Horses fed meal contaminated with mycotoxins performing short-term exercises / Efeitos das micotoxinas na performance de equinos submetidos a exercícios de curta duração

This research was carried out to evaluate the effect of mycotoxins on the performance of horses through physiological parameters, and hematology and serum biochemistry analyses. The essay lasted 40 days, with 12 days for adaptation and 28 days of experimentation. In the experimental stage, the horses were distributed in a completely randomized design, with three treatments with four animals each. The treatments used were 0 (control), 50 ppb and 100 ppb of Aflatoxin B1 (AFB1) added to a concentrate in a basal diet. The basal diet contained mycotoxins from feedstuffs naturally contaminated. The exercise test was performed over the 21th day of the experimental stage. The exercise consisted in an interval training test with a warm-up of 17 mins at a trot followed by three gallops of 450m/min. The heart rate was monitored between the gallops. Before the exercise test and immediately after the third gallop, the physiological and blood parameters were evaluated, and continued up to 48 hours after the exercise. The results of the physiological, hematological and biochemical parameters were submitted to analysis of variance (ANOVA) and compared by the Tukey test at 5% of significance. The presence of AFB1 in the diet influenced the alkaline phosphatase activity, which presented higher values in horses fed diet with inclusion of 100 ppb AFB1, suggesting a hepatotoxic activity associated with the others mycotoxins naturally present in the feedstuffs.(AU)

Esta pesquisa foi conduzida para avaliar o efeito de micotoxinas no desempenho de equinos com avaliações fisiológicas e análises hematológicas e da bioquímica sérica. O ensaio durou 40 dias, com 12 dias de adaptação e 28 dias de experimentação. Na fase experimental, os equinos foram distribuídos em delineamento inteiramente casualizado em três tratamentos, com quatro animais cada. Os tratamentos utilizados foram 0 (controle), 50 ppb e 100 ppb de Aflatoxina B1 (AFB1) adicionada ao concentrado de uma dieta basal. A dieta basal continha alimentos naturalmente contaminados por micotoxinas. O teste de desempenho foi executado no 21º dia da fase experimental por meio de teste intervalado consistindo em aquecimento ao trote por 17 minutos, seguido de três galopes de 450m/min. A frequência cardíaca (FC) foi monitorada entre os galopes. Antes do exercício e imediatamente após o terceiro galope, os parâmetros fisiológicos e sanguíneos foram avaliados e continuaram sendo monitorados até 48 horas após o exercício. Os resultados dos parâmetros fisiológicos, hematológicos e bioquímicos foram submetidos à análise de variância (ANOVA) e comparados pelo teste de Tukey a 5% de significância. A presença de AFB1 na dieta influenciou a atividade da fosfatase alcalina, que apresentou valores mais elevadas na dieta com inclusão de 100 ppb de AFB1, sugerindo uma atividade hepatotóxica associada às outras micotoxinas naturalmente presentes nos alimentos.(AU)

Occurrence, antifungal susceptibility, and virulence factors of opportunistic yeasts isolated from Brazilian beaches

BACKGROUND Opportunistic pathogenic yeast species are frequently associated with water habitats that have pollution sources of human or animal origin. Candida albicans has already been suggested as a faecal indicator microorganism for aquatic environments. OBJECTIVES The goal of this study was to investigate the occurrence of C. albicans and other opportunistic yeasts in sand and seawater samples from beaches in Brazil to assess their correlation with Escherichia coli, and to characterise the pathogenic potential of the yeast isolates. METHODS Opportunistic species (yeasts that grow at 37ºC) were isolated from sand and seawater samples from eight beaches in Brazil during the summer and the winter. Opportunistic yeast species were evaluated for their susceptibility to antifungal drugs, virulence factors, and the in vitro and in vivo biofilm formation. Strains were selected to carry out virulence tests using BALB/c mice. FINDINGS Several water samples could be classified as inappropriate for primary contact recreation in relation to E. coli densities. C. albicans was isolated in low densities. Of the 144 opportunistic yeasts evaluated, 61% displayed resistance or dose-dependent sensitivity to at least one tested drug, and 40% produced proteinase. Strains of C. albicans and Kodamaea ohmeri exhibited the highest rates of adhesion to buccal epithelial cells. All the C. albicans strains that were tested were able to undergo morphogenesis and form a biofilm on catheter fragments in both in vitro and in vivo experiments. It was possible to confirm the pathogenic potential of three of these strains during the disseminated infection test. MAIN CONCLUSIONS The identification of opportunistic yeast species in seawater and sand samples from Brazilian beaches suggest a potential risk to the health of people who use these environments for recreational purposes.

Antimycobacterial and antimalarial activities of endophytic fungi associated with the ancient and narrowly endemic neotropical plant Vellozia gigantea from Brazil

BACKGROUND Endophytic fungi, present mainly in the Ascomycota and Basidiomycota phyla, are associated with different plants and represent important producers of bioactive natural products. Brazil has a rich biodiversity of plant species, including those reported as being endemic. Among the endemic Brazilian plant species, Vellozia gigantea (Velloziaceae) is threatened by extinction and is a promising target to recover endophytic fungi. OBJECTIVE The present study focused on bioprospecting of bioactive compounds of the endophytic fungi associated with V. gigantea, an endemic, ancient, and endangered plant species that occurs only in the rupestrian grasslands of Brazil. METHODS The capability of 285 fungal isolates to produce antimicrobial and antimalarial activities was examined. Fungi were grown at solid-state fermentation to recover their crude extracts in dichloromethane. Bioactive extracts were analysed by chromatographic fractionation and NMR and displayed compounds with antimicrobial, antimycobacterial, and antimalarial activities. FINDINGS Five fungi produced antimicrobial and antimalarial compounds. Extracts of Diaporthe miriciae showed antifungal, antibacterial, and antimalarial activities; Trichoderma effusum displayed selective antibacterial activity against methicillin-resistant Staphylococcus aureus and Mycobacterium intracellulare; and three Penicillium species showed antibacterial activity. D. miriciae extract contained highly functionalised secondary metabolites, yielding the compound epoxycytochalasin H with high antimalarial activity against the chloroquine-resistant strain of Plasmodium falciparum, with an IC50 approximately 3.5-fold lower than that with chloroquine. MAIN CONCLUSION Our results indicate that V. gigantea may represent a microhabitat repository hotspot of potential fungi producers of bioactive compounds and suggest that endophytic fungal communities might be an important biological component contributing to the fitness of the plants living in the rupestrian grassland.

Antifungal and antioxidant activity of fatty acid methyl esters from vegetable oils

ABSTRACT Fatty acid methyl esters (FAMEs) were obtained from vegetable oils of soybean, corn and sunflower. The current study was focused on evaluating the antifungal activity of FAMEs mainly against Paracoccidioides spp., as well as testing the interaction of these compounds with commercial antifungal drugs and also their antioxidant potential. FAMEs presented small IC50 values (1.86-9.42 μg/mL). All three FAMEs tested showed antifungal activity against isolates of Paracoccidioides spp. with MIC values ranging from 15.6-500 µg/mL. Sunflower FAMEs exhibited antifungal activity that extended also to other genera, with an MIC of 15.6 μg/mL against Candida glabrata and C. krusei and 31.2 μg/mL against C. parapsilosis. FAMEs exhibited a synergetic effect with itraconazole. The antifungal activity of the FAMEs against isolates of Paracoccidioides spp. is likely due to the presence of methyl linoleate, the major compound present in all three FAMEs. The results obtained indicate the potential of FAMEs as sources for antifungal and antioxidant activity.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

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.

The influence of ripening period length and season on the microbiological parameters of a traditional Brazilian cheese

The ripening process of Serro Minas cheese, one of the most popular cheeses produced with raw milk in Brazil, was studied over the course of 60 days of ripening during dry and rainy seasons. Brazilian legislation prohibits the production of cheese from raw milk unless it was submitted to a maturation period greater than 60 days. However Minas Serro cheese is sold within a few days of ripening. A total of 100 samples of Serro cheese were obtained from five farms; 50 samples were collected during the dry season (winter in Brazil) and 50 samples were collected during the rainy season (summer in Brazil). From each farm, ten cheeses were collected during each season after two days of ripening. Our results showed high levels of total and fecal coliforms at the beginning of the ripening period (approximately 4 Log MPN/g with 3 days of ripening) that decreased with 60 days of ripening reaching almost 1.5 Log MPN/g. Contamination by coagulase-positive staphylococci was reduced by the end of the ripening period. Salmonella spp. was not detected. The staphylococcal enterotoxins B and C were detected in 1% and 4% of the cheeses, respectively, after 30 days of ripening. These results suggest that the ripening process was not effective in eliminating staphylococcal enterotoxins from the cheese. However, none of the investigated strains of Staphylococcus spp. isolated from Serro cheese produced enterotoxins A, B, C or D. The high pathogen and coliform levels at the beginning of the ripening process for the cheese produced during both seasons indicate the need for improvement of the sanitation of the manufacturing conditions.

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.

Water quality and diversity of yeasts from tropical lakes and rivers from the Rio Doce basin in Southeastern Brazil

Yeast communities were assessed in 14 rivers and four lakes from the Doce River basin in Brazil, during the rainy and dry seasons of the years 2000 and 2001. Water samples were collected at the subsurface in all sites. The following physical and chemical parameters were measured: temperature, dissolved oxygen, pH, electrical conductivity, total phosphorus, ortho-phosphate, ammonium, nitrate, nitrite and total nitrogen and the counts of faecal coliforms and heterotrophic bacteria were carried out to characterize the aquatic environmental sampled. The yeast counts were higher in aquatic environments with the highest counts of coliform and heterotrophic bacteria. These environments receive a high influx of domestic and industrial waste. A total of 317 isolates identified in forty eight yeast species were recorded in the sites sampled and the specie Aureobasidium pullulans were found in eleven out of eighteen sites sampled and some opportunistic pathogens such as the yeast species Candida krusei were isolated only in the polluted rivers with a positive correlation with the biotic and abiotic parameters that indicate sewage contamination.

The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7 percent produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4ºC and 20ºC, indicating that they could be metabolically active in the sampled substrates.

Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaça and minas cheese production using culture-independent 16s rRNA gene sequence analysis

We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaça and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6 percent of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8 percent of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaça samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9 percent of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75 percent of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9 percent of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent.

Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil

One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia, Palicourea tetraphylla, Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis, Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2 percent) exhibited at least one biological activity. Seventeen extracts (14 percent) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8 percent) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60 percent and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9 percent) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria, Arthrinium, Cochliobolus, Colletotrichum, Penicillium, Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.

Identification of lactic acid bacteria associated with traditional cachaça fermentations

During the production of traditional cachaça (alembicïs cachaça), contamination of the fermented must is one of the factors leading to economic losses in the beverage manufacturing industry. The diversity of bacterial populations and the role of these microorganisms during the cachaça production process are still poorly understood in Brazil. In our work, the fermentation process was followed in two distilleries located in the state of Minas Gerais. The objective of this work was to identify the populations of lactic acid bacteria present during cachaça fermentation using physiological and molecular methods. Lactic acid bacteria were isolated in high frequencies during all of the fermentative processes, and Lactobacillus plantarum and L. casei were the most prevalent species. Other lactic acid bacteria were found in minor frequencies, such as L. ferintoshensis, L. fermentum, L. jensenii, L. murinus, Lactococcus lactis, Enterococcus sp. and Weissella confusa. These bacteria could contribute to the increase of volatile acidity levels or to the production of compounds that could influence the taste and aroma of the beverage.

Yeast communities in two Atlantic rain Forest fragments in Southeast Brazil / Comunidades de leveduras em dois fragmentos de Mata Atlântica no sudeste do Brasil

We studied the yeast communities associated with fruits, mushrooms, tree exudates, and flies of the genus Drosophila, in two Atlantic Rain Forest fragments in state of Minas Gerais, Brazil. A total of 456 samples were collected from Rio Doce State Park and 142 from Ecological Station of Universidade Federal de Minas Gerais. From these samples, 608 yeast isolates were obtained, belonging to 71 different species. Among the yeasts isolated from Rio Doce State Park, 17 isolates were recovered from fruits, 12 from mushrooms, 13 from tree exudates, and 299 from Drosophila spp. In the Ecological Station of Universidade Federal de Minas Gerais, 24 isolates were recovered from fruits and 243 from Drosophila spp. Distinct communities of yeast were observed in Drosophila flies, fruits, mushrooms and tree exudates. The highest number of yeast species was recovered from Drosophila flies suggesting that flies are the natural vectors of these microorganisms.

O objetivo deste trabalho foi estudar as comunidades de leveduras associadas a frutos, cogumelos, exudatos de árvores e moscas do gênero Drosophila, em dois fragmentos de Mata Atlântica no Estado de Minas Gerais, Brasil. Foram coletadas 456 amostras no Parque Estadual do Rio Doce e 142 na Estação Ecológica da Universidade Federal de Minas Gerais. Destas amostras foram obtidas 608 isolados de levedura, distribuídas em 71 espécies. Entre os isolados obtidos a partir do Parque Estadual do Rio Doce, 17 foram provenientes de frutos, 12 de cogumelos, 13 de exudatos de árvores e 299 de Drosophila spp. A Estação Ecológica da Universidade Federal de Minas Gerais possibilitou a obtenção de 24 isolados de frutos e 243 de Drosophila spp. Foram observadas comunidades distintas de leveduras associadas a Drosophila, frutos, cogumelos e exudatos de árvores. O maior número de espécies foi obtido em drosófilas, sugerindo que estas moscas são vetores naturais destes microrganismos.

Incidence and anatomic localization of oral candidiasis in patients with AIDS hospitalized in a public hospital in Belo Horizonte, MG, Brazil

The objective of this study was to evaluate the incidence, anatomic localization and yeast species isolated from each clinical type of oral candidiasis. The clinical samples were obtained from 67 patients with AIDS with CD4 cell counts below 200 cells/mm³ and hospitalized in a public hospital (Eduardo de Menezes Hospital) in the city of Belo Horizonte, MG, Brazil. Yeasts were isolated using Chromagar® Candida. The results show that 50.7 percent of these patients had oral candidiasis. The pseudomembranous form was the most frequent clinical manifestation of oral candidiasis, followed by the erythematous and angular cheilite forms. The most common site of these clinical forms of oral candidiasis was the tongue. Candida albicans was the most common yeast species isolated from the lesions. However, other species were also found to be associated with these forms of oral candidiasis.

Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays

Probiotics are viable defined microorganisms (bacteria or yeasts) that exert a beneficial effect on the health of the host when ingested in adequate amounts. Screening for such biotherapeutic agents is commonly performed by in vitro assays simulating gastrointestinal environment to determine the ability to survive in the digestive tract. In the present study, the possibility of extrapolation of data obtained in in vitro assays to in vivo conditions was studied using five Saccharomyces cerevisiae strains isolated from Brazilian Atlantic rain forest. Trehalose contents and survival after exposure to a combination of physiological stresses generally found in the gastrointestinal tract of humans were determined for the five yeasts and compared to the behavior of Saccharomyces boulardii, a well-known probiotic. The results were completed with the colonization capacity of the gastrointestinal tract of gnotobiotic mice by these yeast strains. Some results obtained by in vitro assays are not confirmed by in vivo experiments, indicating that the extrapolation cannot be always done.

Probióticos são definidos como microrganismos (bactérias e leveduras) que exercem um efeito benéfico na saúde do hospedeiro quando ingeridos em quantidades adequadas. A seleção desses agentes bioterapêuticos normalmente é feita por testes in vitro simulando o ambiente gastrointestinal que determina a capacidade de sobrevivência no trato digestivo. Neste trabalho, a possibilidade de extrapolação dos dados obtidos nos testes in vitro para as condições in vivo foi estudada utilizando cinco linhagens de Saccharomyces cerevisiae isoladas da floresta Atlântica brasileira. O conteúdo de trealose e a sobrevivência após a exposição a diversos estresses fisiológicos geralmente encontrados no trato gastrointestinal de humanos foram determinados para as cinco linhagens e os resultados comparados com a Saccharomyces boulardii, um probiótico conhecido. Esses resultados foram completados com a capacidade de colonização do trato gastrointestinal de camundongos gnotobióticos pelas leveduras. Pelos resultados obtidos, concluimos que os testes in vitro não são confirmados pelos ensaios in vivo, indicando que essa extrapolação não pode sempre ser feita.

Biological control of Penicillium italicum, P. digitatum and P. expansum by the predacious yeast Saccharomycopsis schoenii on oranges

In this study we evaluated the ability of Saccharomycopsis schoenii Nadson and Krassiln (UWO-PS 80-91) as biocontrol agent against plant pathogenic filamentous fungi P. expansum Link (UFMG 01-2002), P. italicum Wehmer (LCP 61.1199), and P. digitatum (Pers.: Fr.) (LCP 984263, LCP 68175 and LCP 4354). S. schoenii was able to reduce disease severity in oranges inoculated with all fungi. Among the phytopathogens, P. digitatum LCP4354 was the most virulent whereas P. digitatum LCP 68175 was the most susceptible to predation. The yeast was able to survive for 21 days on the fruit surface and did not produce lesions on oranges. Production of antagonistic substances by S. schoenii was not detected using standard techniques. Our results point to the potential use of S. schoenii to control postharvest phytopathogens in fruits.

Este estudo avaliou a capacidade de levedura Saccharomycopsis schoenii Nadson & Krassiln (UWO-PS 80-91) em controlar o crescimento dos fungos fitopatogênicos Penicillium expansum Link (UFMG 01-2002), P. italicum Wehmer (LCP 61.1199), e P. digitatum (Pers.: Fr.) (LCP 984263, LCP 68175 e LCP 4354). S. schoenii reduziu a severidade da doença em laranjas inoculadas com todos os fitopatógenos testados. Entre estes fitopatógenos, P. digitatum LCP4354 apresentou a maior virulência enquanto que P. digitatum LCP 68175 foi o mais suscetível à predação. A levedura foi capaz de permanecer viável, sem produzir lesões na superfície dos frutos por 21 dias. Outra característica desejável observada foi a ausência de produção de substâncias antagonistas. Sendo assim, este trabalho evidência o potencial de utilização da levedura S. schoenii em protocolos de controle biológico de doenças pós-colheita em laranjas.

Genes associated with pathogenicity of avian Escherichia coli (APEC) isolated from respiratory cases of poultry / Genes associados à patogenicidade de Escherichia coli patogênica para aves (APEC) isoladas de frangos de corte com sintomatologia clínica respiratória

The virulence mechanisms of avian pathogenic Escherichia coli (APEC) have been continually studied and are believed to be multi-factorial. Certain properties are primarily associated with virulent samples and have been identified in avian isolates. In this study a total of 61 E. coli, isolates from chicken flocks with respiratory symptomatology, were probed by Polimerase Chain Reation (PCR) for the presence of genes responsible for the adhesion capacity, P fimbria (papC) e F11 fimbria (felA), colicin production (cvaC), aerobactin presence (iutA), serum resistance (iss), temperature-sensitive hemagglutinin (tsh), and presence of K1 and K5 capsular antigens (kpsII). The iss gene was detected in 73,8 percent, tsh in 55,7 percent, iutA in 45,9 percent, felA in 39,3 percent, papC in 24,3 percent, cvaC in 23 percent and kpsII in18 percent.

Os mecanismos de virulência das amostras de Escherichia coli potencialmente patogênicas para aves (APEC) têm sido continuamente estudados e acredita-se ser multifatorial. Certas propriedades são associadas primariamente a amostras virulentas e vêm sendo identificadas em amostras de E. coli isoladas de aves. Neste estudo um total de 61 amostras de E. coli, isoladas de frangos de corte com problemas respiratórios, foram testadas através da Reação em Cadeia da Polimerase (PCR), para a presença dos genes responsáveis pela capacidade de adesão, fimbria P (papC) e fimbria F11 (felA), produção de colicinas (cvaC), presença de aerobactina (iutA), resistência sérica (iss), hemaglutinina temperatura sensível (tsh) e presença de dos antígenos capsulares K1 e K5 (kpsII). O gene iss foi detectado em 73,8 por cento, tsh em 55,7 por cento, iutA em 45,9 por cento, felA em 39,3 por cento, papC em 24,3 por cento, cvaC em 23 por cento e kpsII em 18 por cento.

Monitoring Saccharomyces cerevisiae populations by mtDNA restriction analysis and other molecular typing methods during spontaneous fermentation for production of the artisanal cachaça

An ecological study on Saccharomyces cerevisiae populations in spontaneous fermentation has been conducted in three vats of a cachaça distillery in Minas Gerais, Brazil. Ninety-seven yeast isolates were collected at the beginning, the middle and at the end of the production period, and were identified by standard methods. Differentiation between the indigenous S. cerevisiae strains isolated was performed by mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR, and PCR fingerprint using an intron splice primer. Analysis of the mtDNA restriction profiles revealed 12 different patterns, 11 corresponding to indigenous yeasts (I to XI) and one (XII) to a commercial strain of the bakery yeast. Pattern II (53.6 percent of the population) and pattern IV strains were present in all the vats. Pattern IV strain raised from the middle to the end of the period reaching proportions near those of pattern II strain. PCR methods allowed the differentiation of 41 molecular profiles. Both methods showed population fluctuation of S. cerevisiae strains along the period of cachaça production and among different vats of the distillery.

Um estudo ecológico das populações de Saccharomyces cerevisiae em fermentações espontâneas foi conduzido em três dornas de uma destilaria de cachaça em Minas Gerais, Brasil. Noventa e sete isolados foram coletados no início, meio e final do período de produção, e identificados por métodos padrões. A diferenciação entre as linhagens isoladas de S. cerevisiae indígenas foi feita pela analise de restrição do DNA mitocondrial (mtDNA), RAPD-PCR, e PCR por impressão digital do DNA utilizando um iniciador complementar a sítios de processamento de íntron. As análises dos perfis de restrição do mtDNA mostraram a ocorrência de 12 perfis diferentes, sendo 11 correspondentes as leveduras indígenas (I ao XI) e um (XII) a uma linhagem comercial de levedura de panificação. Linhagens com o perfil II (53,6 por cento da população) e o perfil IV estiveram presentes em todas as dornas. A linhagem com perfil IV aumentou do meio para o final do período de fermentação, alcançando proporções próximas a aquelas encontradas para a linhagem com o perfil II. Os métodos baseados em PCR permitiram a diferenciação de 41 perfis moleculares. Ambos os métodos mostraram flutuações populacionais nas linhagens de S. cerevisiae durante o período de produção da cachaça e entre as diferentes dornas da destilaria.