Identificación y selección de Saccharomyces cerevisiae nativas para mejorar el proceso productivo del Pisco a partir de uva Quebranta

El Pisco es el destilado del Perú, elaborado a partir de mostos recientemente fermentados con uvas criollas denominadas "pisqueras". Las levaduras son los microorganismos clave en la fermentación y el uso de cepas nativas seleccionadas presenta ventajas competitivas para la tipicidad del producto, así como también para la estandarización del producto y el control microbiológico del proceso. El objetivo fue identificar y seleccionar las cepas de levaduras nativas para la producción del Pisco de uva Quebranta aisladas de procesos productivos de Pisco en el valle de Ica. Para ello, se emplearon técnicas microbiológicas y moleculares mediante el análisis de ITS1-5.8S-ITS2 PCR-RFLP para la identidad taxonómica. La evaluación de las cepas para producir Pisco consistió en el análisis fisicoquímico y organoléptico del destilado obtenido con las cepas seleccionadas. Se evaluaron 3 aislados para la producción de Pisco identificados como Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49 y UNA SC - 54, de los cuales la cepa UNA SC-49 destacó por mostrar aptitudes enológicas diferentes a las otras cepas. Este trabajo constituye el primer registro de levaduras nativas del Perú para mostos procedentes de uva Quebranta.

El Pisco stands as Peru's distilled spirit, crafted from recently fermented musts derived from native grape varieties known as "pisqueras". Yeasts serve as decisive microorganisms in the fermentation process, and the utilization of selected native strains confers competitive advantages for product typicity, standardization, and microbiological control within the production process. The aim was to identify and select native yeast strains for Quebranta grape Pisco production, isolated from Pisco production processes in the Ica Valley. Microbiological and molecular techniques were employed, utilizing ITS1-5.8S-ITS2 PCR-RFLP analysis for taxonomic identification. The assessment of yeast strains for Pisco production involved the physicochemical and organoleptic analysis of the distilled product obtained using the selected strains. Three isolates were evaluated for Pisco production, identified as Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49, and UNA SC - 54. Among these, the UNA SC-49 strain stood out due to displaying oenological characteristics distinct from the other strains. This work is the first documentation of native yeasts in Peru for musts derived from Quebranta grapes.

Evaluación de la formación de biopelículas en aislamientos bacterianos y fúngicos por el método semicuantitativo de microtitulación con cristal violeta y el cualitativo de agar con rojo Congo / Assessment of biofilms formation of bacterial and fungal isolates using qualitative Congo red agar and semiquantitative crystal violet microtiter methods

Introducción. El 65 % de las infecciones humanas son producidas por bacterias o levaduras, cuya capacidad de formar biopelículas las hace más resistentes a los antimicrobianos y antifúngicos. Objetivo. Determinar la capacidad de formación de biopelículas en aislamientos bacterianos y fúngicos por medio de los métodos cuantitativo de microtitulación con cristal violeta y cualitativo de cultivo en agar con rojo Congo. Materiales y métodos. Con el método cuantitativo, se utilizaron los medios de cultivo infusión cerebro-corazón, tripticasa de soya y Müeller-Hinton para aislamientos bacterianos; para levaduras, se usaron caldo infusión cerebro-corazón y Sabouraud dextrosa. Para el método cualitativo de cultivo en agar, se utilizaron los mismos medios de cultivo más una solución con 3 % de rojo Congo y 10 % de dextrosa. Cómo método de referencia, se utilizó la propuesta de Stepanovic et al. Resultados. Se evaluaron 103 aislamientos bacterianos y 108 de levaduras. No es recomendable sustituir el caldo infusión cerebro-corazón por los caldos tripticasa de soya y Müeller-Hinton en el método cuantitativo, para evaluar la formación de biopelículas en los aislamientos bacterianos. El medio Sabouraud dextrosa, en caldo y agar, puede sustituir al de infusión de cerebro-corazón para evaluar la formación de biopelículas en levaduras, tanto por el método cuantitativo como por el cualitativo. Conclusión. El estudio de las biopelículas en el laboratorio de microbiología, a partir del método cualitativo de cultivo en agar con rojo Congo, es un procedimiento sencillo, rápido y de bajo costo, que proporciona información útil para el diagnóstico y la terapéutica de infecciones persistentes causadas por bacterias y levaduras.

Introduction. Sixty-five percent of human infections are caused by bacteria or yeasts able to form biofilms. This feature makes them more resistant to antimicrobials and antifungals. Objective. To determine biofilm formation capacity of bacterial and fungal isolates by quantitative crystal violet microtiter and qualitative Congo red agar methods. Materials and methods. Brain-heart infusion, trypticase soy broth and Müeller-Hinton culture media were used in bacterial isolates for the quantitative method; brain-heart infusion broth and Sabouraud dextrose were used for yeasts. The same culture media plus 3% Congo red and 10% dextrose were used to apply the qualitative method in agar. The proposal by Stepanovic, et al. was used as a reference method. Results. We evaluated 103 bacterial isolates and 108 yeasts isolates. We did not recommend substitute brain-heart infusion broth for trypticase soy and Müeller-Hinton broths for biofilm formation assessment in bacterial isolates using the quantitative method. Sabouraud dextrose medium, both broth and agar, can replace brain-heart infusion to assess biofilm formation in yeasts, quantitatively and qualitatively. Conclusion. The study of biofilms in the microbiology laboratory, using Congo red agar qualitative method, is a simple, fast, and inexpensive procedure that provides precise information for the diagnosis and treatment of persistent infections caused by bacteria and yeasts.

Biosynthesis of natural products by non-conventional yeasts / 生物工程学报

Non-conventional yeasts such as Yarrowia lipolytica, Pichia pastoris, Kluyveromyces marxianus, Rhodosporidium toruloides and Hansenula polymorpha have proven to be efficient cell factories in producing a variety of natural products due to their wide substrate utilization spectrum, strong tolerance to environmental stresses and other merits. With the development of synthetic biology and gene editing technology, metabolic engineering tools and strategies for non-conventional yeasts are expanding. This review introduces the physiological characteristics, tool development and current application of several representative non-conventional yeasts, and summarizes the metabolic engineering strategies commonly used in the improvement of natural products biosynthesis. We also discuss the strengths and weaknesses of non-conventional yeasts as natural products cell factories at current stage, and prospects future research and development trends.

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

Estudio piloto de caracterización microbiológica de los billetes que circulan en la ciudad de Bucaramanga, Colombia / Pilot Study of Microbiological Characterization of Banknotes Circulating in the City of Bucaramanga, Colombia / Estudo piloto de caracterização microbiológica das notas em circulação na cidade de Bucaramanga, Colômbia

Introducción. Los billetes son un potencial medio de transmisión de microorganismos capaces de producir enfermedades. Es el caso del Staphylococcus aureus, una bacteria distribuida por toda América Latina, causante de infecciones y resistente a antibióticos de uso común. El objetivo del estudio es realizar una caracterización bacteriana y fúngica de billetes circulantes en la ciudad de Bucaramanga, y en especial identificar algunos que puedan relacionarse con problemas de salud pública. Metodología. Estudio observacional y cuantitativo, con una muestra de 50 billetes (5 diferentes denominaciones de 2 fechas de emisión). Se identificaron y cuantificaron los microorganismos mediante siembra en caldo peptona, posteriormente en agar Reasoner´s 2A (R2A), nutritivos y selectivos, además del uso de técnicas de índice analítico de perfil (API) y microscopia óptica. Se realizó un análisis estadístico de correlación de variables mediante el software Statistical Package for the Social Sciences (SPSS). Resultados. Se identificaron 21 géneros y 12 especies de bacterias, así como 3 géneros y 2 especies de hongos filamentosos, entre ellos algunos que pueden ocasionar infecciones como Klesiella, Enterobacter, Listeria, Staphylococcus, Cryptococcus y Aspergillus. Discusión. En relación con estudios internacionales, en este trabajo se identificaron menos tipologías de microorganismos, lo cual se explica en razón a las limitaciones propias de las técn icas utilizadas y del nivel de contaminación local. Conclusión. Se pudo establecer que el grado de contaminación microbiana no depende significativa o consistentemente de la fecha de emisión ni de la denominación; pero la identificación de patógenos sugiere plantear medidas para limitar su transmisión por esta vía.

Introduction. Banknotes are a potential means of transmission of microorganisms capable of producing diseases. This is the case of Staphylococcus aureus, a bacterium distributed throughout Latin America, which causes infections and is resistant to commonly used antibiotics. The objective of the study is to perform a bacterial and fungal characterization of banknotes circulating in the city of Bucaramanga, and especially to identify some that may be related to public health problems. Methodology. Observational and quantitative study, with a sample of 50 banknotes (5 different denominations of 2 issue dates). Microorganisms were identified and quantified by seeding in peptone broth, then on Reasoner's 2A agar (R2A), nutrient and selective, in addition to the use of analytical profile index (API) and light microscopy techniques. A statistical analysis of correlation of variables was performed using the Statistical Package for the Social Sciences (SPSS) software. Results. Twenty-one genera and 12 species of bacteria were identified, as well as 3 genera and 2 species of filamentous fungi, including some that can cause infections such as Klesiella, Enterobacter, Listeria, Staphylococcus, Cryptococcus, and Aspergillus. Discussion. In comparison with international studies, this study identified fewer types of microorganisms, which is explained by the limitations of the techniques used and the level of local contamination. Conclusion. It was possible to establish that the degree of microbial contamination does not depend significantly or consistently on the date of issue or the denomination; but the identification of pathogens suggests that measures should be taken to limit their transmission by this ro ute.

Introdução. As notas são um meio potencial de transmissão de microrganismos capazes de produzir doenças. É o caso do Staphylococcus aureus, bactéria distribuída por toda a América Latina, causadora de infecções e resistente aos antibióticos comumente utilizados. O objetivo do estudo é realizar uma caracterização bacteriana e fúngica das notas em circulação na cidade de Bucaramanga e, principalmente, identificar algumas que possam estar relacionadas a problemas de saúde pública. Metodologia. Estudo observacional e quantitativo, com uma amostra de 50 notas (5 denominações diferentes de 2 datas de emissão). Os microrganismos foram identificados e quantificados por meio de semeadura em caldo peptonado, depois em ágar Reasoner 2A (R2A), nutritivo e seletivo, além da utilização de índice de perfil analítico (API) e técnicas de microscopia óptica. Foi realizada uma análise estatística de correlação das variáveis por meio do software Statistical Package for the Social Sciences (SPSS). Resultados. Foram identificados 21 gêneros e 12 espécies de bactérias, além de 3 gêneros e 2 espécies de fungos filamentosos, incluido alguns que podem causar infecções como Klesiella, Enterobacter, Listeria, Staphylococcus, Cryptococcus e Aspergillus. Discussão. Em relação aos estudos internacionais, neste trabalho foram identificados menos tipos de microrganismos, o que se explica pelas limitações das técnicas utilizadas e pelo nível de contaminação local. Conclusão. Foi possível estabelecer que o grau de contaminação microbiana não depende de forma significativa ou consistente da data de emissão ou da denominação; mas a identificação de patógenos sugere considerar medidas para limitar sua transmissão por essa via.

Conteo de bacterias y levaduras en imágenes digitales

RESUMEN Fundamento: en los laboratorios de microbiología, la identificación y conteo de microorganismos es un procedimiento habitual. Aunque existen en el mercado equipos que posibilitan su realización de manera automática o semiautomática, son muy costosos, por lo cual esta tarea, difícil e irritante para los ojos, la siguen realizando los expertos de manera tradicional mediante la observación de las muestras en los microscopios, con la consiguiente variabilidad entre ellos. Objetivo: proponer un nuevo método para el conteo de bacterias y levaduras en imágenes digitales, bajo diferentes magnificaciones, tomadas a bioproductos de origen microbiano obtenidos por fermentación. Métodos: el sensor empleado para la toma de imágenes de las muestras fue una cámara digital modelo HDCE-X, con un sensor CMOS de ½", con una resolución de 2592 píxeles por 1944 píxeles (5 Mp). Se emplearon dos tipos de magnificaciones: magnificación 40x (PL40, 0.65 apertura numérica and 0.17 de distancia de trabajo) y magnificación 100x (HI plan 100/1.25 con inmersión de aceite). El método propuesto se basa en técnicas de procesamiento digital de imágenes, utilizando herramientas como la detección de contornos, operaciones morfológicas y análisis estadístico, y fue desarrollado en lenguaje Python con empleo de la biblioteca OpenCV. Resultados: la detección y conteo de bacterias se logró con una exactitud y precisión aceptable, en ambos casos por encima de 0,95; no en el caso de las levaduras cuya exactitud y precisión fueron menores, alrededor de 0,78 y 0,86 respectivamente. Se proponen flujos de trabajo basados en técnicas de procesamiento digital de imágenes, fundamentalmente en detección de contornos, operaciones morfológicas y análisis estadístico. Conclusiones: el método posee una efectividad aceptable para el contexto y depende de las características que presenten las imágenes.

ABSTRACT Background: In microbiology laboratories, the identification and counting of microorganisms is a common procedure; and although there is a variety of equipment on the market that possibility to carry out these processes automatically or semi-automatically, it is usually expensive to many laboratories. These are some of the reasons why this arduous and difficult task is still performed in many laboratories by experts in the traditional way, through the observation of samples in microscope, consuming a great time and having variations in the results between experts. Objective: The present work aims to propose a new method for counting bacteria and yeasts in digital images, taken under different magnifications, of microbial bioproducts obtained by fermentation. Methods: The sensor used to take images of the samples was a digital camera model HDCE-X, with a ½" CMOS sensor, with a resolution of 2592 pixels by 1944 pixels (5 Mp). Two types of magnifications were used: 40x magnification (PL40, 0.65 numerical aperture and 0.17 working distance) and 100x magnification (HI plan 100/1.25 with oil immersion). The proposed method is based on digital image processing technics, using tools as contour detection, morphological operations and statistical analysis, and was developed in Python language using the OpenCV library. The work also presents a comparison with the results obtained using ImageJ software for the same purpose. Results: the detection and count of bacteria was achieved with an acceptable accuracy and precision, in both cases above 0.95; not in the case of yeasts whose accuracy and precision was lower, around 0.78 for accuracy and 0.86 for precision. Workflows based on digital image processing techniques are proposed, using tools as contour detection, morphological operations and statistical analysis. Conclusions: the method has an acceptable effectiveness for the context and depends on the characteristics presented by the images.

Usos de la levadura Pichia pastoris en la producción de proteínas recombinantes / Uses of Pichia pastoris yeast in the production of recombinant proteins

La levadura metilotrófica Pichia pastoris (clasificada actualmente como Komagataella phaffii) es una de las más importantes para la producción de proteínas heterólogas. En el trabajo se presenta un análisis de las principales características que se ponen de manifiesto en la expresión de proteínas recombinantes expresadas en este microorganismo. Se describen las cepas disponibles para la transformación y producción de proteínas recombinantes expresadas en Pichia pastoris, los principales vectores comerciales para la expresión, los promotores más eficientes, los marcadores seleccionables, la señal de secreción, los métodos usados en las transformaciones genéticas y los patrones de glicosilación que se presentan. Se brindan recomendaciones generales acerca de los parámetros de bioprocesos como la composición del medio, el pH, la temperatura, la velocidad de aireación, la inducción y las estrategias de alimentación para alcanzar altos valores de productividad. Se presentan los resultados de las aplicaciones de Pichia pastoris en la producción de dos vacunas en Cuba, la vacuna contra la hepatitis B y la vacuna para el control de la garrapata(AU)

Pichia pastoris metylotrofic yeast (currently classified as Komagataella phaffii) is one of the most important yeast for the production of heterologous proteins. The work presents an analysis of the main characteristics that are marked in the production of recombinant proteins expressed in Pichia pastoris. It describes the strains available for the transformation and production of recombinant proteins expressed in P. pastoris, the main commercial vectors for expression, the most efficient promoters, selectable markers, the secretion signal, the methods used in genetic transformations and glycosylation patterns that occur. General recommendations are provided on bioprocess parameters such as media composition, pH, temperature, aeration velocity, induction, and feeding strategies to achieve high productivity values. The results of Pichia pastoris applications for the production of two vaccines in Cuba, the hepatitis B vaccine and the tick control vaccine are shown(AU)

Advances in metabolic engineering of non-conventional yeasts / 生物工程学报

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.

Yeast microbiota of free-ranging amphibians and reptiles from Caatinga biome in Ceará State, Northeast Brazil: High pathogenic potential of Candida famata / Leveduras da microbiota de anfíbios e répteis do bioma Caatinga no Ceará, Nordeste do Brasil: alto potencial patogênico de Candida famata

ABSTRACT: Studies on the fungal microbiota of reptiles and amphibians are necessary to better understand of host-microbe interactions and the establishment of fungal disease in these animals. However, these studies are limited. The present researchidentified yeasts from free-ranging reptiles and amphibians from the Caatinga biome andevaluated the virulence factors production, the antifungal susceptibility in planktonic and biofilm growth and the pathogenicity of Candida famata isolates. Twenty-nine isolates of the genera Candida, Cryptococcus and Rhodotorula were identified by phenotypic and/or molecular methods and production of hydrolytic enzymes in vitro by these genera of fungi was evaluated. In addition, susceptibility of planktonic cells and biofilms to azoles and amphotericin B was evaluated. The pathogenicity of C. famata, the most prevalent yeast species isolated, was evaluated using Caenorhabditis elegans model. C. famata was the most prevalent yeast in amphibian and reptilian microbiota. Phospholipase and protease production was observed in 18/29 and 11/29 of the yeast isolates, respectively, while 100% formed biofilms. Itraconazole presented high minimal inhibitory concentrations against C. famata and C. tropicalis. Amphotericin B reduced the biomass and metabolic activity of biofilms. C. famata induced the mortality of C. elegans. In conclusion, reptiles and amphibians are colonized by yeasts capable of producing important virulence factors, especially by Candida spp. that present low susceptibility to azoles which may result from imbalances in ecosystem. Finally, C. famata isolated from these animals presented high pathogenicity, showing the importance of the study of reptile and amphibians fungal microbiota.

RESUMO: Estudos sobre a microbiota fúngica de répteis e anfíbios são necessários para melhor compreender as interações hospedeiro-microrganismo e o estabelecimento de doenças fúngicas nesses animais. No entanto, esses estudos são limitados. O objetivo da presente pesquisa foi identificar leveduras isoladas de répteis e anfíbios do bioma Caatinga e avaliar a produção de fatores de virulência, a sensibilidade a antifúngicos no crescimento planctônico e de biofilme e a patogenicidade de Candida famata. Vinte e nove isolados dos gêneros Candida, Cryptococcus e Rhodotorula foram identificados por métodos fenotípicos e/ou moleculares e a produção de enzimas hidrolíticas in vitro por esses gêneros de fungos foi avaliada. Além disso, foi avaliada a suscetibilidade de células planctônicas e biofilmes a azólicos e anfotericina B. A patogenicidade de C. famata, a espécie de levedura isolada mais prevalente, foi avaliada usando Caenorhabditis elegans. C. famata foi a levedura mais prevalente na microbiota de anfíbios e répteis. A produção de fosfolipase e protease foi observada em 18/29 e 11/29 dos isolados de levedura, respectivamente, enquanto 100% formaram biofilmes. O itraconazol apresentou altas concentrações inibitórias mínimas contra C. famata e C. tropicalis. A anfotericina B reduziu a biomassa e atividade metabólica dos biofilmes. C. famata induziu a mortalidade de C. elegans. Em conclusão, répteis e anfíbios são colonizados por leveduras capazes de produzir importantes fatores de virulência, especialmente por cepas de Candida spp. que apresentam baixa suscetibilidade a azólicos que podem resultar de desequilíbrio no ecossistema. Por fim, C. famata isolados desses animais apresentaram alta patogenicidade, mostrando a importância do estudo da microbiota fúngica de répteis e anfíbios.

Prevalence, antifungal susceptibility and virulence determinants of oral yeast species isolated from immunodeficient patients in Northeastern Brazil / Prevalência, suscetibilidade a antifúngicos e determinantes de virulência de leveduras orais isoladas de pacientes imunodeficientes do Nordeste do Brasil / Prevalencia, susceptibilidad antifúngica y determinantes de virulencia de levaduras orales aisladas de pacientes inmunodeficientes del Noreste de Brasil

Justificativa e Objetivos: A candidíase oral tem uma ocorrência comum em pacientes imunocomprometidos. No entanto, outras infecções emergentes tornaram-se cada vez mais habituais. O objetivo deste estudo foi investigar a prevalência, os determinantes de virulência e a suscetibilidade a antifúngicos de leveduras que colonizam a mucosa de pacientes imunocomprometidos na região Nordeste do Brasil. Métodos: A amostra foi composta por 60 pacientes HIV positivos atendidos no Serviço de Atendimento Especializado/Hospital Dia do Hospital Universitário Prof. Alberto Antunes, vinculado à Universidade Federal de Alagoas. As amostras foram coletadas em regiões subgengivais e semeadas em CHROMagar para confirmação presuntiva de Candida spp., seguido por PCR e sequenciamento. Além disso, testamos os determinantes de virulência fosfolipase e protease e avaliamos in vitro a concentração inibitória mínima dos antifúngicos anfotericina B e fluconazol. Este projeto foi aprovado pelo Comitê de ética em pesquisa do Centro de Estudos Superiores de Maceió. Resultados: Aproximadamente 63% dos pacientes foram colonizados por leveduras. A espécie C. albicans foi predominante, enquanto as espécies de Candida não-albicans representaram 49% dos isolados, sendo C. dubliniensis e C. parapsilosis as mais comuns. Entretanto, C. intermedia, Bullera penniseticola e Naganishia liquefaciens também foram encontrados. Os determinantes da virulência protease e/ou fosfolipase também foram produzidos por Candida spp. e alguns isolados oportunistas incomuns como Kodamaea ohmeri, N. liquefaciens e Saitozyma podzolica. Além disso, a maioria dos isolados de Candida spp. e algumas espécies oportunistas incomuns apresentaram altos valores de concentração inibitória mínima. Conclusão: Os resultados obtidos indicam que C. albicans continua a ser a espécie predominante na cavidade oral de pacientes imunodeficientes e, juntamente com outras espécies incomuns, pode apresentar alta resistência aos antifúngicos testados.(AU)

Background and Objectives: Oral candidiasis has a common occurrence in immunocompromised patients. However, other emergent infections have become increasingly common. The aim of this study was to investigate the prevalence, virulence determinants and the antifungal susceptibility of yeast colonizing the mucosa of immunocompromised patients in Northeastern Brazil. Methods: Samples from sixty HIV-positive patients seen at the Specialized Service / Hospital Dia - Hospital Universitário Prof. Alberto Antunes from the Federal University of Alagoas were collected from subgingival sites and seeded on CHROMagar for presumptive confirmation of Candida spp. followed by PCR and sequencing. In addition, we tested virulence determinants, phospholipase and protease and evaluated in vitro the Minimum Inhibitory Concentration of antifungals amphotericin B and fluconazole. This project was approved by the Research Ethics Committee of the Center for Higher Studies in Maceió. Results: Approximately 63% of the patients were colonized by yeasts, with C. albicans as the predominant species, while non-Candida albicans species accounted for 49% of the isolates, with C. dubliniensis and C. parapsilosis being the commonest, but C. intermedia, Bullera penniseticola and Naganishia liquefaciens were also found. The virulence determinants protease and/or phospholipase were also produced by Candida spp. and some uncommon opportunistic isolates such as Kodamaea ohmeri, N. liquefaciens and Saitozyma podzolica. Furthermore, most of Candida spp. strains and some uncommon opportunistic species showed high values of minimal inhibitory concentration. Conclusion: Results obtained indicate that C. albicans continues to be the predominant species in oral cavity of immunodeficient patients and along with other unusual species may present high resistance to the antifungals tested.(AU)

Justificación y Objetivos: La candidiasis oral acomete con frecuencia a pacientes inmunocomprometidos. Sin embargo, otras infecciones emergentes se han vuelto cada vez más comunes. El objetivo de este estudio fue investigar la prevalencia, la producción de determinantes de virulencia y la susceptibilidad a antifúngicos de levaduras que colonizan la mucosa de pacientes inmunocomprometidos en la región Nordeste de Brasil. Métodos: Se colectaron muestras de sesenta pacientes VIH positivos atendidos en el Servicio de Atención Especializado/Hospital Día del Hospital Universitario Prof. Alberto Antunes, vinculado a la Universidad Federal de Alagoas. Se colectaron las muestras en las regiones subgingivales y las sembraron en CHROMagar para la presunta confirmación de Candida spp. seguido de PCR y secuenciación. Además, analizamos los determinantes de virulencia fosfolipasa y proteasa y evaluamos in vitro la concentración mínima inhibitoria de los antifúngicos anfotericina B y fluconazol. Este proyecto fue aprobado por el Comité de Ética en Investigación del Centro de Estudios Superiores de Maceió. Resultados: Aproximadamente el 63% de los pacientes fueron colonizados por levaduras, y la C. albicans fue la especie predominante, mientras que las especies de Candida no-albicans representaron el 49% de los aislamientos, de las cuales la C. dubliniensis y la C. parapsilosis fueron las más comunes. Sin embargo, también se encontraron C. intermedia, Bullera penniseticola y Naganishia liquefaciens. Los determinantes de virulencia de proteasa y/o fosfolipasa también fueron producidos por Candida spp. y algunos aislados oportunistas inusuales como Kodamaea ohmeri, N. liquefaciens y Saitozyma podzolica. Además, la mayoría de los asilados de Candida spp. y algunas especies oportunistas inusuales mostraron valores altos de concentración mínima inhibitoria. Conclusión: Los resultados obtenidos indican que C. albicans continúa siendo la especie predominante en la cavidad oral de pacientes inmunodeprimidos y, junto con otras especies poco comunes, puede presentar una alta resistencia a los antifúngicos evaluados.(AU)

Sensitivity to antifungals by Candida spp samples isolated from cases of chronic atrophic candidiasis (CAC) / Sensibilidade a antifúngicos por amostras de Candida spp isoladas de casos de candidíase atrófica crônica (CAC)

Abstract The treatment of choice for chronic atrophic candidiasis (CAC), also known as denture stomatitis, is topical antifungal therapy. This study aimed to isolate, identify, and assess the antifungal susceptibility of Candida species from mucosal sites in denture wearers with a diagnosis of CAC and determine the prevalence of associated variables. The sample consisted of 44 patients wearing complete or partial dentures who had a clinical diagnosis of CAC. Using sterile cotton swabs, specimens were collected from the oral mucosa of all patients and grown at 30ºC for 48 h in CHROMagar Candida, as a means of isolating and screening the species. The complementary identification of the species was performed using the VITEK 2 automated system (BioMérieux), as well as the determination of their susceptibility to antifungal agents. Data were analyzed using the chi-square test. STATA 13.1 was used for statistical analysis (α = 5%). Of 44 patients with CAC, 33 (75%) had lesions classified as Newton type II. Yeasts were isolated in 38 cases. The most prevalent species was Candida albicans. None of the isolates were resistant to the antifungals tested. Our findings suggest that current indications for antifungal agents are appropriate. Also, antifungal susceptibility testing and proper fungal identification can help dentists to determine the optimal course of treatment for CAC.

Resumo O tratamento de escolha para candidíase atrófica crônica (CAC), também conhecida como estomatite protética, é a terapia antifúngica tópica. Este estudo teve como objetivo isolar, identificar e avaliar a susceptibilidade antifúngica de espécies de Candida de locais mucosos em portadores de prótese com diagnóstico de CAC e determinar a prevalência de variáveis associadas. A amostra consistiu em 44 pacientes portadores de próteses completas ou parciais que tiveram um diagnóstico clínico de CAC. Usando swab estéril, foram coletados espécimes da mucosa oral de todos os pacientes e cultivados a 30ºC durante 48 h em CHROMagar Candida, como forma de isolamento e triagem das espécies. A identificação complementar das espécies foi realizada no sistema automatizado VITEK 2 (BioMérieux), bem como a determinação da susceptibilidade delas a agentes antifúngicos. Os dados foram analisados usando o teste do qui-quadrado. O STATA 13.1 foi utilizado para análise estatística (α = 5%). Dos 44 pacientes com CAC, 33 (75%) apresentaram lesões classificadas como Newton tipo II. As leveduras foram isoladas em 38 casos. A espécie mais prevalente foi Candida albicans. Nenhum dos isolados foi resistente aos antifúngicos testados. Nossas descobertas sugerem que as indicações atuais para os agentes antifúngicos são apropriadas. Além disso, testes de susceptibilidade antifúngicos e identificação fúngica adequada podem ajudar os dentistas a determinar o curso ótimo de tratamento para CAC.

Thermotolerance of Histoplasma capsulatum at 40 °C predominates among clinical isolates from different Latin American regions

ABSTRACT The yeast phase of 22 Histoplasma capsulatum clinical isolates from Mexico, Argentina, Colombia, and Guatemala and three reference strains, one from Panama and two from the United States of America (USA), were screened for thermosensitivity characteristics using different analyses. Growth curves at 0, 3, 6, 12, 24, and 30 h of incubation at 37 and 40 °C, the growth inhibition percentage at 40 °C, and the doubling time at 37 and 40 °C were determined for all yeasts studied. Most of the isolates examined exhibited thermotolerant phenotypes at 40 °C, whereas a thermosensitive phenotype at 40 °C was only detected in the Downs reference strain from the USA. Growth inhibition values lower than 33.8% supported the predominance of the thermotolerant phenotype at 40 °C. The doubling time means found for the different isolates were 5.14 h ± 1.47 h at 37 °C and 5.55 h ± 1.87 h at 40 °C. This is the first report to underscore the predominance of thermotolerant and delayed doubling time phenotypes in H. capsulatum clinical isolates from different regions of Latin America.

The secreted acid trehalase encoded by the CgATH1 gene is involved in Candida glabrata virulence

BACKGROUND Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

Multicenter study of Candida species in oral mucosa of different patients: analysis of 711 strains and literature review

This study aimed to identify Candida spp. collected from oral mucosa and maintained in culture collections, correlating the findings with the medical history of patients and comparing with data from the literature over the past five years. Seven hundred and eleven oral Candida spp. isolates, collected between 2013 and 2017, were selected and identified using traditional and molecular methods. In addition, a literature review was performed with the key words: "Oral", "Candida" and "Yeast". Seven species of the genus Candida: were identified C. albicans(73.3%); C. tropicalis (9.3%); C. parapsilosis (8.2%); C. glabrata (3.9%); C. guilliermondii(2.8%); C. krusei (1.7%) and C. lusitaniae (0.3%). The strains identified as C. albicans were submitted to molecular methods using specific primers and of these, 5.8% were identified as C. dubliniensis strains. The greatest diversity of strains was found in patients presenting no systemic diseases or HIV +, while the highest percentage of strains of Candidanon-albicanswere observed in cancer patients. This study reports a representative distribution of Candidaspecies among individuals exhibiting distinct clinical conditions, in order to contribute to the design of future research on details of aspects involved in the infections caused by these microorganisms. The correct identification of oral Candida strains contributes to a realistic epidemiological approach and future clinical protocols against these pathogens

Disentangling the genetic bases of Saccharomyces cerevisiae nitrogen consumption and adaptation to low nitrogen environments in wine fermentation

The budding yeast Saccharomyces cerevisiae has been considered for more than 20 years as a premier model organ- ism for biological sciences, also being the main microorganism used in wide industrial applications, like alcoholic fermentation in the winemaking process. Grape juice is a challenging environment for S. cerevisiae , with nitrogen deficiencies impairing fermentation rate and yeast biomass production, causing stuck or sluggish fermentations, thus generating sizeable economic losses for wine industry. In the present review, we summarize some recent efforts in the search of causative genes that account for yeast adaptation to low nitrogen environments, specially focused in wine fermentation conditions. We start presenting a brief perspective of yeast nitrogen utilization under wine fermentative conditions, highlighting yeast preference for some nitrogen sources above others. Then, we give an outlook of S. cerevisiae genetic diversity studies, paying special attention to efforts in genome sequencing for population structure determination and presenting QTL mapping as a powerful tool for phenotype-genotype correlations. Finally, we do a recapitulation of S. cerevisiae natural diversity related to low nitrogen adaptation, specially showing how different studies have left in evidence the central role of the TORC1 signalling pathway in nitrogen utilization and positioned wild S. cerevisiae strains as a reservoir of beneficial alleles with potential industrial applications (e.g. improvement of industrial yeasts for wine production). More studies focused in disentangling the genetic bases of S. cerevisiae adaptation in wine fermentation will be key to determine the domestication effects over low nitrogen adaptation, as well as to definitely proof that wild S. cerevisiae strains have potential genetic determinants for better adaptation to low nitrogen conditions.

Isolation of Malassezia spp. in HIV-positive patients with and without seborrheic dermatitis

Abstract Background Malassezia, a skin saprophyte, is frequently isolated from patients with seborrheic dermatitis, which is one of the most common dermatoses in HIV-infected patients. Its role in pathophysiology has not been defined. Objective To determine whether patients living with HIV and seborrheic dermatitis have more Malassezia than those without seborrheic dermatitis. Method This is an descriptive, observational, prospective cross-sectional study to which all adult patients living with HIV that attend the infectious disease outpatient clinic at the Dr. Manuel Gea González General Hospital were invited. Patients presenting with scale and erythema were included in Group 1, while patients without erythema were included in Group 2. Samples were taken from all patients for smear and culture. Results Thirty patients were included in each group. All patients with seborrheic dermatitis had a positive smear, with varying amounts of yeasts. In the control group, 36.7% of patients had a negative smear. The results are statistically significant, as well as the number of colonies in the cultures.Study limitations The study used a small sample size and the subspecies were not identified. Conclusions Patients with clinical manifestations of seborrheic dermatitis have larger amounts of Malassezia. Further studies need to be performed to analyze if the greater amount is related to imbalances in the microbiota of the skin.

Compêndio de métodos e de boas práticas em coleção de cultura de leveduras do Instituto de Biologia do Exército / Compendium of methods and good practices in yeast culture collection of the Brazilian Army Biology Institute

Objetivo: O Compêndio de Métodos e de Boas Práticas em Coleção de Cultura de Leveduras do Instituto de Biologia do Exército (IBEx) foi elaborado com o propósito de operacionalizar as atividades de pesquisa, garantindo o desempenho seguro e confiável dos seus objetivos regimentais. Métodos: A Coleção de Cultura de Leveduras do IBEx foi criada a partir de cepas isoladas de quadros de candidíase sistêmica, através da utilização de metodologias manuais e automatizadas para autenticação das mesmas, com o propósito de garantir a pesquisa científica e atividades de ensino. Resultados: O Instituto adequou e modernizou seus laboratórios de pesquisa criando o Centro de Estudos em Biodefesa, com Nível de Biossegurança NB-3. Com base na necessidade de organização para atender às demandas da defesa biológica, o IBEx vem aprimorando sua estrutura para dominar e garantir novas tecnologias de identificação e manejo dos microrganismos envolvidos em Bioterrorismo. Conclusão: A compilação de metodologias na forma de um Compêndio proporcionou a operacionalização da Coleção de Cultura do IBEx.

Objective: The Compendium of Methods and Good Practices in Yeast Culture Collection of the Brazilian Army Biology Institute (IBEx) was elaborated with the purpose of operationalizing the research activities, guaranteeing the safe and reliable performance of its regimental objectives. Methods: The IBEx Yeast Culture Collection was created from isolated strains of systemic candidiasis, through the use of manual and automated methodologies to authenticate them, in order to guarantee scientific research and teaching activities. Results: The Institute adapted and modernized its research laboratories by creating the Center for Biodefense Studies, with Biosafety Level NB-3. Based on the need for organization to meet the demands of biological defense, the IBEx has been improving its structure to dominate and guarantee new technologies of identification and management of the microorganisms involved in Bioterrorism. Conclusion: The compilation of methodologies in the form of a Compendium provided the operationalization of the IBEx Culture Collection

Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

Background: In industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze­thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain. Results: Elite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze­thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/ v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield. Conclusions: A multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze­thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.

National production of certified reference fungal cultures / Producción nacional de cultivos fúngicos de referencia certificados

Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short-and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20 ±5 °C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry.

Los cultivos microbianos de referencia (CR) son imprescindibles para el control de calidad interno de los laboratorios. Asegurar su producción requiere de procedimientos estandarizados (IRAM 14950:2016 e ISO 17034:2016) realizados en un laboratorio reconocido y acreditado. El objetivo de este estudio fue producir cultivos fúngicos de referencia en discos de papel, a partir de un panel de cultivos autóctonos caracterizados por dos métodos de referencia, trazables a nivel taxonómico de especie, homogéneos y estables. Se produjeron CR de 14 especies circulantes en Argentina (7 de levaduras y 7 de hongos miceliales), depositadas en la colección de hongos de interés médico (DMic). Los discos de papel fueron embebidos con una suspensión del cultivo por producir, secados y envasados. Se verificó la homogeneidad, viabilidad, identidad y pureza de cada lote. Se evaluó la estabilidad a corto y largo plazo a distintas temperaturas y tiempos de almacenamiento. La caracterización de cada CR nos permitió confirmar su identidad y asegurar su trazabilidad a nivel internacional. Los lotes producidos fueron homogéneos y estables durante 30 meses conservados a -20 ±5 °C. Este método resultó adecuado para producir CR homogéneos y estables, con características fenotípicas y genotípicas correctamente definidas y trazables a nivel internacional. Los procedimientos estandarizados desarrollados en este trabajo pueden ser transferidos para producir CR certificados de otros microorganismos. La provisión de CR que represente cepas regionales permite a los laboratorios producir resultados más confiables con un impacto favorable en el diagnóstico médico, los estudios ambientales y la industria alimenticia.