Testes bioquímicos para identificação de leveduras na medicina veterinária - revisão de literatura / Biochemical tests to yeast identification in veterinary medicine - literature review / Pruebas bioquímicas para identificación de levaduras en la medicina veterinaria - revisión de literatura

As leveduras são fungos de importância à medicina veterinária por causarem doenças infecciosas em diferentes hospedeiros animais. A presente revisão de literatura teve como objetivo relatar os principais testes bioquímicos capazes de auxiliar na identificação de fungos leveduriformes de interesse veterinário e zoonótico. Para o levantamento bibliográfico, foram consideradas 48 publicações científicas selecionadas na área e indexadas nas principais bases de dados, entre os anos de 1988 e 2020. Como resultados, observou-se que oito provas são as mais empregadas na rotina micológica. Devido à baixa variabilidade morfológica das espécies leveduriformes, testes bioquímicos complementares são fundamentais na rotina laboratorial. A análise do perfil bioquímico de leveduras contribui na determinação taxonômica dos fungos a partir de reações químicas, visto que o metabolismo varia de acordo com a espécie, resultando em metabólitos distintos, os quais podem ser avaliados por diferentes provas. Conclui-se que a identificação fenotípica das leveduras é imprescindível no diagnóstico, prognóstico, tratamento e controle de doenças fúngicas e contribui para a manutenção da saúde animal.(AU)

Yeasts are fungi of importance to veterinary medicine because they cause infectious diseases in different animal hosts. This literature review aimed to report the main biochemical tests capable of assisting in the identification of yeast-like fungi of veterinary and zoonotic interest. For the bibliographical survey, 48 selected scientific publications in the area and indexed in the main databases, between the years 1988 and 2020, were considered. As a result, it was observed that eight tests are the most used in the mycological routine. Due to the low morphological variability of yeast species, complementary biochemical tests are fundamental in the laboratory routine. The analysis of the biochemical profile of yeast contributes to the taxonomic determination of fungi based on chemical reactions, since the metabolism varies according to the species, resulting in different metabolites, which can be evaluated by different tests. It is concluded that the phenotypic identification of yeasts is essential in the diagnosis, prognosis, treatment and control of fungal diseases and contributes to the maintenance of animal health.(AU)

Las levaduras son hongos de importancia para la medicina veterinaria porque causan enfermedades infecciosas en diferentes animales huéspedes. Esta revisión de la literatura tuvo como objetivo informar las principales pruebas bioquímicas capaces de ayudar en la identificación de hongos tipo levadura de interés veterinario y zoonótico. Para el levantamiento bibliográfico se consideraron 48 publicaciones científicas seleccionadas en el área e indexadas en las principales bases de datos, entre los años 1988 y 2020. Como resultado se observó que ocho pruebas son las más utilizadas en la rutina micológica. Debido a la baja variabilidad morfológica de las especies de levaduras, las pruebas bioquímicas complementarias son fundamentales en la rutina del laboratorio. El análisis del perfil bioquímico de la levadura contribuye a la determinación taxonómica de los hongos en base a reacciones químicas, ya que el metabolismo varía según la especie, dando como resultado diferentes metabolitos, los cuales pueden ser evaluados mediante diferentes pruebas. Se concluye que la identificación fenotípica de levaduras es fundamental en el diagnóstico, pronóstico, tratamiento y control de enfermedades fúngicas y contribuye al mantenimiento de la salud animal.(AU)

Optimization of the ellagic acid synthesis process at the bioreactor level using non-conventional yeasts

Abstract Ellagic acid (EA) is a phenolic biomolecule. For its biosynthesis, a source of ellagitannins is required, such as strawberries and yeasts, as precursors of the tannase and ß-glucosidase enzymes responsible for hydrolysis of ellagitannins. Two experimental mixture designs were applied., varying the yeast concentration and the number of ellagitannins in the culture medium, evaluating the enzymatic activity and ellagic acid biosynthesis. Aiming to find the optimal compositions of the non-conventional yeasts assessed in the research to biosynthesize ellagic acid feasibly and efficiently using a response surface performing the statistical analysis in the StatGraphics® program for obtaining a higher yield and optimizing the ellagic acid synthesis process, the results indicate that the strains Candida parapsilosis ITM LB33 and Debaryomyces hansenii ISA 1510 have a positive effect on the synthesis of ellagic acid, since as its concentration increases in the mixture the concentration of ellagic acid in the medium also increases; on the other hand, the addition of Candida utilis ITM LB02 causes a negative effect, resulting in the compositions of 0.516876, 0.483124 and 2.58687E-9 respectively, for a treatment under the same conditions, an optimal value of ellagic acid production would be obtained. With an approximate value of 7.33036 mg/mL

Pharmaceutical co-crystals of posaconazole for improvement of physicochemical properties

Abstract Posaconazole exerts an extended spectrum of antifungal activity against various strains of clinically relevant moulds and yeasts. In recent years, antifungal triazole posaconazole has become increasingly important for the prophylaxis and treatment of systemic mycoses. After oral administration of posaconazole, absolute bioavailability has been estimated to range from 8% to 47%. Pharmaceutical co-crystallization is a promising approach for improving dissolution rate or manipulating other physical properties of API. The objective of this study is to improve the dissolution rate of posaconazole by co-crystallization. A 1:1 stoichiometric co-crystals of adipic acid were prepared by solvent assisted grinding method. The prepared co-crystals were subjected to solid-state characterization by FTIR, PXRD and DSC studies. The physicochemical properties of posaconazole and co-crystals were assessed in terms of melting point, flowability and dissolution rate. The results indicated improvement in flow property and dissolution rate. In vitro dissolution profile of co-crystals showed a significant increased dissolution of posaconazole from initial period in 0.1 N hydrochloric acid solution. The dissolution efficiency for posaconazole-adipic acid co-crystal was 61.65 % against posaconazole, 46.58 %. Thus, co-crystallization can be a promising approach to prepare posaconazole-adipic acid co-crystals with improved physicochemical properties.

Avaliação da interação entre leveduras isoladas de kombucha e bactérias probióticas / Evaluation of the interaction between isolated kombucha yeasts and probiotic bacteria

Um dos maiores desafios no desenvolvimento de produtos probióticos é entender como os microrganismos interagem entre si e com o hospedeiro. Quando falamos em alimentos fermentados tradicionais, este obstáculo aumenta porque a matriz alimentar já possui um microbioma intrínseco. No entanto, também é conhecido que muitos microrganismos podem interagir e cooperar para sobreviver quando condições de estresse são encontradas. Assim, o objetivo deste trabalho foi isolar leveduras de quatro diferentes kombuchas em distintos momentos fermentativos e verificar a influência que leveduras isoladas de kombucha têm na manutenção da viabilidade da bactéria probiótica Bifidobacterium animalis subsp. lactis HN019 em condições de aerobiose. Meyerozyma guilliermondii, Candida albicans, Rhodotorula mucilaginosa e Pichia membranifaciens foram leveduras encontradas nas kombuchas, das quais as duas últimas favoreceram a manutenção da alta viabilidade de HN019 em cocultura por 14 dias. Observou-se a viabilidade da bactéria acima de 9 log ao longo de todo o experimento, o que não foi observado em monocultura. Ademais, utilizou-se de análise de autoagregação, hidrofobicidade, atividade enzimática de proteases e fosfolipases das leveuras para analisar seu potencial patogênico. Observou-se que R. mucilaginosa demonstrou características semelhantes à Saccharomyces cerevisiae subsp. boulardii, e sua interação benéfica com HN019 reforça a possibilidade de que esta levedura seja uma chave para a inserção da bactéria em uma kombucha probiótica. Análises metabólicas foram realizadas e encontrou-se uma vasta diversidade de dipeptídeos, principalmente os compostos de prolina, durante a cocultura da bactéria com as leveduras. Tais dipeptídeos apresentam importantes mecanismos de ação no controle biológico e quorum sensing de bactérias e leveduras, e supostamente regulam a manutenção das relações mutualísticas entre ambos microrganismo

One of the biggest challenges in the development of probiotic products is to understand how microorganisms interact with each other and with the host. When we talk about traditional fermented foods, this obstacle increases because the food matrix already has an intrinsic microbiome. However, it is also known that many microorganisms can interact and cooperate to survive when stressful situations are encountered. Thus, the objective of this work was to isolate yeasts from four different kombuchas at different fermentation times and to verify the influence that yeasts isolated from kombucha have on maintaining the viability of the probiotic bacterium Bifidobacterium animalis subsp. lactis HN019 under aerobic conditions. Meyerozyma guilliermondii, Candida albicans, Rhodotorula mucilaginosa and Pichia membranifaciens were yeasts found in kombuchas, of which the last two favored the maintenance of HN019 high viability in co-culture for 14 days. Bacteria viability above 9 log was observed throughout the experiment, which was not observed in monoculture. In addition, analysis of autoaggregation, hydrophobicity, enzyme activity of proteases and phospholipases of yeasts was used to analyze their pathogenic potential. It was observed that R. mucilaginosa demonstrated characteristics similar to Saccharomyces cerevisiae subsp. boulardii, and its beneficial interaction with HN019 reinforces the possibility that this yeast is a key to the insertion of the bacterium in a probiotic kombucha. Metabolic analysis were performed and a wide diversity of dipeptides, mainly proline-based, was found during the co-culture of the bacteria with the yeasts. Such dipeptides have important mechanisms of action in the biological control and quorum sensing of bacteria and yeast, and supposedly regulate the maintenance of mutualistic relationships between both microorganism

Yeast diversity in Vitis non-vinifera ecosystems / Diversidad de levaduras en ecosistemas de Vitis no-vinifera

The surface of grapes lodges a complex community of yeast species responsible for spontaneous alcoholic fermentation. The study of indigenous Saccharomyces and "non-Saccharomyces" yeasts during grape must fermentation constitutes a major research area in microbial enology. Although there are detailed studies on the microbiota of Vitis vinifera grapes, little is known about the diversity of yeast communities present in non-vinifera Vitis ecosystems (i.e., grapes and spontaneously fermenting grape musts). Potentially scientific and/or enological valuable yeast strains from these non-vinifera Vitis ecosystems might never be isolated from V. vinifera L. In this updated review, we summarize relevant aspects of the microbial studies conducted on V. non-vinifera grapes and spontaneously fermenting grape musts.

La superficie de las uvas aloja una comunidad compleja de especies de levaduras responsables de la fermentación alcohólica espontánea. El estudio de estas levaduras Saccharomyces y «no-Saccharomyces¼ durante la fermentación del mosto de uvas constituye un área relevante de investigación microbiológica en enología. Si bien existen estudios detallados de la microbiota de uvas de Vitis vinifera L., poco se sabe sobre la diversidad de comunidades de levaduras presentes en ecosistemas de Vitis no-vinifera (i.e., uvas y mostos en fermentación espontánea). Cepas de levaduras presentes en ecosistemas de Vitis no-vinífera, con valor potencial científico y/o enológico, podrían no estar presentes en V. vinifera L. En esta revisión actualizada, resumimos los aspectos relevantes de los estudios microbiológicos efectuados en mostos en fermentación espontánea de uvas de V. no-vinifera.

Produção de L-asparaginase pela levedura Leucosporidium muscorum CRM 1648 isolada de sedimento marinho coletado na Península Antártica / L-asparaginase production by the yeast Leucosporidium muscorum CRM 1648 isolated from marine sediments collected in Antarctic Peninsula

L-asparaginase (L-ASNase) é uma enzima com propriedades interessantes para a indústria médica, farmacêutica e de alimentos, que tem recebido atenção especial, inclusive no Brasil, por fazer parte do protocolo de tratamento de distúrbios linfoproliferativos, como a leucemia linfoblástica aguda (LLA). No mercado desde a década de 1970, as enzimas de origem bacteriana enfrentam algumas limitações por provocarem reações adversas graves em quase 80% dos pacientes em tratamento. Nesse contexto, L-ASNases provenientes de leveduras se destacam como alternativa, por serem mais próximas às congêneres humanas. A Antártica ainda é um ambiente pouco explorado, com grande diversidade de microrganismos com potencial para a produção de moléculas biológicas de interesse industrial. Nesse contexto, 150 leveduras isoladas de amostras de sedimento marinho coletadas na Península Antártica como parte do projeto MICROSFERA (PROANTAR/CNPq) foram avaliadas para a produção de L-ASNase. A triagem resultou em 9 isolados produtores, dos quais 7 pertencem ao gênero Leucosporidium. A linhagem L. muscorum CRM 1648 foi a que produziu mais enzima (540 U.L-1), com maior produtividade (5,6 U.L-1.h-1) e, por isso, foi alvo deste estudo. A análise univariada de fontes de carbono e nitrogênio indicou maior crescimento desse microrganismo e produção de L-ASNase em meio CD com extrato de levedura, prolina e sacarose. Ureia, cloreto de amônio e sulfato de amônio resultaram em baixa ou nenhuma produção da enzima, sugerindo que a metabolização de fontes de nitrogênio por essa linhagem está sob a influência do fenômeno de repressão catabólica pelo nitrogênio (RCN). Dois delineamentos experimentais do tipo fatorial completo resultaram em um aumento de 10 vezes na produção e produtividade da enzima (4582,5 U.L-1 e 63,6 U.L-1.h-1, respectivamente). A análise univariada da concentração inicial de inóculo (X0), pH inicial do meio, temperatura e adição de água do mar mostrou que a melhor condição para a produção foi: pH = 5,5 ou 6,5, cultivo a 15°C com adição de água do mar (25-50% m/v). A variável X0 não foi significativa nas concentrações avaliadas. Cultivos em biorreator (batelada) foram conduzidos em quatro diferentes níveis de oxigênio dissolvido (OD): (1) OD não controlado e abaixo de 20%, (2) OD não controlado e acima de 20%, (3) OD controlado em 80% e (4) OD controlado em 20%. Os resultados mostraram que OD é fator limitante para o crescimento de L. muscorum CRM 1648 e produção de L-ASNase por essa levedura e deve ser mantido acima de 35% para maior produção da enzima.Neste trabalho, a composição do meio e condições de cultivo foram estabelecidas para favorecer a produção de uma nova L-ASNase livre de atividade glutaminásica por levedura adaptada ao frio, abrindo espaço para novos estudos acerca de seu potencial antileucêmico e possível uso como alternativa às enzimas já existentes no mercado no tratamento de LLA

L-asparaginase (L-ASNase) is an enzyme with interesting properties for medical, pharmaceutical and food industry, which has received special consideration, especially in Brazil, for being part of lymphoproliferative disorders treatment, such as acute lymphoblastic leukemia (ALL). Bacterial enzymes are on the market since the 1970s and face some limitations related to theirserious adverse reactions that reach almost 80% of all patients in treatment. In this context, L-ASNases from yeasts are highlighted as important alternative to bacterial enzymes, due to the closerphylogeny to human congeners. Antarctic environment has much to be explored, with a vast diversity of microorganisms with potential to produce biomolecules with industrial interest. A total of 150 yeasts isolated from Antarctic marine sediments as part of MICROSFERA project (PROANTAR/CNPq) were evaluated for L-ASNase production. The screening resulted in 9 producers, 7 species from the genus Leucosporidium. L. muscorum CRM 1648 was the strain that yielded the highest L-ASNase activity (540 U.L-1) and volumetric productivity (5.6 U.L-1.h-1). Carbon and Nitrogen sources were evaluated by a method of one-factor at a time (OFAT). From the gather results, sucrose, yeast extract and proline resulted in a maximal growth and highest enzyme production.The absence or low production of L-ASNase in medium with urea, ammonium chloride and ammonium sulfate suggests the presence of nitrogen catabolic repression (NCR). Carbon and nitrogen concentration were evaluated by full factorial design and yielded about ten times higher enzyme and volumetric productivity (4582.5 U.L-1 and 63.6 U.L-1.h-1, respectively). Initial inoculum concentration (X0), initial pH, temperature and concentration of seawater in the culture were evaluated by OFAT analysis and the best condition for L-ASNase production was: pH = 5.5 or 6.5, at 15 °C with addition of seawater (25-50 wt%). X0 was not considered a significant variable. Bioreactor assays (in batch regime) were performed in four different dissolved oxygen (DO) levels: (1) without DO control (DO remained under 20%), (2) without DO control (DO remained above 20%), (3) DO controlled at 80%, and (4) DO controlled at 20%.The results showed that DO is a key factor for growth of L. muscorum CRM 1648 and production of L-ASNase by this yeast and should be maintained above 35% for higher production of this enzyme.At this work, the medium and culture conditions were established to support the production of a novel glutaminase-free L-ASNase by a cold adapted yeast, opening a new path for further studies regarding its antileukemic potential and possible use as an alternative for ALL treatment

Saccharomyces cerevisiae populations and other yeasts associated with indigenous beers (chicha) of Ecuador

ABSTRACT Chicha, a type of beer made mainly with maize or cassava, is a traditional fermented beverage of the Andean region. There have only been a few studies on yeasts associated with chicha fermentation, and the species diversity occurring during the production of this beverage is not known. The objective of this study was to determine the biodiversity of yeasts in chicha, and to characterize the Saccharomyces cerevisiae populations associated with the production of chicha de jora, seven-grain chicha, chicha de yuca, and chicha de morocho in Ecuador. The molecular diversity of S. cerevisiae populations was determined by restriction polymorphism mitochondrial profiles. The beverages were characterized based on their physicochemical parameters. Twenty-six species were identified, and the most prevalent species were S. cerevisiae and Torulaspora delbrueckii. Other yeast species were isolated at low frequencies. Among 121 isolates of S. cerevisiae, 68 different mtDNA molecular profiles were identified. These results showed that chichas are fermented by a high number of different strains of S. cerevisiae. Some other species provided a minor contribution to the fermentation process. The chicha presented generally similar physicochemical parameters to those observed for other traditional fermented beverages, and can be considered as an acid fermented beverage.

The isolation of pentose-assimilating yeasts and their xylose fermentation potential

ABSTRACT For the implementation of cellulosic ethanol technology, the maximum use of lignocellulosic materials is important to increase efficiency and to reduce costs. In this context, appropriate use of the pentose released by hemicellulose hydrolysis could improve de economic viability of this process. Since the Saccharomyces cerevisiae is unable to ferment the pentose, the search for pentose-fermenting microorganisms could be an alternative. In this work, the isolation of yeast strains from decaying vegetal materials, flowers, fruits and insects and their application for assimilation and alcoholic fermentation of xylose were carried out. From a total of 30 isolated strains, 12 were able to assimilate 30 g L-1 of xylose in 120 h. The strain Candida tropicalis S4 produced 6 g L-1 of ethanol from 56 g L-1 of xylose, while the strain C. tropicalis E2 produced 22 g L-1 of xylitol. The strains Candida oleophila G10.1 and Metschnikowia koreensis G18 consumed significant amount of xylose in aerobic cultivation releasing non-identified metabolites. The different materials in environment were source for pentose-assimilating yeast with variable metabolic profile.

Isolation and characterization of yeasts from fermented apple bagasse as additives for ruminant feeding

Abstract Solid-state fermentation can be used to produce feeds for ruminants, which can provide an enriched population of yeasts to improve ruminal fermentation. Fermentation of apple bagasse was performed to obtain a yeast-rich product, with the objective of isolating, identifying, and characterizing yeast strains and testing their capability to enhance in vitro ruminal fermentation of fibrous feeds. Yeasts were isolated from apple bagasse fermented under in vitro conditions, using rumen liquor obtained from cannulated cows and alfalfa as a fibrous substrate. A total of 16 new yeast strains were isolated and identified by biochemical and molecular methods. The strains were designated Levazot, followed by the isolate number. Their fermentative capacity was assessed using an in vitro gas production method. Strain Levazot 15 (Candida norvegensis) showed the greatest increase in gas production (p < 0.05) compared with the yeast-free control and positively affected in vitro ruminal fermentation parameters of alfalfa and oat straw. Based on these results, it was concluded that the Levazot 15 yeast strain could be potentially used as an additive for ruminants consuming high-fiber diets. However, further studies of effects of these additives on rumen digestion, metabolism, and productive performance of ruminants are required.

Isolation and characterization of phenol degrading yeasts from wastewater in the coking plant of Zarand, Kerman

Abstract Phenol and phenolic compounds are environmental pollutants present in industrial wastewaters such as coal tar, oil refineries and petrochemical plants. Phenol removal from industrial effluents is extremely important for the protection of environment. Usually, phenol degradation is carried out by physicochemical methods that are costly and produce hazardous metabolites. Recently, phenol biodegradation has been considered. Yeasts are the most important phenol biodegraders. In this study, the phenol-degrading yeast from environmental samples (soil and wastewater) was isolated from the coking plant of Zarand, Kerman. Then total heterotrophic yeasts were counted. The soil samples had higher rates of yeast degrader, in comparison to wastewater samples. After three passages, four yeasts (K1, K2, K7 and K11) that had the highest growth rate were selected for further study. Also, these yeasts were able to remove phenol measured by Gibbs reagent. The effect of four different concentrations of phenol (50, 125, 200 and 275) mg L−1 was measured and three degradation patterns in these yeasts were observed. The hydrophobicity and emulsification activity were measured in all eleven yeasts. Finally, strong yeasts in phenol degrading yeasts were identified by molecular method using amplification of 18S rRNA gene region. The sequencing results showed that these isolated yeasts belonged to Candida tropicalis strain K1, Pichia guilliermondii strain K2, Meyerozyma guilliermondii strain K7 and C. tropicalis strain K11.

Levantamento de extratos vegetais com ação anti-Candida no período de 2005-2013 / Survey of plant extracts with anti-Candida activity in the 2005-2013 period

Entre as micoses relevantes em saúde pública destaca-se a candidíase, infecção oportunista que acomete o homem e animais. A enfermidade era considerada pouco frequente na medicina veterinária, porém relatos demonstram um aumento considerável, assim como a resistência aos antifúngicos. Com isso, pesquisas têm sido desenvolvidas visando encontrar substâncias bioativas frente ao gênero Candida. Desta forma, objetivou-se reunir dados das bases Scielo e ScienceDirect com informações entre os anos de 2005-2013 referentes à ação anti-Candida de diferentes extratos vegetais. Foi encontrado um total de 78 famílias e 208 espécies de plantas com atividade frente à Candida spp., destacando-se as famílias Asteraceae, Geraniaceae, Myrtaceae, Fabaceae, Lamiaceae, Rubiaceae, Verbenaceae e Anacardiaceae, com extratos diclorometânicos, aquosos, etanólicos, metanólico, frações e subfrações, sendo as folhas a parte vegetal mais utilizada. As plantas descritas apresentaram ação anti-Candida, porém algumas necessitam concentrações muito altas dos extratos com pequena inibição de crescimento/eliminação destas leveduras, ocorrendo variações, principalmente, quanto ao método de avaliação, tipo de extrato, parte vegetal, e procedência dos isolados fúngicos. Chama a atenção a raridade dos estudos com isolados de animais, principalmente de casos clínicos. Por fim, destacam-se as famílias Asteraceae e Geraniaceae que apresentaram maior número de espécies vegetais com atividade, podendo ser uma fonte de investigação frente à Candida spp.

Among the relevant mycoses in public health, one that stands out is candidiasis, an opportunistic infection that affects humans and animals. The disease was considered uncommon in veterinary medicine, but reports show a significant increase, as well as resistance, to conventional antifungal agents. Therefore, research has been undertaken aimed at finding bioactive substances from plants that fight against Candida. Thus, the objective of this work was to gather the databases SciELO and ScienceDirect with information between the years 2005 and 2013 concerning the anti-Candida activity of different plant extracts. A total of 78 families and 208 species of plants with activity against Candida spp. was found highlighting the Asteraceae, Geraniaceae, Myrtaceae, Fabaceae, Lamiaceae, Rubiaceae, Verbenaceae and Anacardiaceae families, with dichloromethane, aqueous, ethanol and methanol extracts and fractions and subfractions, being the leaf the most used plant part. The plants described showed anti-Candida activity, but some require very high concentrations of the extracts with little growth inhibition / elimination of these yeasts, with variations related mainly to the method of assessment, type of extract, plant parts and origin of the fungal isolates. The rarity of studies with isolates from animals, mainly clinical cases, draws attention. Finally, we highlight the Asteraceae and Geraniaceae families, which had a greater number of plant species with activity and which may be a source of research against Candida spp.

Estudio comparativo entre los sistemas automatizados Vitek YBC® y Microscan Walk Away RYID® con los métodos fenotípicos convencionales para la identificación de levaduras de interés clínico / A comparative study between the Vitek YBC® and Microscan Walk Away RYID® automated systems with conventional phenotypic methods for the identification of yeasts of clinical interest

El objetivo de este trabajo fue comparar la identificación de levaduras de interés clínico por los métodos automatizados Vitek YBC® y Microscan Walk Away RYID® con los métodos fenotípicos convencionales. Se utilizaron 193 aislamientos de levaduras provenientes de muestras clínicas y cinco cepas controles. Todas las levaduras fueron identificadas por los métodos automatizados antes nombrados y los métodos fenotípicos convencionales de asimilación de carbohidratos, visualización de la morfología microscópica con agar harina de maíz y el uso de agar cromogénico. Para evaluar las variables se utilizaron tablas de contingencia de 2×2, Chi cuadrado de Mc Nemar, el índice Kappa, y se calcularon los valores de concordancia, así como los errores mayores y menores de los métodos automatizados. Las levaduras se dividieron en dos grupos: 1) de aislamiento frecuente y 2) de aislamiento poco frecuente. Los sistemas Vitek YBC® y Microscan Walk Away RYID® fueron concordantes en un 88,4 y 85,9% respectivamente, cuando se compararon con los métodos fenotípicos convencionales. Aunque ambos sistemas automatizados se pueden utilizar para la identificación de levaduras, la presencia de errores mayores y menores indica la posibilidad de identificaciones incorrectas, por lo tanto, el operador de estos equipos debe utilizar paralelamente pruebas fenotípicas como la visualización de la morfología microscópica en agar harina de maíz y el agar cromogénico, sobre todo frente a levaduras de aislamiento poco frecuente. Los sistemas automatizados son una herramienta valiosa, sin embargo, la experiencia y el criterio del microbiólogo son una fortaleza importante para asegurar la calidad de los resultados.

The aim of this study was to compare the identification of clinically relevant yeasts by the Vitek YBC® and Microscan Walk Away RYID® automated methods with conventional phenotypic methods. One hundred and ninety three yeast strains isolated from clinical samples and five controls strains were used. All the yeasts were identified by the automated methods previously mentioned and conventional phenotypic methods such as carbohydrate assimilation, visualization of microscopic morphology on corn meal agar and the use of chromogenic agar. Variables were assessed by 2×2 contingency tables, McNemar’s Chi square, the Kappa index, and concordance values were calculated, as well as major and minor errors for the automated methods. Yeasts were divided into two groups: 1) frequent isolation and 2) rare isolation. The Vitek YBC® and Microscan Walk Away RYID® systems were concordant in 88.4 and 85.9% respectively, when compared to conventional phenotypic methods. Although both automated systems can be used for yeasts identification, the presence of major and minor errors indicates the possibility of misidentifications; therefore, the operator of this equipment must use in parallel, phenotypic tests such as visualization of microscopic morphology on corn meal agar and chromogenic agar, especially against infrequently isolated yeasts. Automated systems are a valuable tool; however, the expertise and judgment of the microbiologist are an important strength to ensure the quality of the results.

Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 x 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.

Yeast diversity associated to sediments and water from two Colombian artificial lakes

In Colombia, knowledge of the yeast and yeast-like fungi community is limited because most studies have focused on species with clinical importance. Sediments and water represent important habitats for the study of yeast diversity, especially for yeast species with industrial, biotechnological, and bioremediation potential. The main purpose of this study was to identify and compare the diversity of yeast species associated with sediment and water samples from two artificial lakes in Universidad del Valle (Cali-Colombia). Yeast samplings were performed from fifteen sediment samples and ten water samples. Grouping of similar isolates was initially based on colony and cell morphology, which was then complemented by micro/mini satellite primed PCR banding pattern analysis by using GTG5 as single primer. A representative isolate for each group established was chosen for D1/D2 domain sequencing and identification. In general, the following yeast species were identified: Candida albicans, Candida diversa, Candida glabrata, Candida pseudolambica, Cryptococcus podzolicus, Cryptococcus rajasthanensis, Cryptococcus laurentii, Williopsis saturnus, Hanseniaspora thailandica, Hanseniaspora uvarum, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Torulaspora delbrueckii, Torulaspora pretoriensis, Tricosporon jirovecii, Trichosporon laibachii and Yarrowia lypolitica. Two possible new species were also found, belonging to the Issatchenkia sp. and Bullera sp. genera. In conclusion, the lakes at the Universidad del Valle campus have significant differences in yeast diversity and species composition between them.

Isolation and characterization of lactic acid bacteria and yeasts from the Brazilian grape sourdough

Sourdough is a mixture of flour and water fermented by lactic acid bacteria and yeast, with a large use in bakery products. This study was developed with Brazilian grape (Niagara rosada) sourdough obtained from spontaneous fermentation. The aim of this work was to characterize genotypic and phenotypically lactic acid bacteria and yeasts isolated from sourdough. The phenotypic identification for bacteria and yeasts was performed by using the kit API50CHL and 20CAUX and the genotypic characterization was performed by sequencing method. A total of four isolated strains were analyzed in this study. Two of these strains were phenotypically and genotypic identified as Lactobacillus paracasei and one as Saccharomyces cerevisiae. Another sample phenotypically identified as Candida pelliculosa did not show the same identity by sequencing. It shows the need to use phenotypic and genotypic characterization associated for the correct microorganism identification.

Fermento natural é mistura de farinha e água fermentada por bactérias láticas e leveduras, amplamente utilizada em produtos de panificação. Neste estudo desenvolveu-se um fermento natural de uva brasileira (Niagara rosada), obtido a partir de fermentação espontânea. O objetivo deste trabalho foi caracterizar fenotipicamente e genotipicamente bactérias láticas e leveduras isoladas do fermento natural de uva. A identificação fenotípica para bactéria lática e leveduras foi realizada usando os kits API50CHL e 20CAUX e a caracterização genotípica foi realizada pelo método de sequenciamento. Neste estudo, isolaram-se quatro cepas. Duas cepas foram identificadas fenotipicamente e genotipicamente como Lactobacillus paracasei e outra cepa como Saccharomyces cerevisiae. A outra amostra de levedura, identificada fenotipicamente como Candida pelliculosa, não obteve a mesma identidade com a técnica de sequenciamento. Isso mostra a necessidade do uso da caracterização fenotípica e genotípica em associação para a correta identificação do micro-organismo.

Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species

Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods.

Yeasts and hygienic-sanitary microbial indicators in water buffalo mozzarella produced and commercialized in Minas Gerais, Brazil

The aim of this work was to study the yeast populations and the main hygienic-sanitary microbial indicators in water buffalo mozzarella produced and commercialized in Minas Gerais, Brazil. Forty-two water buffalo mozzarella samples were purchased from retail outlets in Belo Horizonte. In addition, five samples of consecutive starter cultures, curd before acidification, acidified curd and mozzarella were collected at an industry in the city of Oliveira. Only three of the five water samples analyzed were suitable for consumption according to Brazilian sanitary standards. Four milk samples were highly contaminated with fecal coliforms, and did not meet the minimal hygienic-sanitary standards according to Brazilian regulations. Only one sample of buffalo muzzarela purchased from retail outlets exceeded the limit for coagulase-positive Staphylococcus. Eleven samples showed counts of thermotolerant coliforms higher than5x 10³ CFU.g-1, but still lower than the maximum permitted by the Brazilian laws. Salmonella spp. and Listeria monocytogenes were not isolated. Debaryomyces hansenii, Candida lusitaniae and C. parapsilosis were the prevalent yeast species isolated from cheese. Among samples from the production stages, the acidified curd presented the highest numbers of yeasts, with C. catenulata being the most frequent species isolated. Some opportunistic yeast species such as C. guilliermondii, C. tropicalis, C. parapsilosis, C. lusitaniae, C. catenulata, C. rugosa and C. krusei occurred in the mozzarella cheese samples analyzed. The mozzarella cheese presented a low microbial load as compared to other cheese already studied, and the yeast biota included species typical of cheese and also opportunistic pathogens.

Primer caso de fungemia por una levadura relacionada con Candida pseudorugosa / First case of fungemia from a yeast related with Candida pseudorugosa

Se presenta el primer caso de fungemia por una especie de Candida relacionada con Candida pseudorugosa. La identificación de las especies de levaduras es de importancia a nivel epidemiológico y para el tratamiento de los pacientes que cursan una infección por levaduras.

Identificación bacteriana basada en el espectro de masas de proteínas: Una nueva mirada a la microbiología del siglo XXI / Bacterial identification based on protein mass spectrometry: A new insight at the microbiology of the 21st century

Bacterial identification is important for the proper treatment of infected patients hospitalized with serious infections especially in critical care units. Identification by conventional methods used in microbiology laboratories takes at least 16 hours since a culture is positive. The introduction of mass spectrometry, specifically MALDI-TOF MS (matrix-assisted laser desorption/ ionization time-of-flight mass spectrometer) in the microbiology laboratory could mean a radical change in the identification accuracy, turn around time (6 minutes per bacteria) and cost (about 5 times cheaper than conventional identification). Since its introduction in clinical microbiology laboratories in 2008, many reports about its usefulness in identifying microorganisms from colonies, as well as directly from positive blood cultures and urine samples have been published. This review describes MALDI-TOF MS methodology, its identification performance for bacteria (aerobic and anaerobic), mycobacterium and yeasts, its future applications in microbiology and its main disadvantages.

La identificación bacteriana es muy importante en el manejo adecuado de los pacientes infectados, especialmente aquellos con infecciones graves hospitalizados en unidades de pacientes críticos. La identificación por los métodos convencionales utilizados en los laboratorios de microbiología clínica demora al menos 16 horas desde que un cultivo es positivo. La introducción de la espectrometría de masas, específicamente del espectrómetro de masas por tiempo de migración (tiempo de vuelo) con desorción/ionización laser asistida por una matriz (MALDI-TOF MS, por su sigla en inglés matrix-assisted laser desorption/ionization time-of-flight mass spectrometer), en el laboratorio de microbiología podría significar un cambio radical en la precisión de la identificación, el tiempo de detección (6 minutos por bacterias) y el costo (aproximadamente 5 veces más económico que la identificación convencional). Desde su introducción en los laboratorios de microbiología clínica en el año 2008, se han escrito numerosas publicaciones sobre su utilidad en la identificación de microorganismos desde colonias, así como directamente desde hemocultivos positivos y de muestras de orina. Esta revisión describe la metodología de MALDI-TOF MS, su rendimiento en la identificación de bacterias aerobias, anaerobias, micobacterias y levaduras, sus futuras aplicaciones en microbiología y sus principales desventajas.

Species distribution and susceptibility profile of yeasts isolated from blood cultures: results of a multicenter active laboratory-based surveillance study in Argentina

The Mycology Department of the Instituto Nacional de Enfermedades Infecciosas "Dr. C. Malbrán", conducted the Second National Multicenter Survey on Fungemia due to Yeasts in Argentina. The aim was to obtain updated data of the frequency of the causative species encountered and their in vitro susceptibility to seven antifungal agents. Yeast species were identified by micromorphological and biochemical studies. Antifungal susceptibility testing was performed by the reference microdilution method E.Def 7.1 of the European Committee on Antibiotic Susceptibility Testing (EUCAST). A total of 461 viable yeasts were identified. The most frequent species were: Candida albicans (38.4 %), Candida parapsilosis (26 %), Candida tropicalis (15.4 %) and Candida glabrata (4.3 %). Other uncommon species, such as Candida viswanathii (0.6 %), Candida haemulonii (0.4 %), Candida inconspicua (0.2 %) and Candida fermentati (0.2 %) were also isolated. Among the Candida spp., 5.4 % and 1.6 % were resistant to fluconazole and voriconazole, respectively. Itraconazole and caspofungin were the most efficient agents against all Candida spp. tested (MIC < 1 mg/l). For anidulafungin, 21.6 % of C. parapsilosis showed a MIC value of 4 mg/l. Fluconazole was less active against 53.1 % of Cryptococcus neoformans (MIC > 8 mg/l), 75 % of Trichosporon spp., and 100 % of Rhodotorula spp., Geotrichum candidum, Saccharomyces cerevisiae. The global percentage of mortality was 20 %. The presence of uncommon species reinforces the need for performing continuous laboratory surveillance in order to monitor possible changes, not only in the epidemiological distribution of species, but also in the resistance to antifungal drugs.

Distribución de especies y perfil de sensibilidad de levaduras aisladas de hemocultivos: resultados de un estudio multicéntrico de vigilancia de laboratorio en Argentina. El Departamento Micología del Instituto Nacional de Enfermedades Infecciosas "Dr. Carlos G. Malbrán" condujo el segundo estudio multicéntrico nacional sobre funge- mias debidas a levaduras. El objetivo fue obtener datos actualizados sobre la distribución de especies y la sensibilidad in vitro frente a siete antifúngicos. Las levaduras fueron identificadas mediante el estudio de la micromorfología y la realización de pruebas bioquímicas. La determinación de la sensibilidad se realizó según el método de referencia E.Def 7.1 del European Committee on Antibiotic Susceptibility Testing (EUCAST). Se identificaron 461 levaduras. Las especies más frecuentes fueron Candida albicans (38,4 %), Candida parapsilosis (26 %), Candida tropicalis (15,4 %) y Candida glabrata (4,3 %). Se aislaron otras especies menos comunes, como Candida viswanathii (0,6 %), Candida haemulonii (0,4 %), Candida inconspicua (0,2 %) y Candida fermentati (0,2 %). Entre las especies del género Candida, el 5,4 % y el 1,6 % fueron resistentes al fluconazol y al voriconazol, respectivamente. El itraconazol y la caspofungina fueron los antifúngicos más eficaces in vitro frente a las especies de Candida evaluadas (CIM < 1 mg/l). Para la anidulafungina, el 21,6 % de los aislamientos de C. parapsilosis mostraron una CIM de 4 mg/l. El fluconazol fue menos activo para el 53,1 % de los aislamientos de Cryptococcus neoformans (CIM > 8 mg/l), el 75 % de los aislamientos de Trichosporon spp. y el 100 % de los aislamientos de Rhodotorula spp., Geotrichum candidum y Saccharomyces cerevisiae. El porcentaje de mortalidad fue del 20 %. La presencia de especies infrecuentes refuerza la necesidad de realizar la continua vigilancia de laboratorio con el fin de monitorear posibles cambios, no solo en la epidemiología de las especies causantes de fungemia, sino también en la resistencia a los antifúngicos.