Antifungal effect of (R) and (S)-citronellal enantiomers and their predictive mechanism of action on Candida albicans from voriconazole-resistant onychomycoses / Efeito antifúngico dos enantiômeros (R) e (S)-citronelal e seu mecanismo de ação preditivo sobre Candida albicans de onicomicoses resistentes ao voriconazol

Onychomycosis is the most common disease affecting the nail unit and accounts for at least 50% of all nail diseases. In addition, Candida albicans is responsible for approximately 70% of onychomycoses caused by yeasts. This study investigated the antifungal effect of (R) and (S)-citronellal enantiomers, as well as its predictive mechanism of action on C. albicans from voriconazole-resistant onychomycoses. For this purpose, in vitro broth microdilution and molecular docking techniques were applied in a predictive and complementary manner to the mechanisms of action. The main results of this study indicate that C. albicans was resistant to voriconazole and sensitive to the enantiomers (R) and (S)-citronellal at a dose of 256 and 32 µg/mL respectively. In addition, there was an increase in the minimum inhibitory concentration (MIC) of the enantiomers in the presence of sorbitol and ergosterol, indicating that these molecules possibly affect the integrity of the cell wall and cell membrane of C. albicans. Molecular docking with key biosynthesis proteins and maintenance of the fungal cell wall and plasma membrane demonstrated the possibility of (R) and (S)-citronellal interacting with two important enzymes: 1,3-ß-glucan synthase and lanosterol 14α-demethylase. Therefore, the findings of this study indicate that the (R) and (S)-citronellal enantiomers are fungicidal on C. albicans from onychomycoses and probably these substances cause damage to the cell wall and cell membrane of these micro-organisms possibly by interacting with enzymes in the biosynthesis of these fungal structures.

A onicomicose é a doença mais comum que afeta a unidade ungueal e representa pelo menos 50% de todas as doenças ungueais. Além disso, a Candida albicans é responsável por aproximadamente 70% das onicomicoses causadas por leveduras. Nesse estudo, foi investigado o efeito antifúngico dos enantiômeros (R) e (S)-citronelal, bem como seu mecanismo de ação preditivo sobre C. albicans de onicomicoses resistentes ao voriconazol. Para este propósito, foram aplicadas técnicas in vitro de microdiluição em caldo e docking molecular de forma preditiva e complementar para os mecanismos de ação. Os principais resultados deste estudo indicam que C. albicans foi resistente ao voriconazol e sensível aos enantiômeros (R) e (S)-citronelal na dose de 256 e 32 µg/mL respectivamente. Além disso, houve aumento da concentração inibitória mínima (CIM) dos enantiômeros na presença do sorbitol e do ergosterol, indicando que estas moléculas possivelmente afetem a integridade da parede e da membrana celular de C. albicans. O docking molecular com proteínas chave da biossíntese e manutenção da parede celular e da membrana plasmática fúngica, demonstraram a possibilidade do (R) e (S)-citronelal interagir com duas importantes enzimas: 1,3-ß-glucan sintase e lanosterol 14α-demetilase. Portanto, os achados desse estudo indicam que os enantiômeros (R) e (S)-citronelal são fungicidas sobre C. albicans de onicomicoses e provavelmente essas substâncias causem danos a parede e a membrana celular desses microrganismos possivelmente por interagir com as enzimas da biossíntese destas estruturas fúngicas.

Use of different adsorbents in broiler diets naturally contaminated by mycotoxins

This study investigated the effects of adding different adsorbent substances to broilers feed naturally contaminated by mycotoxins. Two hundred and eighty male 1-day-old chicks, Cobb Slow® lineage, were distributed in a randomized block design with 4 treatments, 5 repetitions with 14 birds each. The treatments consisted of: T1-basal feed naturally contaminated with mycotoxins. T2-basal feed + Bentonite, Thistle Extract, Yeast Extract, Vitamin E and Choline. T3-basal feed + Bentonite, Thistle Extract, yeast cell wall and Silymarin. T4-basal feed + Bentonite and Algae extract. Performance (weight gain, feed intake, feed conversion) at 7, 14, 21, 28 days were evaluated. At 28 days, a portion of the jejunum was collected in two birds by replicate to study the intestinal morphology. The relative weight of the gizzard, proventricle and total intestine was evaluated. The data obtained were analyzed using the statistical program SAS (9.3). With the use of any adsorbents studied, the performance and liver weight were improved in all evaluated periods. Thus, the inclusion of adsorbents improves the performance of the broiler chickens when the feed is contaminated by mycotoxins.(AU)

Systemic phaeohyphomycosis in a dog caused by Cladophialophora bantiana

Background: Cladophialophora bantiana is a dematiaceous fungus that causes phaeohyphomycosis, a generic term used to describe a variety of unusual mycoses caused by fungi that have melanin in their cell wall. C. bantiana targets the central nervous system, commonly causing localized brain infections that may result in disseminated infections. In Brazil, minimal phaeohyphomycosis data are available, and information about C. bantiana infections in animals, especially canines, is scarce. Thus, the aim of this study was to describe the clinical and pathological aspects of systemic phaeohyphomycosis caused by C. bantiana in a dog. Case: A 1-year-old female Pit Bull presented with weight loss, reduced appetite, and a history of cutaneous lesions on the right thoracic limb; however, clinical evolution was not reported. The bitch had reportedly given birth recently. Physical examination revealed thinness, pale ocular and oral mucosa, submandibular lymph nodes, and enlarged popliteal lymph nodes. The bitch died after convulsive crises during hospitalization. At necropsy, white-yellowish multifocal nodules were observed in the liver and right kidney. The brain featured left cerebral hemisphere asymmetry with blood vessel congestion in the leptomeninges and an irregular brownish focal area on the surface of the right occipital cortex. Cross-sections of the formalin-fi xed brain exhibited compression of the left lateral ventricle and the presence of grayish and friable multifocal areas in the gray matter of the left parietal and right occipital cortices. Fragments of the lesions were collected for histopathological and microbiological examination. Histologically, the lesions were similar, characterized by hepatitis, nephritis, and granulomatous and necrotizing...

Antiadhesion and antibiofilm potential of Fagonia indica from Cholistan desert against clinical multidrug resistant bacteria / Potencial de antiadesão e antibiofilme de Fagonia indica do deserto do Cholistão contra bactérias multirresistentes clínicas

High resistance to antimicrobials is associated with biofilm formation responsible for infectious microbes to withstand severe conditions. Therefore, new alternatives are necessary as biofilm inhibitors to control infections. In this study, the antimicrobial and antibiofilm activities of Fagonia indica extracts were evaluated against MDR clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica has antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against multidrug resistant (MDR) clinical isolates. The extract exhibited its antibiofilm effect by altering adherence and disintegration of bacterial cell wall. Fagonia indica had antibacterial effect as minimum inhibitory concentration (MIC) values ranging from 125 to 500 µg mL-1 and minimum bactericidal concentration (MBC) value was 500-3000 µg mL-1 against MDR isolates. The maximum inhibitory effects of Fagonia indica chloroform extract on biofilm formation was observed on Staphylococcus aureus (71.84%) followed by Klebsiella pneumoniae (70.83%) after 48 hrs showing that inhibition is also time dependent. Our results about bacterial cell protein leakage indicated that MDR isolates treated with chloroform extract of Fagonia indica showed maximum protein leakage of K. pneumoniae (59.14 µg mL-1) followed by S. aureus (56.7 µg mL-1). Cell attachment assays indicated that chloroform extract resulted in a 43.5-53.5% inhibition of cell adherence to a polystyrene surface. Our results revealed that extracts of Fagonia indica significantly inhibited biofilm formation among MDR clinical isolates, therefore, could be applied as antimicrobial agents and cost effective biofilm inhibitor against these MDR isolates.

A alta resistência aos antimicrobianos está associada à formação de biofilme responsável por micróbios infecciosos para suportar condições severas. Portanto, novas alternativas são necessárias como inibidores de biofilme para controlar infecções. Neste estudo, as atividades antimicrobiana e antibiofilme dos extratos de Fagonia indica foram avaliadas contra isolados clínicos MDR. O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica tem efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-1, e valor de concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados clínicos multirresistentes (MDR). O extrato exibiu seu efeito antibiofilme ao alterar a aderência e a desintegração da parede celular bacteriana. Fagonia indica teve efeito antibacteriano com valores de concentração inibitória mínima (CIM) variando de 125 a 500 µg mL-1, e concentração bactericida mínima (MBC) de 500-3000 µg mL-1 contra isolados MDR. Os efeitos inibitórios máximos do extrato de clorofórmio Fagonia indica na formação de biofilme foi observada em Staphylococcus aureus (71,84%), seguido por Klebsiella pneumoniae (70,83%) após 48 horas, mostrando que a inibição também é dependente do tempo. Nossos resultados sobre extravasamento de proteínas de células bacterianas indicaram que isolados MDR tratados com extrato clorofórmico de Fagonia indica apresentaram vazamento máximo de proteínas de K. pneumoniae (59,14 µg mL-1), seguido por S. aureus (56,7 µg mL-1). Ensaios de fixação de células indicaram que o extrato de clorofórmio resultou em uma inibição de 43,5-53,5% da aderência das células a uma superfície de poliestireno. Nossos resultados revelaram que extratos de Fagonia indica inibiram significativamente a formação de biofilme entre isolados clínicos MDR, portanto, poderiam ser aplicados como agentes antimicrobianos e inibidores de biofilme de baixo custo contra esses isolados MDR.

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides

Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. Methods: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. Results: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). Conclusion: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.(AU)

Antifungal activity of liriodenine on agents of systemic mycoses, with emphasis on the genus Paracoccidioides

Endemic systemic mycoses remain a health challenge, since these opportunistic diseases are increasingly infecting immunosuppressed patients. The simultaneous use of antifungal compounds and other drugs to treat infectious or non-infectious diseases has led to several interactions and undesirable effects. Thus, new antifungal compounds should be investigated. The present study aimed to evaluate the activity of liriodenine extracted from Annona macroprophyllata on agents of systemic mycoses, with emphasis on the genus Paracoccidioides. Methods: The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined by the microdilution method. The cellular alterations caused by liriodenine on a standard P. brasiliensis (Pb18) strain were evaluated by transmission and scanning electron microscopy. Results: Liriodenine was effective only in 3 of the 8 strains of the genus Paracoccidioides and in the Histoplasma capsulatum strain, in a very low concentration (MIC of 1.95 µg.mL-1); on yeasts of Candida spp. (MIC of 125 to 250 µg.mL-1), including C. krusei (250 µg.mL-1), which has intrinsic resistance to fluconazole; and in Cryptococcus neoformans and Cryptococcus gattii (MIC of 62.5 µg.mL-1). However, liriodenine was not effective against Aspergillus fumigatus at the studied concentrations. Liriodenine exhibited fungicidal activity against all standard strains and clinical isolates that showed to be susceptible by in vitro tests. Electron microscopy revealed cytoplasmic alterations and damage to the cell wall of P. brasiliensis (Pb18). Conclusion: Our results indicate that liriodenine is a promising fungicidal compound that should undergo further investigation with some chemical modifications.(AU)

A new coumarin derivative, 4-acetatecoumarin, with antifungal activity and association study against Aspergillus spp.

Abstract Fungal infections have become a concern for health professionals, and the emergence of resistant strains has been reported for all known classes of antifungal drugs. Among the fungi causing disease, we highlight those that belong to the genus Aspergillus. For these reasons, the search for new antifungals is important. This study examines the effects of a coumarin derivative, 4-acetatecoumarin (Cou-UMB16) both alone and together with antifungal drugs, and its mode of action against Aspergillus spp. Cou-UMB16 was tested to evaluate its effects on mycelia growth, and germination of Aspergillus spp. fungal conidia. We investigated its possible action on cell walls, on the cell membrane, and also the capacity of this coumarin derivative to enhance the activity of antifungal drugs. Our results suggest that Cou-UMB16 inhibits Aspergillus spp. virulence factors (mycelia growth and germination of conidia) and affects the structure of the fungal cell wall. When applying Cou-UMB16 in combination with azoles, both synergistic and additive effects were observed. This study concludes that Cou-UMB16 inhibits mycelial growth and spore germination, and that the activity is due to its action on the fungal cell wall, and that Cou-UMB16 could act as an antifungal modifier.

A new coumarin derivative, 4-acetatecoumarin, with antifungal activity and association study against Aspergillus spp

Fungal infections have become a concern for health professionals, and the emergence of resistant strains has been reported for all known classes of antifungal drugs. Among the fungi causing disease, we highlight those that belong to the genus Aspergillus. For these reasons, the search for new antifungals is important. This study examines the effects of a coumarin derivative, 4-acetatecoumarin (Cou-UMB16) both alone and together with antifungal drugs, and its mode of action against Aspergillus spp. Cou-UMB16 was tested to evaluate its effects on mycelia growth, and germination of Aspergillus spp. fungal conidia. We investigated its possible action on cell walls, on the cell membrane, and also the capacity of this coumarin derivative to enhance the activity of antifungal drugs. Our results suggest that Cou-UMB16 inhibits Aspergillus spp. virulence factors (mycelia growth and germination of conidia) and affects the structure of the fungal cell wall. When applying Cou-UMB16 in combination with azoles, both synergistic and additive effects were observed. This study concludes that Cou-UMB16 inhibits mycelial growth and spore germination, and that the activity is due to its action on the fungal cell wall, and that Cou-UMB16 could act as an antifungal modifier.(AU)

The protective effects of an adsorbent against oxidative stress in quails fed aflatoxin-contaminated diet

Background: Contamination of crops with aflatoxin is considered a serious global threat to food safety, since potent carcinogenic, teratogenic, mutagenic and immunosuppressive effects of aflatoxins are well recognized. Recently, the use of adsorbents has been linked with protective effects against oxidative stress in several diseases. Thus, the aim of this study was to assess the occurrence of oxidative stress in quails (Coturnix coturnix) fed with aflatoxin-contaminated diet, as well as the protective effect of an adsorbent.Materials, Methods & Results: Twenty-eight quails were divided into four groups (n = 7): diet without additives (control; the group A), diet and adsorbent containing aluminosilicates (the group B), aflatoxin-contaminated diet (200 ppb) (the group C), and aflatoxin-contaminated diet (200 ppb) and adsorbent containing aluminosilicates (the group D). The composition of the adsorbent containing aluminosilicates was 0.3% based on yeast cell wall, silymarin, and bentonite. The animals received feed and water ad libitum during 20 days. At the end of the experimental period, total blood was collected by cardiac puncture in tubes without anticoagulant to obtain serum (centrifuged at 3500 g during 10 min) for later determination of biochemical parameters. The liver was placed in a solution of Tris–HCl 10 mM, pH 7.4 for TBARS (Thiobarbituric acid reactive substances), ROS (Reactive oxygen species), SOD (Superoxide dismutase) and CAT (Catalase) analysis. The hepatic tissue was gently homogenized in a glass potter in specific buffer, homogenated, and centrifuged at 10.000 g at 4ºC for 10 min to yield a supernatant (S1) used for analyses. Homogenate aliquots were stored at -80°C until utilization. Fragments of liver and intestine (5 cm) were collected for histopathological analyses. Between days 15 to 20 of the experiment, group C quails showed clinical signs, such as apathy, creepy feathers and reduced feed intake.[...]

The protective effects of an adsorbent against oxidative stress in quails fed aflatoxin-contaminated diet

Background: Contamination of crops with aflatoxin is considered a serious global threat to food safety, since potent carcinogenic, teratogenic, mutagenic and immunosuppressive effects of aflatoxins are well recognized. Recently, the use of adsorbents has been linked with protective effects against oxidative stress in several diseases. Thus, the aim of this study was to assess the occurrence of oxidative stress in quails (Coturnix coturnix) fed with aflatoxin-contaminated diet, as well as the protective effect of an adsorbent.Materials, Methods & Results: Twenty-eight quails were divided into four groups (n = 7): diet without additives (control; the group A), diet and adsorbent containing aluminosilicates (the group B), aflatoxin-contaminated diet (200 ppb) (the group C), and aflatoxin-contaminated diet (200 ppb) and adsorbent containing aluminosilicates (the group D). The composition of the adsorbent containing aluminosilicates was 0.3% based on yeast cell wall, silymarin, and bentonite. The animals received feed and water ad libitum during 20 days. At the end of the experimental period, total blood was collected by cardiac puncture in tubes without anticoagulant to obtain serum (centrifuged at 3500 g during 10 min) for later determination of biochemical parameters. The liver was placed in a solution of Tris–HCl 10 mM, pH 7.4 for TBARS (Thiobarbituric acid reactive substances), ROS (Reactive oxygen species), SOD (Superoxide dismutase) and CAT (Catalase) analysis. The hepatic tissue was gently homogenized in a glass potter in specific buffer, homogenated, and centrifuged at 10.000 g at 4ºC for 10 min to yield a supernatant (S1) used for analyses. Homogenate aliquots were stored at -80°C until utilization. Fragments of liver and intestine (5 cm) were collected for histopathological analyses. Between days 15 to 20 of the experiment, group C quails showed clinical signs, such as apathy, creepy feathers and reduced feed intake.[...](AU)

The effect of mycotoxins adsorbents beta glucans or montmorillonite on bovine ruminal fermentation in vitro / Impacto dos adsorventes de micotoxinas Beta-glucana ou montmorilonita sobre a fermentação ruminal de bovinos in vitro

Background: The addition of adsorbents in foods has been the strategy used by nutritionists to reduce the toxic effects of mycotoxins in animals. Mycotoxins are found in a range of foods and commonly they present variations in the chemical structure therefore, it has been appropriate to include adsorbents from different sources in the diet of ruminants. However, few researches were conducted in order to better understand the interaction of adsorbents on ruminal fermentation. Our objective in this study was to investigate the possible effects of two adsorbent products on bovine ruminal fermentation. One consists of 65% of -glucan (-glu), originating cell wall of Saccharomyces cerevisiae and used at a concentration of 1% and natural sodium montmorillonite (MMT) at a concentration of 5%. Materials, Methods & Results: The effects of -glu adsorbents (1%) and MMC (5%) in culture medium that simulated ruminal fermentation were evaluated. Bottles, with a capacity of 120 mL, were used to be filled with substrate such as maize and ryegrass hay ground, nutrient solution (medium of Menke), liquid extracted rumen fistulated bovine and the two adsorbents evaluated, totaling 50 mL. The experiment was conducted with three treatments, named after: control (Cont), -glu and MMT. In the control treatment adsorbents were not added. Six replicates were used for each treatment and two trials were conducted. One of the tests aimed to determine the fermentation kinetics by means of the gas production after 72 h incubation and quantifying the production of methane (CH4) at 48h. While another test aimed to quantify the production of short chain fatty acids (SCFA) - acetic, propionic and butyric acid - and the production of ammonia (NH3) in 24 h incubation. [...](AU)

The effect of mycotoxins adsorbents beta glucans or montmorillonite on bovine ruminal fermentation in vitro / Impacto dos adsorventes de micotoxinas Beta-glucana ou montmorilonita sobre a fermentação ruminal de bovinos in vitro

Background: The addition of adsorbents in foods has been the strategy used by nutritionists to reduce the toxic effects of mycotoxins in animals. Mycotoxins are found in a range of foods and commonly they present variations in the chemical structure therefore, it has been appropriate to include adsorbents from different sources in the diet of ruminants. However, few researches were conducted in order to better understand the interaction of adsorbents on ruminal fermentation. Our objective in this study was to investigate the possible effects of two adsorbent products on bovine ruminal fermentation. One consists of 65% of -glucan (-glu), originating cell wall of Saccharomyces cerevisiae and used at a concentration of 1% and natural sodium montmorillonite (MMT) at a concentration of 5%. Materials, Methods & Results: The effects of -glu adsorbents (1%) and MMC (5%) in culture medium that simulated ruminal fermentation were evaluated. Bottles, with a capacity of 120 mL, were used to be filled with substrate such as maize and ryegrass hay ground, nutrient solution (medium of Menke), liquid extracted rumen fistulated bovine and the two adsorbents evaluated, totaling 50 mL. The experiment was conducted with three treatments, named after: control (Cont), -glu and MMT. In the control treatment adsorbents were not added. Six replicates were used for each treatment and two trials were conducted. One of the tests aimed to determine the fermentation kinetics by means of the gas production after 72 h incubation and quantifying the production of methane (CH4) at 48h. While another test aimed to quantify the production of short chain fatty acids (SCFA) - acetic, propionic and butyric acid - and the production of ammonia (NH3) in 24 h incubation. [...]

A quitosana como fungistático no crescimento micelial de Rhizoctonia solani Kuhn / Chitosan as fungistatic mycelial growth of Rhizoctonia solani Kuhn

Rhizoctonia solani é um fungo causador de tombamento de plântulas em várias espécies vegetais. A quitosana é um polímero derivado do processo de desacetilação da quitina, a qual é encontrada em grande quantidade na carapaça de crustáceos, insetos e parede celular de fungos. A quitosana tem sido testada para diversos usos, inclusive no controle de fitopatógenos em agricultura, já que apresenta atividade antimicrobiana, para controle de patógenos. O presente trabalho teve como objetivo avaliar o efeito fungistático de diferentes concentrações de quitosana (0; 0,25; 0,5; 1 e 2%) no crescimento micelial do fungo R. solani in vitro. Os resultados obtidos demonstraram efeito significativo de quitosana nas diferentes concentrações utilizadas, na redução do crescimento micelial de R. solani. Observou-se também aumento do efeito fungistático da quitosana conforme o aumento da dose.(AU)

Rhizoctonia solani is a fungus that causes damping-off of seedlings in various plant species. Chitosan is a polymer derived from the process of desacetylation of chitin, which is found in large quantities in the exoskeleton of crustaceans, insects and fungal cell wall. Chitosan has been tested for various uses, including the control of plant pathogens in agriculture, since it presents antimicrobial activity to control pathogens. This study aimed to evaluate the fungistatic effect of different chitosan concentrations (0; 0.25; 0.5; 1 and 2%) in mycelial growth in vitro of the fungus R. solani. The results showed a significant effect of different concentrations of chitosan, in reduccing the mycelial growth of R. solani. It was also observed increased fungistatic effect with increasing of the concentration.(AU)

DIGESTIBILIDADE E BALANÇO DE NITROGÊNIO EM CORDEIROS SUPLEMENTADOS COM CEPAS FÚNGICAS AUTÓCTONES DO TRATO DIGESTÓRIO

As gramíneas tropicais no período de seca reduzem a oferta de nutrientes solúveis no conteúdo celular e possuem maior proporção de carboidratos fibrosos da parede celular lignificada. A utilização de aditivos microbianos na alimentação de ruminantes, com o propósito de estimular a digestão no rúmen e a eficiência alimentar poderia contribuir para melhorar a produtividade dos ruminantes alimentados com essas gramíneas. Nesta pesquisa, objetivou-se avaliar os efeitos da suplementação cepas autóctones de Trichoderma longibrachiatum e Rhodotorula mucilaginosa do trato gastrointestinal de ovinos, em dieta para cordeiros alimentados com feno de Urochloa decumbens sobre o consumo, a digestibilidade de nutrientes, a síntese de proteína microbiana, o balanço de nitrogênio e excreção de ureia. O experimento teve duração de 75 dias, sendo os 12 primeiros dias de adaptação às dietas e baias e 63 dias de coleta de dados. Foram utilizados 21 ovinos, mestiços da raça Santa Inês x Dorper, machos, não castrados com idade média de quatro meses e peso corporal médio inicial de (18,80 ± 2,34 kg), distribuídos aleatoriamente em um delineamento inteiramente casualizado (DIC). A relação volumoso concentrado foi 30:70, foi utilizada uma dieta padrão suplementada ou não com a cepa fúngica; sem aditivo microbiano (controle); com o fungo (Trichoderma); e levedura (Rhodotorula). O consumo e digestibilidade de nutrientes, a síntese de proteína microbiana e a retenção de nitrogênio corporal não foram influenciados pela adição dos probióticos (P > 0,05). A eficiência microbiana foi menor (P < 0,05) com o uso da levedura Rhodotorula mucilaginosa. A alta proporção de concentrado na dieta poderia interferir nos possíveis efeitos dos probióticos sobre a utilização da fibra da forragem.

In the dry season, tropical grasses decrease the supply of soluble nutrients in cell content and have a higher proportion of lignified cell wall fibrous carbohydrates. The use of microbial feed additives in ruminants to stimulate rumen digestion and feed efficiency could play the role of improving the productivity of ruminants fed on these grasses. The goal of this study was to evaluate the effects of supplementing autochthonous strains of Trichoderma longibrachiatum and Rhodotorula mucilaginosa from the gastrointestinal tract of sheep on diets fed with Urochloa decumbens hay(on intake), nutrient digestibility, microbial protein synthesis, diet. nitrogen balance and urea excretion. The experiment lasted 75 days, with the first 12 days of adaptation to diets and stalls and 63 days of data collection. Twenty-one male Santa Inês x Dorper crossbred sheep, male, uncastrated, with an average age of four months and initial body weight (18.80 ± 2.34 kg) were randomly distributed in a completely randomized design (IHD). Concentrated forage ratio was 30:70, a standard diet was used supplemented or not with the fungal strain; without microbial additive (control); with the fungus (Trichoderma); and yeast (Rhodotorula). Nutrient intake, digestibility, microbial protein synthesis, and body nitrogen retention were not influenced by adding probiotics (P> 0.05). Microbial efficiency was lower (P <0.05) with the use of Rhodotorula mucilaginosa yeast. High proportions of concentrate in the diet could interfere with the probable effects of probiotics on forage fiber utilization.

Características histomorfológicas e histoquímicas determinantes no diagnóstico da criptococose em animais de companhia / Determining histomorphological and histochemical characteristics in the diagnosis of cryptococosis in companion animals

Seven cases of cryptococosis (six cats and one dog) were studied to establish the determining histomorphological and histochemical characteristics in the histopathological diagnosis of this condition. Additional data related to the epidemiology, clinical aspects, sites of the lesions, and gross findings were obtained from the necropsy and biopsy protocols. Histologically, yeasts were observed inside macrophages or free in the parenchyma, associated with scarse to severe lymphohistioplasmacytic inflammatory reaction. In the hematoxylin-eosin (HE) sections, the yeasts were round, with a central cell containing a nucleus, surrounded by a clear halo (usually non-stained capsule). The techniques of periodic Schiff acid (PAS), Groccot (GMS), and Fontana-Masson (FM) were utilized and demonstrated the wall of the yeast cells. The FM stain showed the melanin present in these cells. The Alcian blue and Mayer's mucicarmin stains showed mainly the yeast polysaccharide capsule. The diameter of the cells ranged from 1.67 to 10.00µm and the full diameter of the encapsulated yeasts varied between 4.17 e 34.16µm. Yeast buddings were better observed through the PAS stain and were narrow based, simple or multiple, mainly in the opposite poles of the cells or forming chains. The definitive diagnosis of cryptococosis was established through the histopathological exam, based on the specific morphology of the agent (encapsulated yeast) and on histochemical proprieties, mostly in the cases without fungal culture.(AU)

Sete casos de criptococose (seis gatos e um cão) foram estudados para estabelecer as características histomorfológicas e histoquímicas determinantes no diagnóstico histopatológico dessa condição. Os dados complementares relacionados à epidemiologia, aos aspectos clínicos, à localização das lesões e às alterações macroscópicas foram obtidos dos protocolos de necropsias e biópsias. Na histologia, as leveduras foram observadas no interior de macrófagos ou livres no parênquima, associadas à reação inflamatória linfo-histioplasmocítica que variou de escassa a acentuada. Pela técnica de hematoxilina-eosina (HE) as leveduras eram arredondadas, com célula central contendo um núcleo, circundada por um halo claro (cápsula geralmente não corada). As técnicas histoquímicas do ácido periódico de Schiff (PAS), Grocott e Fontana-Masson (FM) foram utilizadas e evidenciaram a parede das células das leveduras. Pelo FM observou-se a melanina presente nessas células. As técnicas do azul Alciano e da mucicarmina de Mayer evidenciaram principalmente a cápsula polissacarídica das leveduras. O diâmetro das células das leveduras variou de 1,67 a 10,00µm e o diâmetro total das leveduras encapsuladas variou entre 4,17 e 34,16µm. Os brotamentos foram melhor visualizados através do PAS e ocorreram em base estreita, de forma única ou múltipla, principalmente em polos opostos das células das leveduras ou formando uma cadeia. O diagnóstico definitivo de criptococose foi estabelecido através do exame histopatológico, baseando-se na morfologia característica do agente (levedura encapsulada) e em suas propriedades tintoriais (histoquímicas), principalmente nos casos em que a cultura micológica não foi realizada.(AU)

Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels

Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE), in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase) using a novel substrate, Banana Peels (BP) for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

Identification of genes expressed by Cryptococcus gattii during iron deprivation

Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In the literature, the competition for iron between microbes and mammalian hosts during infection is well documented. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation. A total of 15 and 29 genes were identified as having altered expression levels due to iron depletion after 3 h and 12 h, respectively. Of these, eight genes were identified in both libraries. The transcripts were related to many biological processes, such as cell cycle, ergosterol metabolism, cell wall organization, transportation, translation, cell respiration and the stress response. These data suggest a remodeling of C. gattii metabolism during conditions of iron deprivation.

Uso promissor do óleo essencial de Rosmarinus officinalis L. e Origanum majorana L. na esporotricose cutânea experimental por Sporothrix brasiliensis resistente ao itraconazol, e susceptibilidade de isolados aos óleos vegetais

O itraconazol é o fármaco de eleição no tratamento da esporotricose humana e animal, entretanto, o surgimento de Sporothrix brasiliensis com resistência in vitro tem incentivado os estudos com plantas da família Lamiaceae como fontes promissoras de moléculas antifúngicas. Dessa família, os óleos essenciais de alecrim (Rosmarinus officinalis L.) e manjerona (Origanum majorana L.) têm se destacados pela atividade antifúngica e baixa citotoxicidade. Essa tese objetivou avaliar a atividade dos óleos essenciais de plantas da família Lamiaceae em isolados clínicos de Sporothrix spp. e na esporotricose cutânea experimental por S. brasiliensis resistente ao itraconazol. Quarenta ratos Wistar adultos machos foram inoculados via subcutânea no coxim plantar esquerdo (2×105 células/mL) e, dez dias após, receberam tratamento oral (1 mL) por 30 dias nos seguintes grupos experimentais (n=10): controle com solução salina com propilenoglicol 1% (CONT); itraconazol a 10 mg/kg (ITRA); óleos essenciais de alecrim a 90 mg/ml (ALEC) e manjerona a 30 mg/ml (MANJ). Semanalmente, a evolução clínica era acompanhada e a eutanásia realizada para histopatologia e contagem fúngica (retro-isolamento). Ainda, os óleos foram avaliados por cromatografia gasosa com espectrometria de massas, e o mecanismo de ação pelos ensaios de proteção do sorbitol e efeito do ergosterol. Os resultados revelaram que os animais dos grupos ALEC e MANJ apresentaram remissão dos sinais clínicos, com ausência de edema e exsudato no membro inoculado, ao passo que CONT e ITRA mantiveram os sinais da doença. Ainda, a disseminação do S. brasiliensis para os órgãos foi menos observada no fígado, baço, linfonodos, rins e testículos dos grupos ALEC e MANJ em comparação ao CONT e ITRA (P < 0.05), revelando uma proteção sistêmica a partir da infecção subcutânea. Essa observação foi relacionada com a composição química, cujos compostos prevalentes foram terpineol-4 (34,09%) e -terpineno (14,28%) em manjerona, e 1,8-cineol (47,91%) e cânfora (12,47%) em alecrim. Não houve alteração nos valores da concentração inibitória mínima (CIM) quando adicionado o protetor osmótico, demonstrando ausência de atividade à nível de parede celular, ao passo que o uso do ergosterol elevou os valores de CIM, revelando que ambos óleos apresentaram atividade a nível de complexação com o ergosterol. Esse estudo demonstrou a eficácia do uso oral de alecrim e manjerona no tratamento da esporotricose cutânea experimental, sendo promissores como antifúngicos. Palavras-ch

Itraconazole is the drug of choice for the treatment of sporotrichosis, however, the emergence of Sporothrix brasiliensis with in vitro resistance has encouraged studies with plants of the family Lamiaceae as promising sources of antifungal molecules. From this family, essential oils of rosemary (Rosmarinus officinalis L.) and marjoram (Origanum majorana L.) have been highlighted for their antifungal activities and low cytotoxicity. This thesis aimed to evaluate the effectiveness of the essential oils of the plants from Lamiaceae family in clinical isolates of Sporothrix spp. and in the experimental cutaneous sporotrichosis by itraconazole-resistant S. brasiliensis. Forty male adult Wistar rats were inoculated subcutaneously in the left footpad (2×105 cells/mL) and, ten days later, received oral treatment (1 mL) for 30 days in the following experimental groups (n=10): control with solution saline with 1% propylene glycol (CONT); itraconazole at 10 mg/kg (ITRA); essential oils of rosemary at 90 mg/mL (ROSM) and marjoram at 30 mg/mL (MARJ). The clinical follow-up was done weekly, as well as the euthanasia for histopathology and fungal burden (retro-isolation). Furthermore, the oils were evaluated by gas chromatography with mass spectrometry, and the mechanism of action by the assays of sorbitol protection and ergosterol effect. The results showed that the animals from ALEC and MANJ groups presented remission of the clinical signs, with no edema and exudate in the inoculated limb, whereas CONT and ITRA maintained the signs of the disease. Also, the dissemination of S. brasiliensis to the organs was less observed in the liver, spleen, lymph nodes, kidneys and testicles of the ALEC and MANJ groups compared to the CONT and ITRA (P <0.05), revealing systemic protection from the subcutaneous infection. This observation was related to the chemical composition, whose prevalent compounds were terpineol-4 (34.09%) and -terpinene (14.28%) in marjoram, and 1,8-cineole (47.91%) and camphor (12.47%) in rosemary. There was no change in minimum inhibitory concentration (MIC) values when the osmotic protector was added, showing an absence of activity at the cell wall level, whereas the use of ergosterol increased MIC values, revealing that both oils presented activity at the level of complexion with ergosterol. This study demonstrated the effectiveness of the oral use of rosemary and marjoram in the treatment of experimental cutaneous sporotrichosis, being promising as antifungal.

Antibacterial activity of alfa-terpineol may induce morphostructural alterations in Escherichia coli

The antibacterial effect of α-terpineol from Cinnamomum longepaniculatum (Gamble) N. Chao leaf essential oils were studied with special reference to the mechanism of inhibiting the standard strain of Escherichia coli (CMCC (B) 44102) growth at ultrastructural level. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) and time-kill curves of α-terpineol were determined; Escherichia coli was treated with α-terpineol and observed under a transmission electron microscope. The MIC and MBC values of α-terpineol were all 0.78 µL/mL, and time-kill curves showed the concentration-dependent. Under the transmission electron microscopy (TEM), Escherichia coli exposed to MIC levels of α-terpineol exhibited decreased cell size and irregular cell shape, cell wall and cell membrane were ruptured, nucleus cytoplasm was reduced and nuclear area gathered aside. Results suggest that α-terpineol has excellent antibacterial activity and could induce morphological changes of Escherichia coli.

Screening of thermotolerant and thermophilic fungi aiming Beta-xylosidase and arabinanase production

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of β-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 ºC, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 ºC. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of β-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, β-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 ºC), while the correspondent extracellular activities were favorably secreted from cultures at 30 ºC. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.