Infección por Aspergillus flavus y Rhizopus oryzae complex en paciente con diabetes mellitus / Infection due to Aspergillus flavus and Rhizopus oryzae complex in a patient with diabetes mellitus

La sinusitis micótica es una condición patológica que puede presentarse en pacientes con diabetes mellitus y estar asociada a una crisis hiperglucémica. Es una entidad agresiva con complicaciones locales que incluyen afectación de la órbita y el sistema nervioso central, y compromiso vascular. A pesar del tratamiento quirúrgico y antimicótico, la mortalidad es de hasta el 75 %. Se describe el caso de una paciente con diagnóstico de cetoacidosis diabética y signos de oftalmoplejía unilateral que llevaron al estudio con resonancia magnética del sistema nervioso central; se encontraron signos de sinusitis, meningitis y cerebritis. Los estudios microbiológicos iniciales fueron negativos, y los biomarcadores galactomanano sérico y el antígeno de Cryptococcus también fueron negativos. Tras el manejo quirúrgico, se llegó a la identificación de Aspergillus flavus y Rhizopus spp. en el tejido de los senos paranasales. La paciente recibió tratamiento con posaconazol y, tras dos meses de seguimiento, había presentado mejoría clínica. La infección fúngica dual y la infección por A. flavus son entidades poco frecuentes y de relevancia clínica, sin casos presentados previamente en nuestro país por lo que este corresponde a un caso de interés clínico.

Fungal sinusitis is a pathology that can occur in patients with diabetes mellitus and be associated with a hyperglycemic crisis. It is an aggressive entity with local complications that include involvement of the orbit or the central nervous system, and vascular involvement. Despite surgical and antifungal treatment, mortality raises up to 75%. We report the case of a female patient with a diagnosis of diabetic ketoacidosis and signs of unilateral ophthalmoplegia, which led to the study with magnetic resonance imaging of the central nervous system, finding signs of sinusitis, meningitis, and cerebritis. Initial microbiological studies were negative, and biomarkers such as serum galactomannan and Cryptococcus antigen were also negative. After surgical management and the identification of Aspergillus flavus and Rhizopus spp. in sinus tissue, the patient received treatment with posaconazole and after two months of follow-up she presented clinical improvement. Dual fungal infection and infection by A. flavus are uncommon and clinically relevant entities, with no cases previously reported in our country, therefore this corresponds to a case of clinical interest.

Fungal decomposition of chicken-feather waste in submerged and solid-state fermentation / Decomposição fúngica de resíduos de pena de frango em fermentação submersa e de estado sólido

Poultry industry is expanding rapidly and producing million tons of feather waste annually. Massive production of keratinaceous byproducts in the form of industrial wastes throughout the world necessitates its justified utilization. Chemical treatment of keratin waste is proclaimed as an eco-destructive approach by various researchers since it generates secondary pollutants. Keratinase released by a variety of microbes (bacteria and fungi) can be used for the effective treatment of keratin waste. Microbial degradation of keratin waste is an emerging and eco-friendly approach and offers dual benefits, i.e., treatment of recalcitrant pollutant (keratin) and procurement of a commercially important enzyme (keratinase). This study involves the isolation, characterization, and potential utility of fungal species for the degradation of chicken-feather waste through submerged and solid-state fermentation. The isolated fungus was identified and characterized as Aspergillus (A.) flavus. In a trial of 30 days, it was appeared that 74 and 8% feather weight was reduced through sub-merged and solid-state fermentation, respectively by A. flavus. The pH of the growth media in submerged fermentation was changed from 4.8 to 8.35. The exploited application of keratinolytic microbes is, therefore, recommended for the treatment of keratinaceous wastes to achieve dual benefits of remediation.

A indústria avícola está se expandindo rapidamente e produzindo milhões de toneladas de resíduos de penas anualmente. A produção massiva de subprodutos queratinosos na forma de resíduos agrícolas e industriais em todo o mundo exige sua utilização justificada. O tratamento químico de resíduos de queratina é proclamado como uma abordagem ecodestrutiva por vários pesquisadores, uma vez que gera poluentes secundários. A queratinase liberada por uma variedade de micróbios (bactérias e fungos) pode ser usada para o tratamento eficaz de resíduos de queratina. A degradação microbiana de resíduos de queratina é uma abordagem emergente e ecológica e oferece benefícios duplos, ou seja, tratamento de poluente recalcitrante (queratina) e obtenção de uma enzima comercialmente importante (queratinase). Este estudo envolve o isolamento, caracterização e utilidade potencial de espécies de fungos para a degradação de resíduos de penas de frango por meio da fermentação submersa e em estado sólido. O fungo isolado foi identificado e caracterizado como Aspergillus (A.) flavus. Em um ensaio de 30 dias, constatou-se que 74% e 8% do peso das penas foram reduzidos por A. flavus, respectivamente, por meio da fermentação submersa e em estado sólido. O pH do meio de crescimento em fermentação submersa foi alterado de 4,8 para 8,35. A aplicação explorada de micróbios queratinolíticos é, portanto, recomendada para o tratamento de resíduos ceratinosos para obter benefícios duplos de remediação.

Aflatoxins and fungal community distribution during harvesting and processing of Platycladi Semen / 中国中药杂志

This study aimed to analyze aflatoxins content and fungal community distribution in the harvesting and processing of Platycladi Semen, and explore the key link that affects aflatoxins contamination. The related Platycladi Semen samples of different maturity periods(cone non-rupture period, early rupture, and complete rupture period) and different processing periods(before drying, during 2-d drying, during 7-d drying, before and after seed scale removal, before and after peeling, 1 d after color sorting, and 7 d after color sorting) were collected for identifying the fungal community composition on sample surface by ITS amplicon sequencing. Then the content of aflatoxins B_1, B_2, G_1 and G_2 was determined by HPLC-MS/MS. The results showed that during the harvesting of Platycladi Semen from cone non-rupture to complete rupture, aflatoxins were only detected in the seed scale and seed coat, with aflatoxin G_2 in the seed scale and aflatoxin B_1 in the seed coat. During the drying, with the prolongation of drying time, aflatoxins B_1 and G_2 were detected simultaneously in the seed scale, aflatoxin B_1 in the seed coat, and low-content aflatoxin B_1 in the seed kernel. During subsequent processing, the aflatoxin content in seed kernel during subsequent processing was slighted increased. As demonstrated by fungal detection, Aspergillus flavus was not present during the harvesting of Platycladi Semen, but present during the drying and processing. Its content in the seed coat during the drying process was relatively higher. In short, Platycladi Semen should be harvested as soon as possible after it becomes fully mature. Drying process is the key link of preventing aflatoxin contamination. It is advised to build a sunlight room or adopt similar settings, standardize the operations in other processes, and keep the surrounding environment clean to minimize aflatoxin contamination.

Buffaloes (Bubalus bubalis) mycotic eye infection: reported herd cases in Thi-Qar province, Iraq / Infecção ocular micótica em búfalos (Bubalus bubalis): relato de casos em rebanho na província de Thi-Qar, Iraque

Buffaloes are one of the important farm animals in the south of Iraq and play an essential economical role mainly acting as dairy, meat, and draft animals. This study intended to diagnose buffalo mycotic eye infections in Thi-Qar province/Iraq. Some 250 buffaloes in the herd of 3,700 animals suffered from eye infections from December 2017 to November 2018. Eye swabs were collected from each infected eye of the affected buffaloes of both sexes before treatment. The animals were in different age groups. All samples were transferred to the laboratory in transfer media, and cultured on Sabouraud dextrose (SDA) agar with and without 0.05 g/mL and 0.4 g/mL chloramphenicol and cycloheximide, respectively. Later, the agars were incubated at 25oC and 37oC. The total percentage of eye infection was (6.75%), constituting (49.2%) mycotic infections. The predominant clinical manifestations that appeared on the infected buffaloes were eye inflammation represented by congestion, lacrimation, the opacity of cornea and edema, and reduced productivity of the infected animals. Different fungal isolates were identified from the samples including Aspergillus fumigates, Aspergillus flavus, Aspergillus niger, Penicillium spp., Alternaria spp., Fusarium spp., Candida spp., Cladosporium spp., Rhodotorula spp., Mucor spp. and Rhizopus spp. Calves buffaloes below one-year-old were more prone to mycotic infection than one-year-old or more. Additionally, male buffaloes were more susceptible to infection than females. In conclusion, this study isolated various types of fungus from the inflamed eyes of buffaloes. Fungal eye infection and the potential risk factors for fungal keratitis in buffaloes were also investigated. The study also approved the rapid diagnosis of fungi by direct microscopic detection and culture. The author recommends future studies including large numbers of the buffalo herd in Iraq to determine the epidemiology of this condition in the country.(AU)

Os búfalos são um dos animais de fazenda mais importantes no sul do Iraque e desempenham um papel econômico essencial, atuando principalmente na produção de leite, carne e como animal de tração. Este estudo objetivou diagnosticar infecções oculares micóticas em búfalos na província de Thi-Qar, Iraque. 250 búfalos no rebanho de um total de 3700 animais apresentaram infecção ocular durante o período compreendido entre dezembro de 2017 e novembro de 2018. Os esfregaços oculares foram colhidos dos olhos infectados dos búfalos afetados de ambos os sexos antes do tratamento. Os animais estavam em diferentes faixas etárias. Todas as amostras foram transferidas para o laboratório por meio de transferência e cultivadas em Ágar Sabouraud e Dextrose (SDA) com e sem 0,05 g/mL e 0,4 g/mL de cloranfenicol e cicloheximida, respectivamente. Posteriormente, os ágares foram incubados a 25ºC e 37ºC. A porcentagem total de infecção ocular foi de 6,75%, representando 49,2% de infecção micótica. As manifestações clínicas predominantes nos búfalos infectados foram inflamação ocular com congestão, lacrimejamento, opacidade da córnea e edema. Os animais acometidos também apresentaram redução de produtividade. Diferentes isolados de fungos foram identificados a partir das amostras, incluindo Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Penicillium spp., Alternaria spp., Fusarium spp., Candida spp., Cladosporium spp., Rhodotorula spp., Mucour spp. e Rizopus spp. Búfalos com menos de um ano de idade foram mais propensos a infecções micóticas do que com um ano ou mais. Além disso, os búfalos machos foram mais suscetíveis a infecção do que as fêmeas. Em conclusão, este estudo registrou o isolamento de vários tipos de fungos em olhos inflamados de búfalos. Além disso, a infecção ocular por fungos e os fatores de risco potenciais para ceratite fúngica em búfalos também foram observados. O estudo também aprovou o diagnóstico rápido de fungos por detecção microscópica direta e cultura. O autor recomenda outro estudo futuro, incluindo um grande número de rebanhos de búfalos no Iraque para determinar a epidemiologia desta condição no país.(AU)

In vitro antibacterial activity of endophytic fungus Aspergillus flavus IBRL-C8 from Senna siamea against Gram-bacteria

Aims@#To evaluate the antibacterial efficacy of ethyl acetate extract of Aspergillus flavus IBRL-C8 against Gram-positive and Gram-negative bacteria.@*Methodology and results@#In this experiment, an endophytic fungus which identified as A. flavus IBRL-C8 was extracted using ethyl acetate and methanol, from Senna siamea, prior to in vitro antibacterial test on eight Gram-bacteria. The results were significantly more enunciated to the ethyl acetate extract since the Gram-bacteria signified 9.0 to 20.0 mm of inhibition zones on Muller Hinton Agar (MHA) during disc diffusion assay. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extract were ranged from 125-1000 µg/mL and 125-2000 µg/mL, respectively. Time-kill assay depicted the ethyl acetate extract of A. flavus IBRL-C8 exceptionally retarded methicillin-resistant Staphylococcus aureus (MRSA) and also manifested extended antibacterial activity. The maximum reduction in cell numbers occurred at 2MIC concentration (250 µg/mL) during the interval time of 16 h. The malformations noticed from microscopic observations where the transformation of structural annihilation from regular spherical morphology to non-spherical shape with an irregular surface and also disruption around the cell membrane when the MRSA treated with ethyl acetate extract of A. flavus IBRL-C8. @*Conclusion, significance and impact of study@#This study proposed the ethyl acetate extract of A. flavus IBRL-C8 as a potential antibacterial agent against MRSA infection, which can be useful in pharmaceutical application.

Monitoramento de fungos anemófilos no ambiente de uma biblioteca no Município de São José do Rio Preto - Sp, Brasil / Anemophilic fungi monitoring in a library environment in the city of São Jose do Rio Preto, Brazil

Os fungos desempenham vários papéis que impactam a humanidade de diversas maneiras. Suas características metabólicas são importantes na biotecnologia, porém, tais microrganismos podem desencadear alguns problemas de saúde pública e até mesmo serem letais. Objetivo: detectar a presença de fungos no acervo de uma biblioteca no município de São José do Rio Preto. Metodologia: foram coletadas quarenta amostras nas superfícies inanimadas (livros, estantes, documentos, mapas, artigos e revistas) das principais salas da biblioteca com o auxílio de swabs umedecidos em solução salina estéril, posteriormente encaminhados ao laboratório de Biomedicina da Universidade Paulista ­ UNIP. As amostras foram semeadas em meio de cultura ágar Sabouraud Dextrose (SDA), tendo adicionado cloranfenicol e incubadas a 30 °C. Foi realizada a colônia gigante em todas as cepas crescidas em SDA para a realização da técnica de microcultivo para a identificação dos fungos, de acordo com o Manual de Detecção e Identificação dos Fungos de Importância Médica da Agência Nacional de Vigilância Sanitária. Resultados: Houve positividade em trinta e uma amostras (78%) e em quatro delas foi observado mais de um tipo de colônia (13%). Das vinte e duas superfícies de livros analisadas, foram isolados e identificados: Aspergillus flavus, Aspergillus niger, Cunninghamella sp., Cladosporium sp., Curvularia sp., Mucor sp. e Nigrospora sp. Nas oito superfícies de estantes: Aspergillus flavus, Aspergillus niger, Aspergillus versicolor, Penicillium sp. e Scopulariopsis sp. e, nos dez documentos: Aspergillus nidulans, Aspergillus sp., Cladosporium sp., Cunninghamella sp. e Trichoderma sp. Conclusão: Os fungos encontrados estão amplamente distribuídos no ambiente como solo e ar e, por diversos fatores, instalam-se em locais como bibliotecas. Em condições favoráveis, podem infectar o homem e causar perdas patrimoniais para os acervos.

Fungi play many roles that impact humankind in different ways. Their metabolic characteristics are important in biotechnology; however, these microorganisms can trigger some public health problems or may even be lethal. Objective: detect the presence of fungi in the collection of a public library in the city of São José do Rio Preto, Brazil. Methods: a total of forty samples were collected from inanimate surfaces (books, shelves, documents, maps, articles and magazines) located in the main rooms of the library with swabs soaked in sterile saline solution and sent to the Universidade Paulista ­ UNIP laboratories. The samples were plated in Sabouraud Dextrose Agar (SDA) supplemented with chloramphenicol and incubated at 30 °C. The colonies that grew in SDA were isolated in Potato Dextrose Agar for performing the slide culture technique for the identification of the fungi, performed according to the Manual of Detection and Identification of Fungi of Medical Importance from the Brazilian Health Surveillance Agency (ANVISA). Results: Thirty-one samples (78%) were positive, and in four of them more than one fungus genus was observed (13%). From the twenty-two book surfaces analyzed, the following fungi were isolated and identified: Aspergillus flavus, Aspergillus niger, Cunninghamella sp., Cladosporium sp., Curvularia sp., Mucor sp. and Nigrospora sp. On the eight shelves: Aspergillus flavus, Aspergillus niger, Aspergillus versicolor, Penicillium sp. and Scopulariopsis sp. The ten documents analyzed presented the following fungi: Aspergillus nidulans, Aspergillus sp., Cladosporium sp., Cunninghamella sp. and Trichoderma sp.. Conclusion: These fungi are widely distributed in the environment such as in the soil and air, and due to several factors, they colonize public places, such as libraries. In favorable conditions, they may infect humans and cause diseases.

Incidência de aflatoxinas B1, B2, G1 e G2 em amendoins / Incidence of aflatoxins B1, B2, G1 and G2 in peanuts

Micotoxinas são substâncias tóxicas produzidas por fungos e encontradas nos alimentos. As micotoxinas mais tóxicas são as aflatoxinas, produzidas, principalmente por Aspergillus flavus. Estudos realizados no país demonstraram alta incidência dessas micotoxinas em produtos de amendoim, que representa risco à saúde da população. O objetivo do estudo foi avaliar a incidência de aflatoxinas B1, B2, G1 e G2 em amostras de amendoins comercializados na região Nordeste do Estado de São Paulo nos períodos de 1994-2001 e 2016-2017. O método utilizado para analisar as amostras no primeiro período foi extração líquido-líquido e cromatografia em camada delgada e no segundo foi utilizando colunas de imunoafinidade, cromatografia líquida com derivatização pós- coluna e detector por fluorescência. No levantamento de 1994-2001 das 82 amostras, 39% tiveram contaminação de aflatoxinas variando de 11 a 1556 µg/kg com 37% das amostras contendo níveis maiores que 20 µg/kg, enquanto na pesquisa de 2016-17, das 56 amostras, 38% apresentaram contaminação destas toxinas variando de 0,09 a 60,40 µg/kg com 13% das amostras contendo níveis maiores que 20 µg/kg. Os resultados dos dois períodos estudados indicam que houve uma diminuição na incidência e nível das aflatoxinas estudadas, embora esta contaminação em amendoim permaneça um problema de saúde pública. (AU)

Mycotoxins are toxic compounds produced by fungi found in food. The most toxic mycotoxins are the aflatoxins produced mainly by Aspergillus flavus . Studies carried out in Brazil showed a high incidence of these mycotoxins in peanut products, a fact that represents public health problems. The aim of the study was to evaluate aflatoxins B1, B2, G1, and G2 in samples of peanuts sold in cities of the Northeast of the State of São Paulo in the period from 1994 to 2001 and from 2016 to 2017. The samples of the first period were analyzed using liquid-liquid extraction and thin-layer chromatography and the second using immunoaffinity columns, post-column derivative liquid chromatography and fluorescence detector. In the 1994-2001 survey, among 82 samples, 39% presented aflatoxins contamination ranging from 11 to 1556 µg/kg with 37% with levels greater than 20 µg/kg whereas, in the 2016-17 survey, 38% of the 56 samples presented contamination of aflatoxins ranging from 0.09 to 60.40 µg/kg and 7 samples 13% containing aflatoxins levels higher than 20 µg/kg. The results indicated there was a decrease in the incidence and level of aflatoxins, but the contamination of aflatoxins in peanuts remains a public health problem. (AU)

Incidência de aflatoxinas B1, B2, G1 e G2 em amendoins / Incidence of aflatoxins B1, B2, G1 and G2 in peanuts

Micotoxinas são substâncias tóxicas produzidas por fungos e encontradas nos alimentos. As micotoxinas mais tóxicas são as aflatoxinas, produzidas, principalmente por Aspergillus flavus. Estudos realizados no país demonstraram alta incidência dessas micotoxinas em produtos de amendoim, que representa risco à saúde da população. O objetivo do estudo foi avaliar a incidência de aflatoxinas B1, B2, G1 e G2 em amostras de amendoins comercializados na região Nordeste do Estado de São Paulo nos períodos de 1994-2001 e 2016-2017. O método utilizado para analisar as amostras no primeiro período foi extração líquido-líquido e cromatografia em camada delgada e no segundo foi utilizando colunas de imunoafinidade, cromatografia líquida com derivatização pós-coluna e detector por fluorescência. No levantamento de 1994-2001 das 82 amostras, 39% tiveram contaminação de aflatoxinas variando de 11 a 1556 μg/kg com 37% das amostras contendo níveis maiores que 20 μg/kg, enquanto na pesquisa de 2016-17, das 56 amostras, 38% apresentaram contaminação destas toxinas variando de 0,09 a 60,40 μg/kg com 13% das amostras contendo níveis maiores que 20 μg/kg. Os resultados dos dois períodos estudados indicam que houve uma diminuição na incidência e nível das aflatoxinas estudadas, embora esta contaminação em amendoim permaneça um problema de saúde pública.

Mycotoxins are toxic compounds produced by fungi found in food. The most toxic mycotoxins are the aflatoxins produced mainly by Aspergillus flavus. Studies carried out in Brazil showed a high incidence of these mycotoxins in peanut products, a fact that represents public health problems. The aim of the study was to evaluate aflatoxins B1, B2, G1, and G2 in samples of peanuts sold in cities of the Northeast of the State of São Paulo in the period from 1994 to 2001 and from 2016 to 2017. The samples of the first period were analyzed using liquid-liquid extraction and thin-layer chromatography and the second using immunoaffinity columns, post-column derivative liquid chromatography and fluorescence detector. In the 1994-2001 survey, among 82 samples, 39% presented aflatoxins contamination ranging from 11 to 1556 μg/kg with 37% with levels greater than 20 μg/kg whereas, in the 2016-17 survey, 38% of the 56 samples presented contamination of aflatoxins ranging from 0.09 to 60.40 μg/kg and 7 samples 13% containing aflatoxins levels higher than 20 μg/kg. The results indicated there was a decrease in the incidence and level of aflatoxins, but the contamination of aflatoxins in peanuts remains a public health problem.

El factor de transcripción bZIP Afap1 afecta al estrés oxidativo y la biosíntesis de aflatoxinas en Aspergillus flavus / The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus

Abstract Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap 1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H 2O 2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H 2O 2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB 1 in the mutant strains was approximately V of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus. ©2018 Published by Elsevier España, S.L.U. on behalf of Asociación Argentina de Microbiología. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/ licenses/by-nc-nd/4.0/).

Resumen La aflatoxina es un metabolito secundario cancerígeno producido principalmente por Aspergillus flavus y Aspergillus parasiticus, que pone en riesgo grave a la salud de los humanos y los animales. El estrés oxidativo es una respuesta de defensa común, y es sabido que las especies reactivas de oxígeno (ROS) pueden inducir la síntesis de una serie de metabolitos secundarios, incluida la aflatoxina. Empleando mutantes carentes del gen afap1 se evaluó el papel de Afap1 en el estrés oxidativo y la síntesis de aflatoxinas. El crecimiento de las cepas mutadas se vio significativamente inhibido con el aumento de la concentración de H 2O 2, la inhibición fue completa a 40mmol/l. Sin embargo, en el análisis cuantitativo por HPLC, la concentración de la aflatoxina AFBi aumentó con el aumento de la concentración de H 2O 2 hasta 10mmol/l. Tras un análisis apoyado en la información provista por la herramienta NCBI BLAST, se supuso que Afap1, un factor de transcripción de la cremallera de leucina básica (bZIP), estaba asociado con el estrés oxidativo en este hongo. El tratamiento con 5mmol/l de H 2O 2 inhibió completamente el crecimiento de las cepas mutantes en afap1, pero no afectó el crecimiento de la cepa CA14PTs (cepa no mutada). Además, la concentración de AFB 1 en las cepas mutadas fue de aproximadamente 1/4 de la observada en CA14PTs. Estos resultados sugieren que Afap1 juega un papel clave en la regulación del estrés oxidativo y la producción de aflatoxinas en A. flavus.

Antibacterial and Antifungal Activities of Compounds Isolated from Gymnosporia Royleana (Celastraceae) / Actividades antibacterianas y antifúngicas de compuestos aislados de Gymnosporia Royleana (Celastraceae)

ABSTRACT Objective: In the present study, five new source compounds isolated from aerial parts of Gymnosporia royleana (G royleana) were screened for antibacterial and antifungal activities. Methods: Extraction from plant material was carried out using cold maceration technique. Isolation of pure compounds was accomplished through repeated column chromatography of different fractions obtained from crude extract and using silica gel as stationary phase. Their structures were established via advanced spectroscopic techniques along with the spectral data previously reported for these compounds. Dilution method was used for the evaluation of antimicrobial potential of the compounds against various microbial strains. Results: Among the tested compounds, Gymnosporin B displayed moderate antimicrobial activity against Escherichia Coli (E coli), Staphylococcus aureus (S aureus), Candida albicans (C albicans) and Aspergillus flavus (A flavus) [minimum inhibitory concentration (MIC) range; 32-64 μg/mL]. Similarly, Gymnosporin C also showed moderate activity against E coli and S aureus (MIC; 32 μg/mL each) as well as weak activity against C albicans and A flavus (MIC; 64 μg/mL each). However, Royaflavone showed moderate antibacterial activity against S aureus only (MIC; 32 μg/mL). Antimicrobial activity of the rest of the compounds was weak and negligible. Conclusion: The present study has provided fascinating results of antimicrobial activities of the isolated compounds. However, the broad antimicrobial spectrum of Gymnosporin B and Gymnosporin C demands for further exploration of these triterpenes, both on the basis of mechanism and quantitative structure-activity relationship.

RESUMEN Objetivo: En el presente estudio, cinco nuevos compuestos de origen aislados de partes aéreas de Gymnosporia royleana (G royleana) fueron tamizados en sus actividades antibacterianas y antifúngicas. Métodos: La extracción de material vegetal se realizó mediante la técnica de maceración en frío. El aislamiento de compuestos puros se logró a través de la cromatografía en columna repetida de diversas fracciones obtenidas del extracto crudo y usando gel de silicona como fase estacionaria. Sus estructuras fueron establecidas mediante técnicas espectroscópicas avanzadas junto con los datos espectrales previamente reportados para estos compuestos. El método de dilución fue usado para evaluar el potencial antimicrobiano de los compuestos contra diversas cepas microbianas. Resultados: Entre los compuestos sometidos a prueba, Gymnosporina B mostró una actividad antimicrobiana moderada contra Escherichia Coli (E coli), Staphylococcus aureus (S aureus), Candida Albicans (C albicans) y Aspergillus flavus (A flavus) [rango de concentración inhibitoria mínima (CIM); 32 - 64 μg/mL]. De manera similar, Gymnosporina C también mostró actividad moderada contra E coli y S aureus (CIM; 32 μg/mL cada uno) así como débil actividad frente a C albicans y A flavus (CIM; 64 μg/mL cada uno). Sin embargo, Royaflavone mostró actividad antibacteriana moderada sólo frente a S aureus (CIM; 32 μg/mL). La actividad antimicrobiana del resto de los compuestos fue débil e insignificante. Conclusión: El presente estudio ha proporcionado resultados interesantes acerca de las actividades antimicrobianas de los compuestos aislados. Sin embargo, el amplio espectro antimicrobiano de la Gymnosporina B y la Gymnosporina C exige una mayor exploración de estos triterpenos, tanto sobre la base del mecanismo como a partir de la relación cuantitativa estructura-actividad.

Transcriptome and proteome analyses of resistant preharvest peanut seed coat in response to Aspergillus flavus infection

BACKGROUND: The infection of peanut (Arachis hypogaea L.) seed coat by the pathogenic fungus Aspergillus flavus has highly negative economic and health impacts. However, the molecular mechanism underlying such defense response remains poorly understood. This study aims to address this issue by profiling the transcriptomic and proteomic changes that occur during the infection of the resistant peanut cultivar J11 by A. flavus. RESULTS: Transcriptomic study led to the detection of 13,539 genes, among which 663 exhibited differential expression. Further functional analysis found the differentially expressed genes to encode a wide range of pathogenesis- and/or defense-related proteins such as transcription factors, pathogenesis-related proteins, and chitinases. Changes in the expression patterns of these genes might contribute to peanut resistance to A. flavus. On the other hand, the proteomic profiling showed that 314 of the 1382 detected protein candidates were aberrantly expressed as a result of A. flavus invasion. However, the correlation between the transcriptomic and proteomic data was poor. We further demonstrated by in vitro fungistasis tests that hevamine-A, which was enriched at both transcript and protein levels, could directly inhibit the growth of A. flavus. Conclusions: The results demonstrate the power of complementary transcriptomic and proteomic analyses in the study of pathogen defense and resistance in plants and the chitinase could play an important role in the defense response of peanut to A. flavus. The current study also constitutes the first step toward building an integrated omics data platform for the development of Aspergillus-resistant peanut cultivars

Desarrollo y capacidad de remoción de los pesticidas atrazina, clorpirifós y endosulfán por parte de cepas de Aspergillus sección Flavi no toxigénicas / Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5,10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25 °C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p <0.001) correlation (r = 0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

En este estudio se evaluó los efectos in vitro de 3 concentraciones de atrazina, clorpirifós y endosulfán sobre los parámetros de crecimiento de 4 cepas no toxigénicas de Aspergillus sección Flavi. También se evaluó la capacidad de las cepas de remover los pesticidas. Los parámetros de crecimiento se ensayaron en medio agar extracto de suelo suplementado con 5, 10 y 20mg/l de cada pesticida y acondicionado a -0.70, -2.78, -7.06 y -10.0 de potencial de agua (MPa). Los ensayos de remoción se realizaron en medio Czapek Dox con 20mg/l de cada pesticida bajo condiciones óptimas de crecimiento (-2.78 de MPa y 25 °C). Los niveles residuales de atrazina, clorpirifós y endosulfán se detectaron en un sistema HPLC con detección por fluorescencia. La fase de latencia de las cepas disminuyó significantemente en presencia de los pesticidas, indicando una rápida adaptación a dichas condiciones. La velocidad de crecimiento se incrementó considerablemente dependiendo de la concentración de pesticida. Las cepas Aspergillus oryzae AM1 y AM2 mostraron porcentajes elevados de degradación de atrazina (aproximadamente el 90%), seguidos por endosulfán (56 y 76%) y clorpirifós (50 y 73%). Se observó una correlación (r = 0.974) significante (p <0.001) entre el porcentaje de pesticida removido y la velocidad de crecimiento. Este estudio muestra que cepas no-toxigénicas de Aspergillus sección Flavi aisladas de suelos agrícolas desarrollan eficientemente en presencia de altas concentraciones de atrazina, clorpirifós y endosulfán en un amplio rango de MPa. Además, presentan capacidad de remover in vitro altos niveles de pesticidas en corto tiempo.

Chemical composition and antifungal activity of propolis on Aspergillus flavus / Composição química e atividade antifúngica de própolis sobre Aspergillus flavus

Research has indicated the antifungal activity of ethanol extracts from propolis (EEP) on fungi of the genera Phakopsora, Colletotrichum, and Cercospora. Here, chemical compositions and antioxidant activity of three EEP (propolis from Scaptotrigona polysticta stingless bee and two types produced by Apis mellifera - red and brown) were evaluated and their action against Aspergillus flavus was investigated. Ash, dry extract, total phenolic and total flavonoid contents were determined. Phenolic composition was evaluated by high performance liquid chromatography, with using the following reference substances: gallic acid, caffeic acid, -coumaric acid, ferulic acid, quercetin, kaempferol, and apigenin. Tests on mycelial growth, sporulation, and germination of spores were performed to assess the biological activity of the EEP on A. flavus. All EEP showed low dry extract content (<11%) with adequate amounts of ash (<5%). The red EEP (Apis mellifera) showed the highest contents of total phenolic and total flavonoid (5.38 and 2.77 g 100 g-1), while the highest recorded antioxidant activity was exhibited by brown EEP (92.9%). The EEP of S. polysticta presented higher levels of -coumaric acid (10.99 mg g-1), while red and brown extracts from A. mellifera stood out with the highest levels of quercetin (27.26 mg g-1) and gallic acid (5.88 mg g-1), respectively. No extract was effective in inhibiting mycelial growth and sporulation of A. flavus, but red EEP inhibited spore germination. On the basis of these results, it is suggested that the inhibitory effect of red EEP on spore germination may be associated with increased levels of flavonoids found in it as compared with the other extracts investigated here.

Outros estudos apontaram a ação antifúngica dos extratos etanólicos de própolis (EEP) sobre fungos do gênero Phakopsora, Colletotrichum e Cercospora. Neste estudo, avaliou-se a composição química e a atividade antioxidante de três EEP (própolis produzido pela abelha sem ferrão Scaptotrigona polysticta e dois tipos de própolis produzida pela Apis melífera: marrom e vermelha) e sua ação contra o Aspergillus flavus. Foram determinados os teores de cinzas, extrato seco, fenólicos e flavonoides totais nos EEP. A composição fenólica foi determinada por cromatografia líquida de alta eficiência, empregando-se as seguintes substâncias de referência: ácido gálico, ácido p-cumárico, ácido ferrúlico, quercitina, kaempferol e apigenina. Para avaliar a atividade biológica dos EEP sobre A. flavus foram realizados os testes de crescimento micelial, esporulação e germinação de esporos. Todos os EEP apresentaram baixos teores de extrato seco (< 11%), porém quantidades de cinzas adequadas (< 5%). O EEP vermelha apresentou os maiores teores de fenólicos e flavonoides totais (5,38 e 2,77 g 100g-1), enquanto que a maior atividade antioxidante registrada foi do EEP marrom (92,9%). O EEP de S. polysticta apresentou maiores teores de ácido p-cumárico (10,99 mg g-1), enquanto que os de A. mellifera, vermelha e marrom, destacaram-se com os maiores níveis de quercetina (27,26 mg g-1) e de ácido gálico (5,88 mg g-1), respectivamente. Nenhum extrato foi eficaz na inibição do crescimento micelial e esporulação de A. flavus, porém observou-se inibição na germinação de esporos pelo EEP vermelha. Sugere-se que o efeito inibitório na germinação de esporos pode estar relacionado com os maiores níveis de flavonoides encontrados na própolis vermelha, quando comparado com os outros extratos investigados.

Differential role of gpaB and sidA gene expressions in relation to virulence in Aspergillus species from patients with invasive aspergillosis

Abstract The virulence genes in invasive aspergillosis (IA) have not been analyzed adequately. The present study was designed to evaluate the expression of gpaB and sidA genes, which are important virulence genes in Aspergillus spp. from bronchoalveolar lavage (BAL) samples. Direct examination and culture on Czapek Agar and Sabouraud Dextrose Agar media were performed for 600 BAL specimens isolated from patients with possible aspergillosis. A Galactomannan ELISA assay was also carried out. The expression levels of the gpaB and sidA genes in isolates were analyzed using quantitative real-time PCR (qRT-PCR). We identified 2 species, including Aspergillus flavus (A. flavus) and Aspergillus fumigatus (A. fumigatus) in 25 positive samples for invasive aspergillosis as validated using GM-ELISA. A. flavus is the main pathogen threatening transplant recipients and cancer patients worldwide. In this study, A. flavus had low levels of the gpaB gene expression compared to A. fumigatus (p = 0.006). The highest sidA expression was detected in transplant recipients (p = 0.05). There was no significant correlation between sidA expression and underlying disease (p = 0.15). The sidA and gpaB gene expression patterns may provide evidence that these virulence genes play important roles in the pathogenicity of Aspergillus isolates; however, there are several regulatory genes responsible for the unexpressed sidA and gpaB genes in the isolates.

In vitro antifungal susceptibility of clinical and environmental isolates of Aspergillus fumigatus and Aspergillus flavus in Brazil

ABSTRACT The in vitro susceptibility of 105 clinical and environmental strains of Aspergillus fumigatus and Aspergillus flavus to antifungal drugs, such as amphotericin B, azoles, and echinocandins was evaluated by the broth microdilution method proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Following the EUCAST-proposed breakpoints, 20% and 25% of the clinical and environmental isolates of A. fumigatus, respectively, were found to be resistant to itraconazole (Minimal Inhibitory Concentration, MIC > 2.0 mg/L). Voriconazole showed good activity against A. fumigatus and A. flavus strains, except for one clinical strain of A. fumigatus whose MIC was 4.0 mg/L. Posaconazole (≤0.25 mg/L) also showed appreciable activity against both species of Aspergillus, except for six A. fumigatus strains with relatively higher MICs (0.5 mg/L). The MICs for Amphotericin B ranged from 0.06 to 1.0 mg/L for A. fumigatus, but were much higher (0.5-8.0 mg/L) for A. flavus. Among the echinocandins, caspofungin showed a geometric mean of 0.078 and 0.113 against the clinical and environmental strains of A. flavus, respectively, but had elevated minimal effective concentrations (MECs) for seven of the A. fumigatus strains. Anidulafungin and micafungin exhibited considerable activity against both A. fumigatus and A. flavus isolates, except for one environmental isolate of A. fumigatus that showed an MEC of 1 mg/L to micafungin. Our study proposes that a detailed investigation of the antifungal susceptibility of the genus Aspergillus from different regions of Brazil is necessary for establishing a response profile against the different classes of antifungal agents used in the treatment of aspergillosis.

Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens Against Aspergillus and Penicillium spp. Predominant in Stored Rice Grains: Study II

In our previous studies, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15 have been shown to be antagonistic to Aspergillus flavus in stored rice grains. In this study, the biocontrol activities of these strains were evaluated against Aspergillus candidus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum, which are predominant in stored rice grains. In vitro and in vivo antifungal activities of the bacterial strains were evaluated against the fungi on media and rice grains, respectively. The antifungal activities of the volatiles produced by the strains against fungal development and population were also tested using I-plates. In in vitro tests, the strains produced secondary metabolites capable of reducing conidial germination, germ-tube elongation, and mycelial growth of all the tested fungi. In in vivo tests, the strains significantly inhibited the fungal growth in rice grains. Additionally, in I-plate tests, strains KU143 and AS15 produced volatiles that significantly inhibited not only mycelial growth, sporulation, and conidial germination of the fungi on media but also fungal populations on rice grains. GC-MS analysis of the volatiles by strains KU143 and AS15 identified 12 and 17 compounds, respectively. Among these, the antifungal compound, 5-methyl-2-phenyl-1H-indole, was produced by strain KU143 and the antimicrobial compounds, 2-butyl 1-octanal, dimethyl disulfide, 2-isopropyl-5-methyl-1-heptanol, and 4-trifluoroacetoxyhexadecane, were produced by strain AS15. These results suggest that the tested strains producing extracellular metabolites and/or volatiles may have a broad spectrum of antifungal activities against the grain fungi. In particular, B. megaterium KU143 and P. protegens AS15 may be potential biocontrol agents against Aspergillus and Penicillium spp. during rice grain storage.

Effect of Temperature and Relative Humidity on Growth of Aspergillus and Penicillium spp. and Biocontrol Activity of Pseudomonas protegens AS15 against Aflatoxigenic Aspergillus flavus in Stored Rice Grains

In this study, we evaluated the effect of different temperatures (10, 20, 30, and 40 °C) and relative humidities (RHs; 12, 44, 76, and 98%) on populations of predominant grain fungi (Aspergillus candidus, Aspergillus flavus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum) and the biocontrol activity of Pseudomonas protegens AS15 against aflatoxigenic A. flavus KCCM 60330 in stored rice. Populations of all the tested fungi in inoculated rice grains were significantly enhanced by both increased temperature and RH. Multiple linear regression analysis revealed that one unit increase of temperature resulted in greater effects than that of RH on fungal populations. When rice grains were treated with P. protegens AS15 prior to inoculation with A. flavus KCCM 60330, fungal populations and aflatoxin production in the inoculated grains were significantly reduced compared with the grains untreated with strain AS15 regardless of temperature and RH (except 12% RH for fungal population). In addition, bacterial populations in grains were significantly enhanced with increasing temperature and RH, regardless of bacterial treatment. Higher bacterial populations were detected in biocontrol strain-treated grains than in untreated control grains. To our knowledge, this is the first report showing consistent biocontrol activity of P. protegens against A. flavus population and aflatoxin production in stored rice grains under various environmental conditions of temperature and RH.

Diversity and Bioactive Potential of Culturable Fungal Endophytes of Medicinal Shrub Berberis aristata DC.: A First Report

Bioactive natural compounds, isolated from fungal endophytes, play a promising role in the search for novel drugs. They are an inspiring source for researchers due to their enormous structural diversity and complexity. During the present study fungal endophytes were isolated from a well-known medicinal shrub, Berberis aristata DC. and were explored for their antagonistic and antioxidant potential. B. aristata, an important medicinal shrub with remarkable pharmacological properties, is native to Northern Himalayan region. A total of 131 endophytic fungal isolates belonging to eighteen species and nine genera were obtained from three hundred and thirty surface sterilized segments of different tissues of B. aristata. The isolated fungi were classified on the basis of morphological and molecular analysis. Diversity and species richness was found to be higher in leaf tissues as compared to root and stem. Antibacterial activity demonstrated that the crude ethyl acetate extract of 80% isolates exhibited significant results against one or more bacterial pathogens. Ethyl acetate extract of Alternaria macrospora was found to have potential antibacterial activity. Significant antioxidant activity was also found in crude ethyl acetate extracts of Alternaria alternata and Aspergillus flavus. Similarly, antagonistic activity of the fungal endophytes revealed that all antagonists possessed inhibition potential against more than one fungal pathogen. This study is an important step towards tapping endophytic fungal diversity for bioactive metabolites which could be a step forward towards development of novel therapeutic agents.

Serum Cytokine Profile in Patients with Chronic Rhinosinusitis with Nasal Polyposis Infected by Aspergillus flavus

BACKGROUND: Fungi, especially Aspergillus flavus, can cause chronic rhinosinusitis with nasal polyposis and modulate host innate immune components. The objective of this study was to examine the serum levels of T helper (Th) cell subset Th1, Th2, and Th17 cytokines and total IgE in patients having chronic rhinosinusitis with nasal polyposis and Aspergillus flavus infection. METHODS: A case-control study including 40 patients with chronic rhinosinusitis with nasal polyposis and 20 healthy controls was conducted. Aspergillus flavus infection was confirmed by standard potassium hydroxide (KOH) testing, culture, and PCR. Serum samples of all patients and controls were analyzed for various cytokines (interleukins [IL]-1β, IL-2, IL-4, IL-6, IL-17, IL-21, IL-27, TGF-β) and total IgE by ELISA. Data from patients with Aspergillus flavus infection and healthy volunteers were compared using the independent t-test and non-parametric Mann-Whitney U test. RESULTS: Aspergillus flavus infection was found in 31 (77.5%) patients with chronic rhinosinusitis with nasal polyposis. IL-1β, IL-17, IL-21, and TGF-β serum levels were significantly higher in these patients than in controls; however, IL-2, IL-4, IL-6, and IL-27 levels were lower. Compared with nine (22.5%) patients without Aspergillus flavus infection, IL-17 level was higher while IL-2 level was lower in patients with Aspergillus flavus infection. Total IgE was significantly higher in patients with Aspergillus flavus infection than in controls. CONCLUSIONS: High levels of IL-17 and its regulatory cytokines in patients with chronic rhinosinusitis with nasal polyposis infected by Aspergillus flavus raise a concern about effective disease management and therapeutic recovery. Surgical removal of the nasal polyp being the chief management option, the choice of post-operative drugs may differ in eosinophilic vs. non-eosinophilic nasal polyposis. The prognosis is likely poor, warranting extended care.