NATIVE AMAZONIAN FUNGI TO CONTROL TERMITES Nasutitermes sp. (BLATTODEA: TERMITIDAE) / Hongos natives para el control de Nasutitermes sp. (Blattodea: Termitidae)

ABSTRACT Termites of the Nasutitermes genus are considered one of the main urban and agroforestry pests in Brazil, where the main method of control is the application of pesticides. The uso of entomopathogenic fungi to reduce the population of this plague in the environment could be use as an alternative. The goal of this study was to evaluate the virulence of isolates native Amazonian fungi belonging to the Tolypocladium endophyticum, Metarhizium anisopliae and Metarhizium marquandii species for the control of Nasutitermes sp. The strains of T. endophyticum (4.439), M. anisopliae (4.443) and M. marquandii (4.472) with their respective isolation codes, were evaluated using suspensions at concentrations of 105, 106, 107 and 108 conidia/mL against the termites. The fungi were characterized to species level by molecular analysis. The greatest virulence was registered with T. endophyticum (4.439), with a mortality of 100 % on the 4th day of treatment for all analyzed concentrations. The M. anisopliae strain (4.443) proved to be efficient, causing a mortality of 100 % on the 7th and 6th days at dilutions of 107 and 108 conidia/mL, respectively. Rates lower than 100 % were registered with M. marquandii (4.472). Therefore, the three fungal strains showed virulence against the termites Nasutitermes sp. In this study, the fungi Tolypocladium endophyticum and Metarhizium marquandii are reported for the first time for the biological control of pests, indicating the potential of native Amazonian fungi for the biological control of thermites Nasutitermes sp.

RESUMEN Las termitas del género Nasutitermes son consideradas una de las principales plagas urbanas y agroforestales de Brasil, donde el principal método de control es la aplicación de pesticidas. Los hongos entomopatógenos pueden ser usados como alternativa para reducir la población de esta plaga en el medio ambiente. Por tanto, el objetivo de este estudio fue evaluar la virulencia de aislados de hongos nativos de suelo amazónico pertenecientes a las especies Tolypocladium endophyticum, Metarhizium anisopliae y Metarhizium marquandii para el control de Nasutitermes sp. Las cepas de T. endophyticum (4,439), M. anisopliae (4,443) y M. marquandii (4,472) con sus respectivos códigos de aislamiento, fueron evaluadas utilizando suspensiones a concentraciones de 105, 106, 107 y 108 conidios/mL contra las termitas. Los hongos se caracterizaron mediante análisis moleculares para confirmar la especie. La mayor virulencia se registró con el hongo T. endophyticum (4,439), con una mortalidad del 100 % al cuarto día de tratamiento para todas las concentraciones analizadas. La cepa M. anisopliae (4,443) demostró ser eficiente, causando una mortalidad del 100 % al sexto y septimo días a las diluciones de 107 y 108 conidios / mL, respectivamente. Se registraron tasas inferiores al 100 % con M. marquandii (4,472). Por tanto, las tres cepas de hongos mostraron virulencia contra las termitas Nasutitermes sp. En este estudio, los hongos Tolypocladium endophyticum y Metarhizium marquandii son reportados por primera vez para el control biológico de plagas, indicando el potencial de hongos nativos de suelo amazónico para el control biológico de termitas Nasutitermes sp.

Antibacterial activity of fractions and isolates of Maytenus guianensis Klotzsch ex Reissek (Celastraceae) Chichuá Amazon

Abstract INTRODUCTION This aim of this study was to evaluate the antibacterial activity of fractions and isolates of Maytenus guianensis, a plant species used in Amazonian folk medicine. METHODS A disk diffusion technique was used to investigate the antibacterial potential. RESULTS The hexanic fractions and tingenone B isolate showed inhibitory effects against Staphylococcus aureus and Streptococcus pneumoniae. CONCLUSIONS These results indicate the antibacterial potential of this species and will enable future studies to identify novel therapeutic alternatives from this species.

Metabolites from endophytic Aspergillus fumigatus and their in vitro effect against the causal agent of tuberculosis / Metabolitos de Aspergillus fumigatus endofítico e seu efeito in vitro contra o agente causal da tuberculose

ABSTRACT Tuberculosis (TB) remains one of the most deadly communicable infectious diseases, causing 1.4 million deaths in 2015 worldwide due to many conditions, including the inadequate treatment and the emergence of multidrug-resistant strains of the causal agent, Mycobacterium tuberculosis. Therefore, drugs developed from natural sources, as microorganisms and plant extracts, are a frequent target for the research and discovery of antimicrobial compounds. The current study started the characterization of compounds produced by an Aspergillus fumigatus isolated from copaíba (Copaifera multijuga) that efficiently inhibits M. tuberculosis by releasing the compounds into the fermentation broth under specific culture conditions. A preliminary assay was carried out with a correlate species, M. smegmatis, aiming to detect an antimicrobial effect related to A. fumigatus fermentation broth. The direct use of this substrate in antibiosis assays againstM. tuberculosis H37Rv strain (ATCC 27294) allowed the detection of antimicrobial activity with a minimal inhibitory concentration of 256 μg mL-1, demonstrating that purification processes developed by the Biotage Flash Chromatography System are robust and reliable techniques for purification of compounds from natural sources. Also, this chromatographic system can be used in combination with specific biochemical tests, improving the search for reliable results. We conclude that this fraction can express a broad action range, inhibiting both Mycobacterium species used as target organisms.

RESUMO A tuberculose continua a ser uma das doenças infecciosas transmissíveis mais mortais, causando 1,4 milhão de mortes em 2015 em todo o mundo devido a vários fatores, incluindo o tratamento inadequado e o surgimento de cepas multirresistentes do agente causal, Mycobacterium tuberculosis. Portanto, as drogas desenvolvidas a partir de fontes naturais, como micro-organismos e extratos de plantas, são um alvo freqüente para a pesquisa e descoberta de compostos antimicrobianos. O presente estudo foi um ponto de partida para caracterizar compostos produzidos por um Aspergillus fumigatus isolado de copaíba (Copaifera multijuga) que inibe eficientemente M. tuberculosis, liberando os compostos no caldo de fermentação em condições de cultura específicas. Realizou-se um ensaio preliminar com uma espécie correlata, M. smegmatis, com o objetivo de detectar um efeito antimicrobiano relacionado ao caldo de fermentação de A. fumigatus. O uso direto deste substrato em ensaios de antibiose contra a estirpe H37Rv de M. tuberculosis (ATCC 27294) permitiu a detecção de atividade antimicrobiana com uma concentração inibitória mínima de 256 μg mL-1, demonstrando que os processos de purificação desenvolvidos pelo Biotage Flash Chromatography System são técnicas robustas e confiáveis para purificar compostos de fontes naturais. Além disso, este sistema cromatográfico pode ser usado em combinação com testes bioquímicos específicos, melhorando a busca de resultados confiáveis. Concluímos que esta fração pode expressar uma ampla gama de ação, inibindo ambas as espécies de Mycobacterium utilizadas como organismos-alvo.

Effects of culture filtrates of endophytic fungi obtained from Piper aduncum L. on the growth of mycobacterium tuberculosis

Substances that inhibit the growth of Mycobacterium tuberculosis could potentially be used as antibiotics. These substances could also be added to test culture media to improve the speed of tuberculosis diagnosis. The aim of this work was to investigate the influence of culture filtrates of endophytic fungi isolated from P. aduncum L. on the growth of M. tuberculosis. To achieve this objective, the following methodology was used: a) endophytic fungi were isolated from the leaves and stems of P. aduncum L.; b) the isolated fungi were submitted to submerged bioprocessing; c) culture filtrates from the bioprocess were assayed to evaluate their effect on the growth of M. tuberculosis. We isolated 315 fungal types, which represented 85 morphologies, from different parts of P. aduncum L. The bioassays were performed on 82 culture filtrates and 6 plant extracts and resulted in the detection of 1 culture filtrate that stimulated the growth of M. tuberculosis and 15 that inhibited microbial growth. None of the phytochemical extracts had an effect on the growth of M. tuberculosis. In conclusion, we observed that the endophytic fungi isolated from P. aduncum L. (Piperaceae) produced extracellular metabolites (present in the culture filtrate) that affect the growth of M. tuberculosis. These compounds have the potential to be used as antimicrobials or in the diagnosis of tuberculosis.

PKO: alternative method for isolating mycobacteria from sputum

We elaborated an alternative culture method, which we denominated PKO (initials in tribute of respect to Petroff, Kudoh and Ogawa), for isolating Mycobacterium tuberculosis from sputum for diagnosis of pulmonary tuberculosis (TB), and to compare its performance with the Swab and Petroff methods. For the technique validation, sputum samples from patients suspected of pulmonary TB cases were examined by acid-fast microscopy (direct and concentrated smear), PKO, Swab and Petroff methods. We found that Petroff and PKO methods have parity in the effectiveness of M. tuberculosis isolation. However, by the PKO method, 65 percent of isolated strains were detected in a period of £15 days, while by the Petroff method the best detection was in an interval of 16-29 days (71 percent). In positive smear samples, the average time of PKO isolation is only superior to the one related for Bactec 460TB. In conclusion, the exclusion of the neutralization stage of pH in the PKO reduces the manipulation of the samples, diminishes the execution time of the culture according to the Petroff method and facilitates the qualification of professionals involved in the laboratorial diagnosis of Tuberculosis.

Foi elaborado um método de cultivo alternativo, denominado por nós PKO (iniciais referentes à Petroff, Kudoh e Ogawa), para o isolamento do Mycobacterium tuberculosis em amostras de escarro para o diagnóstico da tuberculose pulmonar (TB). Para validação da técnica, amostras de escarro de pacientes suspeitos de TB foram submetidas aos métodos de baciloscopia (direta e pós-concentração), PKO, Swab e Petroff. A análise comparativa entre o método de Petroff e o PKO mostrou paridade de resultados em relação ao isolamento e número de colônias de M. tuberculosis. Porém, pelo método PKO, 65 por cento das cepas isoladas foi detectada em um período £15 dias, enquanto que pelo método de Petroff a melhor detecção ocorreu em um intervalo de 16-29 dias (71 por cento). O tempo médio de isolamento pelo PKO é somente superior ao sistema comercial Bactec 460TB em amostras positivas na baciloscopia. A exclusão da etapa de neutralização de pH no método PKO reduz a manipulação das amostras, diminui o tempo de execução do cultivo em relação ao de Petroff e facilita o treinamento de profissionais que realizam o diagnóstico laboratorial da TB.