Estudio comparativo entre aislados clínicos y no clínicos de S. cerevisiae y su papel como patógeno emergente.

RESUMEN La levadura Saccharomyces cerevisiae es la especie más utilizada desde un punto de vista biotecnológico. En la industria agroalimentaria interviene en la elaboración de pan y bebidas alcohólicas como el vino y la cerveza, además se emplea como suplemento dietético y como agente probiótico bajo el nombre de S. cerevisiae var. boulardii. A pesar de sus propiedades beneficiosas, S. cerevisiae se considera actualmente una levadura patógena oportunista emergente de baja virulencia, capaz de causar infecciones principalmente en hospedadores inmunodeprimidos. Con el fin de conocer el posible papel de esta levadura como patógeno emergente, el presente trabajo aborda un estudio comparativo entre aislados clínicos y no clínicos de S. cerevisiae desde diferentes puntos de vista. En primer lugar, la caracterización mediante técnicas moleculares de un gran número de aislados clínicos y su comparación con aislados no clínicos, ha sido de gran ayuda para esclarecer el origen de colonización en infecciones por S. cerevisiae. Dicha caracterización junto con un estudio filogenético de secuencias del gen COX2, evidenciaron dos posibles casos de colonización exógena por parte de cepas de panadería y la cepa probiótica S. cerevisiae var. boulardii. Por otro lado, los estudios de rasgos fenotípicos asociados con virulencia de esta levadura, mostraron que la capacidad de crecimiento a 42º C, el crecimiento pseudohifal y niveles altos en la actividad fosfolipasa, son rasgos de virulencia que marcan la diferencia entre los aislados clínicos y las cepas industriales. Dicho estudio junto con los ensayos en sistemas murino de infección sistémica, han permitido establecer el potencial patógeno de algunas cepas de S. cerevisiae. Finalmente se han observado diferentes grados de activación de las MAP quinasas Slt2 y Kss1 entre los aislados clínicos y no clínicos incluidos en este estudio, además de detectarse la presencia de dos proteínas Slt2 para un grupo de aislados clínicos. El análisis de la secuencia del gen SLT2 ha revelado que estos aislados tienen más de dos alelos que se diferencian en el tamaño, como consecuencia de la variación en un gran número de repeticiones de los trinucleótidos CAA/que codifican glutaminas. Estos estudios pueden ser de gran interés para las industrias de alimentos que incluyen a S. cerevisiae en sus preparados, así como en el ámbito hospitalario, advirtiendo del peligro que conlleva la administración de productos que contengan dicha levadura, a individuos inmunodeprimidos. __________________________________________________________________________________________________Saccharomyces cerevisiae has traditionally been used in industrial fermentative processes, beer and wine production, baking, and as a nutritional supplement or even as a probiotic. This is the case of the probiotic strain of this species, S. cerevisiae var. boulardii which has been widely used in Europe to treat several types of diarrhoea. Although S. cerevisiae and S. boulardii have commonly been considered as safe microorganism, this concept has changed due to an increased number of human infections. Consequently, S. cerevisiae is now considered an emerging opportunistic pathogen that can cause clinically relevant infections, mainly associated with immunocompromised patients. With the aim of know the possible role of this yeast as an opportunistic pathogen, the present work tackles a comparative study between clinical and no clinical isolates of S. cerevisiae from different point of view. This study could be of great interest for all food industry that include S. cerevisiae inside their preparation, just as in the hospital environment, warning of the risk that entail the administration of products which contain the above mentioned yeast to immunosuppressed person

Microbiological Characterization of the Biofilms Colonizing Bioplastics in Natural Marine Conditions: A Comparison between PHBV and PLA

Biodegradable polymers offer a potential solution to marine pollution caused by plastic waste. The marine biofilms that formed on the surfaces of poly(lactide acid) (PLA) and poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were studied. Bioplastics were exposed for 6 months to marine conditions in the Mediterranean Sea, and the biofilms that formed on their surfaces were assessed. The presence of specific PLA and PHBV degraders was also studied. PHBV showed extensive areas with microbial accumulations and this led to higher microbial surface densities than PLA (4.75 vs. 5.16 log CFU/cm2 ). Both polymers’ surfaces showed a wide variety of microbial structures, including bacteria, fungi, unicellular algae and choanoflagellates. A high bacterial diversity was observed, with differences between the two polymers, particularly at the phylum level, with over 70% of bacteria affiliated to three phyla. Differences in metagenome functions were also detected, revealing a higher presence of proteins involved in PHBV biodegradation in PHBV biofilms. Four bacterial isolates belonging to the Proteobacteria class were identified as PHBV degraders, demonstrating the presence of species involved in the biodegradation of this polymer in seawater. No PLA degraders were detected, confirming its low biodegradability in marine environments. This was a pilot study to establish a baseline for further studies aimed at comprehending the marine biodegradation of biopolymers

Microbial quality and yeast population dynamics in cracked green table olives' fermentations

Cracked green table olives, from the Manzanilla variety, are a fermented food produced and consumed in Portugal. The objective of the present work was to study the microbiological characteristics and yeast population evolution during the fermentation of cracked green olives. The predominant microorganisms were yeasts while lactic acid bacteria were not detected and a clear decrease of the mould population was observed. At the end of the fermentations, no viable counts of Enterobacteriaceae were found. Yeast isolates were identified by the 5.8S rRNA-ITS region restriction analysis and by sequencing the D1/D2 region of the 26S rRNA gene. During the initial phases of the fermentations a great diversity of yeasts was observed. However, as the processes evolved the biodiversity decreased with the fermentative yeasts Citeromyces matritensis, Zygotorulaspora mrakii and Saccharomyces cerevisiae becoming the dominant species. The presence of these fermentative yeasts at the end of the production process is associated to a risk of spoilage. The results obtained represent a first attempt towards the comprehension of the microbiota of this type of “Natural olives” that constitute an important component of the Mediterranean diet

Specific microbiota direct the differentiation of Th17 cells in the mucosa of the small intestine

IL-17-producing T-helper cells (Th17) are potent effectors of inflammation, but little is known about the requirements for their differentiation in vivo at steady state. We found that specific commensal microbiota are required for Th17 cell differentiation in the lamina propria (LP) of the small intestine. Differentiation of Th17 cells correlated with presence of Cytophaga-Flavobacter-Bacteroidetes bacteria in the intestine, was independent of TLR, IL-21 or IL-23 signaling, but required appropriate activation of TGF-β. Absence of Th17 cell-inducing bacteria was accompanied by increased Foxp3+ regulatory T cells in the LP. Our results suggest that the composition of the microbiota regulates the Th17:Treg balance in the lamina propria and may thus influence intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases

High-Sensitivity Troponin T and Soluble Form of AXL as Long-Term Prognostic Biomarkers after Heart Transplantation

Cardiac allograft vasculopathy (CAV) is a frequent complication limiting the long-term (>1 year) survival after heart transplantation (HTx). CAV is initiated by endothelial dysfunction and can lead to severe cardiovascular (CV) complications. Since CAV is often clinically silent, biomarkers could help identifying HTx patients at risk of CAV and their severe complications. Evaluate the clinical yield of high-sensitivity cardiac troponin T (hs-cTnT), marker of cardiomyocyte damage, and the soluble form of AXL (sAXL), biomarker of endothelial dysfunction, to assess the prognosis of long-term cardiovascular (CV) events occurring after HTx. 96 patients were evaluated at least > 1 year after HTx. CAV was evaluated by coronary angiography or multisliced tomography, and hs-cTnT and sAXL measured 6 months before or after CAV evaluation. Patients were followed during 42 ± 15 months for a combined end point including cardiac death, angina or acute myocardial infarction, left ventricular ejection fraction 21 ng/L (p<0.001). Hs-cTnT, but not sAXL, measured during the long-term follow-up of HTx patients appears as a helpful biomarker to identify patients at low risk of adverse CV outcomes

Mechanical behaviour of straight and swept tip rotor blades

Consulta en la Biblioteca ETSI Industriales (8906)[EN] The present work has as finality check a mathematical model developed to analyze rotor blades mechanical behavior. In this thesis we have made static and eigenvalue analysis. For it, the mathematical model has been implemented in the computer program Comsol Multiphysics which use The Finite Elements Method to solve problems. To validate the mathematical model we have made calculations in different situations. The main idea is to take tests made in rotor blades and solve it with our mathematical model. Then we have made a comparison between results.Llanos Frutos, CD. (2012). Mechanical behaviour of straight and swept tip rotor blades. http://hdl.handle.net/10251/29235.Archivo delegad

Photodynamic inactivation of Staphylococcus aureus in the presence of aggregation-prone photosensitizers based on BODIPY used at submicromolar concentrations

Two new brominated BODIPYs (1 and 2) bearing amino acid-based chains (l-valine for 1, and dimethyl-l-lysine for 2) were synthesized and characterized. In organic solvents, 1 and 2 were fully soluble and showed the photophysical properties expected for brominated BODIPY dyes, including efficient generation of singlet oxygen (1O2), upon irradiation. In contrast, in aqueous media, both compounds were prone to aggregation and the photo-induced generation of 1O2 was halted. Despite the lack of generation of this reactive species in aqueous media (in cuvette), both 1 and 2 have positive antimicrobial Photodynamic Inactivation (aPDI) effect. The activity against gram-positive Staphylococcus aureus and gram-negative Escherichia coli was determined through the inactivation curves, with a total energy dose of 5.3 J/cm2 (white light LED used as an energy source). Compound 2 was highly active against both gram-positive and gram-negative bacteria (3 log CFU/mL reduction was obtained at 0.16 μM for S. aureus and 2.5–5.0 μM for E. coli), whereas 1 was less effective to kill S. aureus (3 log CFU/mL at 0.32 μM) and ineffective for E. coli. The higher efficiency of 2, as compared to 1, to reduce the population of bacteria, can reside in the presence of a protonatable residue in 2, allowing a more effective interaction of this molecule with the cell walls of the microorganisms. In order to explain the lack of reactivity in pure aqueous media (in cuvette) and the contrasting good activity in the presence of bacterial cells it can be hypothesized that upon interaction with the walls of the microorganisms, the aggregated photosensitizers suffer a disaggregation process restoring the ability to generate 1O2, and hence leading to efficient photodynamic activity against these pathogenic microorganisms, in agreement with the similar effect observed recently for porphyrinoid photosensitizers.Funding for open access charge: CRUE-Universitat Jaume

Development of a Polymeric Film Entrapping Rose Bengal and Iodide Anion for the Light-Induced Generation and Release of Bactericidal Hydrogen Peroxide

A series of poly(2-hydroxyethyl methacrylate) (PHEMA) thin films entrapping photosensitizer Rose Bengal (RB) and tetrabutylammonium iodide (TBAI) have been synthetized. The materials have been characterized by means of Thermogravimetric Analysis (TGA), Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) and UV-vis Absorption spectroscopy. Irradiation of the materials with white light led to the generation of several bactericidal species, including singlet oxygen (1O2 ), triiodide anion (I3 −) and hydrogen peroxide (H2O2 ). 1O2 production was demonstrated spectroscopically by reaction with the chemical trap 2,20 -(anthracene9,10-diylbis(methylene))dimalonic acid (ABDA). In addition, the reaction of iodide anion with 1O2 yielded I3 − inside the polymeric matrix. This reaction is accompanied by the formation of H2O2 , which diffuses out the polymeric matrix. Generation of both I3 − and H2O2 was demonstrated spectroscopically (directly in the case of triiodide by the absorption at 360 nm and indirectly for H2O2 using the xylenol orange test). A series of photodynamic inactivation assays were conducted with the synthesized polymers against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Complete eradication (7 log10 CFU/mL) of both bacteria occurred after only 5 min of white light irradiation (400–700 nm; total energy dose 24 J/cm2 ) of the polymer containing both RB and TBAI. The control polymer without embedded iodide (only RB) showed only marginal reductions of ca. 0.5 log10 CFU/mL. The main novelty of the present investigation is the generation of three bactericidal species (1O2 , I3 − and H2O2 ) at the same time using a single polymeric material containing all the elements needed to produce such a bactericidal cocktail, although the most relevant antimicrobial activity is shown by H2O2 . This experimental approach avoids multistep protocols involving a final step of addition of I−, as described previously for other assays in solution

Predictive value of minimal fungicidal concentration (MFC) of amphotericin B (AMB) in deep-seated infections caused by Candida krusei

Póster presentado en 43rd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy September 14 - 17, 2003; Chicago, IllinoisBackground: It has been reported that AMB MFCs have a wider range and could be better predictors of clinical outcome than MICs in the setting of non-Candida albicans infections. Methods: A 62-year old male patient with acute myeloblastic leukemia, receiving fluconazole prophylaxis, during remission induction chemotherapy developed C. krusei fungemia (CK-18). antifungal therapy consisted on liposomal AMB (3 mg/kg/day) for two weeks followed by standard-dose caspofungin for 4 weeks and then he received, after discharge, itraconazole 200 mg bid for 4 weeks. Four months later, he developed a septic spondilodiscitis and C. krusei (CK-19) was isolated at infection site by fine-needle aspiration. Because of high AMB MFCs, on this occasion a combination of caspofungin and voriconazole, at standard doses, was given for six weeks with favorable clinical response. MICs (100% growth inhibition) were determinated by NCCLS M27-A2, colorimetric, & Etest methods). For MFC, the content of each clear MIC well (0.2 ml) was subcultured onto two Sabouraud dextrose agar plates. Strain characterization was performed by PCR amplification and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene and the sequencing of the D1/D2 regions of the 26S rRNA gene. Results: PCR proved that both strains were C. krusei and the mtDNA restriction analysis and specific PCR of d sequences that they were different strains. AMB MICs (mg/l) for CK-18/CK-19 by M27-A2, Etest and colorimetric methods were: 1, 0.5, 1 / 1, 0.75, 1, respectively and AMB MFCs (mg/l): >16-16. Conclusions: AMB MFCs values for both C. krusei were substantially higher than MICs and could be better predictors of therapeutic failure with AMB and in vitro resistance