mechanisms through transcriptomics. Besides, we studied the possible effects on community composition in biofilms from activated sludge samples. Although the studied enzymes only degraded the AHLs involved in one of the four QS systems of P. aeruginosa, these activities produced the deregulation of the complete QS network. In fact, AuAAC -the enzyme with higher catalytic efficiency- deregulated all the four QS systems. However, both enzymes reduced the biofilm formation and pyocyanin and protease production. The transcriptomic response of P. aeruginosa affected QS related genes, moreover, transcriptomic response to AuAAC affected mainly to QS related genes. Regarding community composition of biofilms, as expected, the abundance of Gram-negative phyla was significantly decreased after enzymatic treatment. These results support the potential use of such AHLs-degrading enzymes as a method to reduce biofilm formation in WWTP membranes and ameliorate bacterial virulence.

[Prognostic significance of hemoperitoneum in peritoneal dialysis]. / Significado pronóstico del hemoperitoneo en diálisis peritoneal.

According to previous reviews, hemoperitoneum episodes appear in 6.1-8.4% of the peritoneal dialysis patients, and they are severe in a 20% of them. Due to the absence of severe hemoperitoneum in our peritoneal dialysis program, we retrospectively reviewed hemoperitoneum non-related with abdominal surgery or catheter placing. We analyzed its incidence, etiology, prognostic and clinical outcome, as well as the possible effect of recurrent hemoperitoneum on peritoneal function. A total of 132 patients were treated in our centre during a period of 173 months. Mean age at the beginning of peritoneal dialysis was 59+/-17.1 years, 43.2% were females, and 22.8% of them were menstruating women. Twenty-two patients had at least one hemoperitoneum episode during follow-up, with an incidence of 17%. The mean time interval between the start of peritoneal dialysis and the first hemoperitoneum episode was 0.66+/-0.94 years (range: 0.01-3.20 years). 73% were women. Most cases (59%) were due to menstruation. Remarkably, all the menstruating women presented hemoperitoneum at least once with a high incidence of recurrent episodes. The other hemoperitoneum episodes were mainly of unknown etiology (32% of patients), being this one the main cause in males. We only observed two more cases: a male who presented hemoperitoneum related to dicumarinic overdose and a female who presented hemoperitoneum due to mesenteric ischemia. All the 22 patients had a favourable outcome, except for the woman with mesenteric ischemia, what represented an incidence of 4.5% of severe hemoperitoneum. No significant association was found between episodes of hemoperitoneum and aspirin treatment, dicumarinic treatment or the presence of coagulopathy. There was no association either between recurrent hemoperitoneum and the number of peritonitis episodes, peritoneal function or technique survival. In conclusion, hemoperitoneum is a common and usually benign problem in peritoneal dialysis patients, frequently due to retrograde menstruation, and no deleterious long-term effects were found in patients with recurrent hemoperitoneum.

The antimicrobial properties of different strains of Lactobacillus spp. isolated from kefir.

The characteristics of 58 strains of Lactobacillus spp. isolated from kefir were studied. These strains were tested for adherence to human enterocyte-like Caco-2 cells, resistance to acidic pH and bile acid, antimicrobial activities against enteropathogenic bacteria and inhibition of Salmonella typhimurium attachment to Caco-2 cells. The best probiotic properties were observed in L. acidophilus CYC 10051 and L. kefiranofaciens CYC 10058. L. kefiranofaciens CYC 10058 produced an exopolysaccharide, which revealed that it was closely related to kefiran, a polysaccharide with antitumoral properties. This is the first in vitro study about the antimicrobial characteristics of the Lactobacillus population of kefir.

Yeasts as biological agents to control Botrytis cinerea.

Yeasts, isolated from different sources, were identified and tested for inhibition using YMA-MB plates seeded with Botrytis cinerea strains. A total of 42 yeast strains of 20 different species were tested in vitro for antagonism against 18 pathogenic B. cinerea strains. Pichia membranifaciens, P. anomala and Debaryomyces hansenii displayed the most important inhibitory effect against Botrytis strains. In small-scale trials, post-harvest application of P. membranifaciens CYC 1106 to apple wounds inhibited B. cinerea CYC 20010. Purified killer toxin from P. membronifaciens CYC 1106 inhibited B. cinerea CYC 20010. Results indicated that certain yeasts, or their toxins such us P. membranifaciens CYC 1106 killer toxin, might have potential as novel agents to control B. cinerea.

Production and characteristics of Debaryomyces hansenii killer toxin.

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 degrees C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.

Nursing protocol for manipulation of haemodialysis catheters.

This study looked at the incidence of infection complications, in relation to central vein catheterisation as a provisional HD access, by means of the establishment of a nursing protocol for the handling of these catheters. Central vein catheterisation is a classical technique in Nephrology.

PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis.

Pichia membranifaciens CYC 1086 secretes a killer toxin (PMKT2) that is inhibitory to a variety of spoilage yeasts and fungi of agronomical interest. The killer toxin in the culture supernatant was concentrated by ultrafiltration and purified to homogeneity by two successive steps, including native electrophoresis and HPLC gel filtration. Biochemical characterization of the toxin showed it to be a protein with an apparent molecular mass of 30 kDa and an isoelectric point of 3.7. At pH 4.5, optimal killer activity was observed at temperatures up to 20 degrees C. Above approximately this pH, activity decreased sharply and was barely noticeable at pH 6. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a variety of fungal and yeast strains. The results obtained suggest that PMKT2 has different physico-chemical properties from PMKT as well as different potential uses in the biocontrol of spoilage yeasts. PMKT2 was able to inhibit Brettanomyces bruxellensis while Saccharomyces cerevisiae was fully resistant, indicating that PMKT2 could be used in wine fermentations to avoid the development of the spoilage yeast without deleterious effects on the fermentative strain. In small-scale fermentations, PMKT2, as well as P. membranifaciens CYC 1086, was able to inhibit B. bruxellensis, verifying the biocontrol activity of PMKT2 in simulated winemaking conditions.

Ion channel activity by Pichia membranifaciens killer toxin.

The cytocidal effect of Pichia membranifaciens killer toxin on Candida boidinii cells was studied. The halotolerant yeast P. membranifaciens CYC 1106 produces a unique 18 kDa killer toxin that exerts its killer activity against C. boidinii IGC 3430 only in the presence of NaCl. Metabolic events associated with the loss of C. boidinii IGC 3430 viability were quantitatively identical to those known to occur with K1 killer toxin-treated sensitive strains of Saccharomyces cerevisiae. The death of sensitive cells was characterized by a leakage of potassium, an influx of sodium and a decrease in intracellular pH. These effects occurred prior to and concomitantly with cell death, indicating that they were primary effects of the action of the toxin. Here we report that this protein forms ion-permeable channels in liposome membranes. These channels are freely permeable to common physiological ions. We suggest that channel formation is the cytotoxic mechanism of action of P. membranifaciens killer toxin. The channels described here are sufficiently non-selective to mediate cell death through a discharge of cellular membrane potential and changes in ionic homeostasis. No specific effects against killer toxin-treated sensitive cells were observed when the cell cycle was analysed.

Killer toxin of Pichia membranifaciens and its possible use as a biocontrol agent against grey mould disease of grapevine.

The use of Pichia membranifaciens CYC 1106 killer toxin against Botrytis cinerea was investigated. This strain exerted a broad-specificity killing action against other yeasts and fungi. At pH 4, optimal killer activity was observed at temperatures up to 20 degrees C. At 25 degrees C the toxic effect was reduced to 70%. The killer activity was higher in acidic medium. Above about pH 4.5 activity decreased sharply and was barely noticeable at pH 6. The killer toxin protein from P. membranifaciens CYC 1106 was purified to electrophoretic homogeneity. SDS-PAGE of the purified killer protein indicated an apparent molecular mass of 18 kDa. Killer toxin production was stimulated in the presence of non-ionic detergents. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a strain of B. cinerea. The symptoms of infection and grey mould observed in Vitis vinifera plants treated with B. cinerea were prevented in the presence of purified P. membranifaciens killer toxin. The results obtained suggest that P. membranifaciens CYC 1106 killer toxin is of potential use in the biocontrol of B. cinerea.

Biology of killer yeasts.

Killer yeasts secrete proteinaceous killer toxins lethal to susceptible yeast strains. These toxins have no activity against microorganisms other than yeasts, and the killer strains are insensitive to their own toxins. Killer toxins differ between species or strains, showing diverse characteristics in terms of structural genes, molecular size, mature structure and immunity. The mechanisms of recognizing and killing sensitive cells differ for each toxin. Killer yeasts and their toxins have many potential applications in environmental, medical and industrial biotechnology. They are also suitable to study the mechanisms of protein processing and secretion, and toxin interaction with sensitive cells. This review focuses on the biological diversity of the killer toxins described up to now and their potential biotechnological applications.

(1-->6)-Beta-D-glucan as the cell wall binding site for Debaryomyces hansenii killer toxin.

AIMS: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin. METHODS AND RESULTS: Various linear (1-->6)-beta-D-glucans of different origins were effective competitive inhibitors of the toxin action. Periodate oxidation and 1H-NMR was used to determine the receptor nature. Affinity chromatography on pustulan-Sepharose column was used to purify D. hansenii killer toxin, probably a 23-kDa protein. The killer toxin character was cureless. CONCLUSIONS: The investigation revealed that the killer toxin was mainly adsorbed by (1-->6)-beta-D-glucans. This is a low molecular weight protein, probably encoded by chromosomal genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The specificity of the killer toxin for its receptor provides an effective means to purify the killer toxin. This study is the first to identify the cell wall binding site of this killer toxin, a toxin with properties of industrial relevance.

Effect of salt on the killer phenotype of yeasts from olive brines.

The killer properties of yeasts isolated from olive brines were examined in the absence and presence of sodium chloride in concentrations of up to 6% (wt/vol). An apparent enhancement of the killing action as the salt concentration increased, as well as changes in the spectra of activity against selected target strains, was observed in a few strains. Culture filtrates from killer strains grown at different NaCl concentrations (0, 3, or 6% [wt/vol]) were tested against sensitive yeasts cultivated under the same conditions. While the sensitivity of the target strain greatly increased in the presence of salt, no significant effect on toxin production was noticed.

(1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin.

The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430). The (1-->6)-beta-D-glucan was purified from cell walls of C. boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor. Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min. Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin.

A mathematical model for toxin accumulation by killer yeasts based on the yeast population growth.

The accumulation of toxin by killer yeast populations is modelled starting from a mechanistic approach that explains the toxin production in terms of yeast population growth, and takes into account the environmental inactivation of the toxin. A modified Richard's general equation for limited growth is used to define the function that describes the toxin produced in relation to the yeast biomass increase. The relationship between the rates of cell and toxin production is explicitly shown, and the implications of the resulting proportionality factor are discussed. The model parameters have been adjusted and the model has been validated using experimental data of growth and toxin accumulation from cultures of Pichia membranaefaciens in two different media. The differences between both types of cultures are analysed on the basis of parameter estimates and the predicted rate of toxin production per cell. The results support the hypothesis that biomass production and toxin synthesis are controlled in different ways; they also suggest that the composition of the medium could have a distinct effect on toxin synthesis. Model assumptions are discussed in comparison with a previous model for killer-sensitive interaction of Saccharomyces cerevisiae strains.

Valor de la TAC en la determinación del tratamiento en pacientes con cáncer de seno maxilar y su repercución en la sobrevida / Value of the TAC in the determination of the treatment in patient with neoplasm of breast maxilar and its repercution in the survival

Objetivos: Evaluar el control local y la sobrevida en pacientes tratados con radioterapia post operatoria. Evaluar el valor de la TAC en la selección de pacientes para tratamiento quirúrgico, evaluar el comportamiento de este tipo de neoplasia en este grupo de pacientes. Resultados: Para este estudio se revisaron 213 historias clínicas de pacientes con cáncer de seno maxilar entre los años 1988 - 1998, en el INEN, de los cuales se registraron 46 casos de seno maxilar tratados con radioterapia post operatoria, lo que corresponde al 21.5 por ciento del total de pacientes con cáncer de seno maxilar, diagnosticados en esta Institución. El control local permanente se obtuvo en el 67.4 por ciento. Compromiso ganglionar cervical al diagnóstico fue de 6.5 por ciento, metástasis a distancia de 13 por ciento. La recurrencia local se registró en el 32.6 por ciento, y la recurrencia cervical en 15.2 por ciento. La sobrevida en este grupo de pacientes fue de 41.3 por ciento a 5 años y la sobrevida específica de 47.8 por ciento. Conclusiones: De acuerdo a los resultados obtenidos en esta serie y comparando con los resultados de estudios realizados anteriormente en la Institución, se observa una mejora significativa en la sobrevida total y sobrevida específica, principalmente por el uso de la TAC para definir criterios de operabilidad, determinar extensión de la enfermedad, permitiendo una mejor selección de pacientes para tratamiento quirúrgico.