In vitro thrombolytic potential of bioactive compounds from marine Streptomyces sp. VITJS4 / Potencial trombolítico in vitro de compostos bioativos de Streptomyces marinhos sp. VITJS4

The most practical approach to reduce morbidity and mortality of coronary heart disease (CHD) is to delay the process of thrombus by usage of clot-dissolving agents. The necessities of such safer compounds are to be critically examined for thrombolytic activity especially, from marine sources. Thrombolytic agents have been investigated as a possible treatment for thrombus. The aim of this study was to investigate the in vitro thrombolytic potential of Streptomyces sp.VITJS4 (NCIM No. 5574); (ACC No: JQ234978.1) active compounds. The fibrin degradation revealed a clear transparent zone of clearance with 500µg/mL concentration showing 24mm hydrolysis. The thrombolytic effect of Streptomyces sp.VITJS4 compounds was also demonstrated in vitro clot lysis assay where The percent of thrombolysis by the crude extract showed 90±1.7% at the concentration of 1000µg/mL, whereas percent of thrombolysis by streptokinase was found 100± 00%%. The bioactive compounds were further studied for spectrophotometric analysis. The UV-VIS profile showed different peaks ranging from 400-700 nm with different absorption respectively. The data confirmed the presence of both analogues with absorption maxima at 210 and 310 nm. A sensitive method using LC-MS technique was optimized for the separation and identification of bioactive metabolites which was indicated by the fingerprints. The results of the LC-MS analysis provided different peaks determining the presence of compounds with different therapeutic activities. The current study refers the bioactive compound as impressive thrombolytic agent for further laboratory study. Further studies should be conducted to ensure the efficacy and safety of different concentration of bioactive compounds for drug development. Hence the results reported perhaps useful for the discovery of novel thrombolytic drugs from marine origin.

A abordagem mais prática para reduzir a morbidade e a mortalidade da doença arterial coronariana (CHD, do inglês coronary heart disease) consiste em retardar o processo de trombo através da utilização de agentes de dissolução de coágulos. As necessidades de tais compostos mais seguros devem ser criticamente examinadas para a atividade trombolítica, especialmente de fontes marinhas. Agentes trombolíticos tem sido estudados como um possível tratamento para o trombo. O objetivo deste estudo foi investigar o potencial trombolítico in vitro dos compostos ativos do Streptomyces sp.VITJS4 (NCIM No. 5574); (ACC No: JQ234978.1). A degradação da fibrina revelou um clara zona livre transparente com concentração de 500µg/mL mostrando uma hidrólise de 24mm. O efeito trombolítico dos compostos de Streptomyces sp.VITJS4 também foi demonstrado no ensaio in vitro de lise dos coágulos em que a percentagem de trombólise pelo extrato bruto mostrou 90±1.7% a uma concentração de 1000µg/mL, enquanto que a percentagem de trombólise pela estreptoquinase foi de 100± 00%. Os compostos bioativos foram estudados posteriormente através da análise espectrofotométrica. O perfil ultra violeta visível (UV-VIS profile, em inglês) mostrou diferentes picos variando entre 400-700 nm com diferentes absorções respectivamente. Os dados confirmaram a presença de ambos os análogos com absorção máxima em 210 e 300 nm. Um método sensível usando a técnica LC-MS (Liquid chromatography­mass spectrometry) foi otimizado para a separação e identificação metabólitos bioativos que foram indicados pelas impressões digitais (?). Os resultados da análise LC-MS forneceram diferentes picos determinando a presença de compostos com diferentes atividades terapêuticas. O estudo atual refere-se ao composto bioativo como um agente trombolítico impressionante para futuros estudos em laboratório. Estudos futuros devem ser conduzidos para assegurar a eficácia e segurança de diferentes concentrações dos compostos bioativos para o desenvolvimento de drogas. Assim, os resultados reportados talvez sejam úteis para a descoberta de novas drogas trombolíticas de origem marinha.

Actividad antimicrobiana y sinérgica de metabolitos producidos por Streptomyces erythrogriseus M10-77 de origen marino / Antimicrobial and synergetic activity of metabolites produced by marine origin Streptomyces erythrogriseus M10-77

Los metabolitos de un actinomiceto de origen marino, identificado como Streptomyces erythrogriseus cepa M10-77, fueron evaluados por su capacidad antimicrobiana y sinérgica con antibióticos convencionales. Las pruebas de antagonismo se realizaron frente a patógenos multidrogorresistentes (MDR) de origen clínico, siendo muy efectivos principalmente frente a especies patógenas de Staphylococcus y Enterococcus. Se determinó la Concentración Mínima Inhibitoria (CMI) de extractos diclorometánicos frente a los patógenos S. aureus 1094, S. epidermidis 1093 y Staphylococcus coagulasa negativo 348, siendo estos valores de 3,9; 15,7 y 1,9 µg/mL respectivamente. Los componentes del extracto diclorometánico fueron fraccionados parcialmente, obteniéndose hasta 4 fracciones orgánicas (I, II, III, IV), las que mostraron actividades inhibitorias de la cepa referencial S. aureus ATCC 43300. Los bioensayos frente a S. aureus meticilino resistente (MRSA) mostraron actividad sinérgica de la fracción II del extracto con antibióticos betalactámicos y aminoglucósidos, resaltando la repotenciación de la actividad de la bencilpenicilina en 128 veces el valor basal; así como de la gentamicina en 8 veces sobre el valor basal. S. erythrogriseus cepa M10-77 resultó ser un productor de metabolitos antibacterianos de alta potencia y con actividad sinérgica con antibióticos de referencia médica.

Metabolites of a marine actinomycete, identified as Streptomyces erythrogriseus M10-77 strain were evaluated for antimicrobial and synergistic activity with conventional antibiotics. Antagonism tests were conducted against multidrug resistant (MDR) pathogens of clinical origin, being very effective mainly against pathogenic Staphylococcus and Enterococcus. Minimum Inhibitory Concentrations (MIC) of dichloromethane extracts were determined against pathogenic S. aureus 1094, S. epidermidis and coagulase-negative Staphylococcus 1093 348, these values being 3.9; 15.7 and 1.9 μg/mL respectively. The components of dichloromethane extracts were fractionated partially yielding four organic fractions (I, II, III, IV), which showed inhibitory activity against the reference strain S. aureus ATCC 43300. The bioassays against S.aureus methicillin-resistant (MRSA ) produced synergistic activity of the extract fraction II with beta-lactams and aminoglycoside antibiotics, highlighting the upgrading of the activity of penicillin at 128 times above the baseline and gentamicin 8-fold above baseline. S. erythrogriseus M10-77 strain proved to be a producer of antibacterial metabolites with high power and synergistic activity with antibiotics of medical use.

Produção de biocompostos com atividade antimicrobiana de Streptomyces sp. ante isolados de mastite caprina / Production of biocompounds with antimicrobial activity of Streptomyces sp. Isolated in face of goat mastitis

Actinomicetos são um dos principais produtores de enzimas, vitaminas e metabólitos secundários, destacando-se o gênero Streptomyces, o qual tem uma ampla capacidade de produção de antibióticos eficazes no combate a diferentes microrganismos, entre eles o Staphylococcus sp. Em virtude dessa eficiência no combate a patógenos, o objetivo deste trabalho foi avaliar a produção de metabólitos com atividade antimicrobiana produzidos por 67 Streptomyces isolados de liquens da região amazônica, ante isolados de mastite caprina (Staphylococcus aureus) do estado de Pernambuco, Brasil. Foi utilizado um planejamento fatorial para avaliar a influência das fontes de carbono (glicose) 0%, 0,5% e 1% e de nitrogênio (farinha de soja) 1%, 2,5% e 4% na produção dos antimicrobianos, bem como das variáveis pH, biomassa e atividade antimicrobiana. Dos Streptomyces estudados, o DPUA 1566 foi o que se destacou por formação de halos de inibição entre 18 e 26mm ante os isolados de mastite caprina. Foi possível verificar que a fonte de carbono inibiu a produção de antimicrobianos quando submetidos a uma concentração de glicose de 1%; com a retirada desta, os Streptomyces apresentaram uma elevada capacidade de produção de metabólitos com atividade antimicrobiana tendo potencial para o tratamento de mastite caprina.

Actinomycetes are a leading producer of enzymes, vitamins and secondary metabolites, especially the genus Streptomyces, which have a large capacity for the production of natural antibiotics and are effective against various micro-organisms including Staphylococcus sp. Due to this efficiency in combating micro-organisms, the aim of this study was to evaluate the production of metabolites with antimicrobial activity produced by Streptomyces isolated from lichens 67 in the Amazonia, compared to isolates from goat mastitis (Staphylococcus aureus) in the state of Pernambuco, Brazil. We used a complete factorial design to evaluate the influence of concentrations of carbon sources (glucose) at 0%, 0.5% and 1% and nitrogen (soybean flour) at 1%, 2.5% and 4% in the production of antimicrobial metabolites, and the influence of the pH, microbial biomass and activity variables. Of the studied Streptomyces DPUA 1566 what stood out was the formation of inhibition halos between 18 to 26 mm compared to the isolates from goats mastitis. It was noted that the carbon source inhibited the production of antimicrobial metabolites when subjected to a glucose concentration of 1%. However, after the discontinuation, Streptomyces showed a high capacity to produce metabolites with antimicrobial activity, which has an excellent potential for the treatment of mastitis in goats.

Condiciones de cultivo que fomentan la producción de sustancias antimicrobianas en actinomicetos patógenos y del suelo / Culture conditions that enhance the production of antimicrobial substances from pathogenic and soil actinomycetes

En el presente estudio fueron evaluadas 300 cepas de actinomicetos (140 de origen clínico y 160 del suelo). La producción de sustancias antimicrobianas del total de las cepas de actinomicetos fue evaluada utilizando los siguientes caldos: Bennett (M1), (M2), y el caldo levadura extracto de malta-dextrosa YMD (M3). Se ajustaron las condiciones óptimas de cultivo para lograr altas concentraciones de los metabolitos bioactivos. El proceso de fermentación fue utilizado en tiempos de incubación que variaron entre 5 hasta 10 días. La mayor producción de metabolitos bioactivos se obtuvo cuando se utilizó el medio de producción M2. La actividad antimicrobiana se vio favorecida con la adición de dextrosa, peptona proteosada y extracto de levadura y fosfato de potasio (K2P04), sulfato de magnesio (MgSO4) x 7H2O y carbonato de calcio (CaCO3). Se relacionaron los patrones de crecimiento, la actividad antimicrobiana y la producción de biomasa en las cepas de actinomicetos en los tres medios de cultivo utilizados. Las cepas que presentaron fuerte actividad antimicrobiana contra la mayoría de las bacterias grampositivas, gramnegativas y hongos, fueron seleccionadas para futuros estudios donde se realizará la extracción, purificación y caracterización de los metabolitos bioactivos producidos.

The present study evaluated 300 actinomyces strains (140 from clinical samples and 160 from soil samples). The production of antimicrobial substances by the total actinomyces strains was evaluated using the following broths: Bennet (M1), (M2), and malt-dextrose yeast extract broth YMD (M3). Cultures were adjusted to optimal conditions in order to obtain high bioactive metabolite concentrations. The fermentation process was used with incubation periods which varied between 5 and 10 days. The highest bioactive metabolite production was obtained when the M2 medium was used. Antimicrobial activity was favored with the addition of dextrose, proteose peptone, yeast extract, potassium phosphate (K2PO4), magnesium sulfate (MgSO4) x 7H2O and calcium carbonate (CaCO2). Growth patterns, antimicrobial activity, and biomass production of the actinomyces were related to the three culture media used. The strains which showed strong antimicrobial activity against most gram positive and gram negative bacteria and fungi were selected for future studies where the bioactive metabolites produced will be extracted, purified, and characterized.

Isolamento e quantificação das populações de bactérias em geral e de actinomicetos presentes no solo / Aislamiento y cuantificación de poblaciones de bacterias en general y de actinomicetos presentes en el suelo / Isolation and quantification of actinomycetes and bacterial populations in general present in soil

O solo é um ecossistema complexo, dinâmico e difícil de ser estudado, a quantidade e diversidade de micro-organismos do solo são imensas. A microbiota do solo é caracterizada pela abundância e diversidade. A atividade microbiana é utilizada como uma maneira de melhor entender os processos de mineralização e observar mais profundamente a intensidade dos fluxos de energia no solo. A fertilidade natural do solo depende, portanto, da dinâmica de matéria orgânica e ciclagem de nutrientes, os quais são catalizados pela biomassa microbiana do solo. Dentre as bactérias existentes no solo, encontram-se as autotróficas, as heterotróficas, as aeróbias, as anaeróbias, as fixadoras de nitrogênio, nitrificantes e desnitrificantes, actinomicetos, entre outras. Estudos ecológicos das populações de actinomicetos são de extrema importância, visto que esses micro-organismos se destacam pela possibilidade de sintetizar vitaminas, substâncias inibidoras da atividade enzimática e antibióticos. A determinação das densidades populacionais na comunidade microbiana dos solos é importante, tanto na identificação de fatores que influenciam o equilíbrio microbiológico dos solos, como na caracterização das relações entre grupos e espécies de microrganismos. Portanto, é de grande relevância a quantificação das bactérias em geral e actinomicetos presentes no solo para que se possa inferir sobre a fertilidade do solo. Estudos dessa natureza podem fornecer subsídios para programas de conservação de áreas florestadas e recuperação de áreas degradadas.

Soil is a complex, dynamic and hard-to-study ecosystem due to the immense quantity and diversity of microorganisms. Soil microbiota is characterized by abundance and diversity. Microbial activity is used as a way to better understand the mineralization processes and have an in-depth visualization of the intensity of energy flows in the soil. The natural fertility of the ground depends, therefore, on the dynamics of organic matter and nutrient recycling, which are catalyzed by the soil microbial biomass. Among the bacteria on the soil, it is possible to find autotrophic, heterotrophic, aerobial, anaerobial, nitrogen-fixing, nitrifying and denitrifying, actinomycetes, and others. Ecological studies of actinomycete populations are of extreme importance, since these microorganisms are outstanding due to their ability to synthesize vitamins, enzymatic activity inhibitors and antibiotics. Determining microbial population densities is important both in the identification of factors which influence the microbiological balance in the soil and in the characterization of relationships among groups and species of microorganisms. Therefore, the quantification of bacteria in general and actinomycetes from the soil is of great importance, since it may enable inferences about soil fertility. Studies of this nature may provide subsidies for conservational programs in forested areas and degraded areas in recovery processes.

El suelo es un ecosistema complejo, dinámico y difícil de ser estudiado, ya que la cantidad y diversidad de microorganismos del suelo son inmensas. La microbiota del suelo se caracteriza por la abundancia y diversidad. La actividad microbiana es utilizada como una forma de entender mejor los procesos de mineralización y visualizar más profundamente la intensidad de los flujos de energía en el suelo. La fertilidad natural del suelo depende, por lo tanto, de la dinámica de materia orgánica y ciclo de nutrientes, los cuales son catalizados por la biomasa microbiana del suelo. Entre las bacterias existentes en el suelo, se encuentran los autótrofos, heterótrofos, aérobicos, anaeróbicos, fijadoras de nitrógeno, nitrificadoras y denitrificadoras, actinomicetos, entre otros. Estudios ecológicos de las poblaciones de actinomicetos son de extrema importancia, ya que esos microorganismos se destacan por la posibilidad de sintetizar vitaminas, sustancias inhibidoras de la actividad enzimática y antibióticos. La determinación de las densidades poblacionales en la comunidad microbiana de los suelos es importante, tanto en la identificación de factores que influencian el equilibrio microbiológico de los suelos, como la caracterización de las relaciones entre grupos y especies de microorganismos. Por lo tanto, es de gran importancia la cuantificación de las bacterias en general y actinomicetos presentes en el suelo para que se pueda inferir sobre la fertilidad del suelo. Estudios de esta naturaleza pueden proporcionar subsidios para programas de conservación de áreas forestales y recuperación de áreas degradadas.

Clasificación e identificación de especies de actinomicetos: Un estudio fenotípico comparativo / Classification and identification of Actinomyces species: A comparative phenotype study

Las pruebas fisiológicas y bioquímicas constituyen dos de las principales metodologías utilizadas, principalmente en los laboratorios de microbiología, para la identificación y diferenciación de los actinomicetos. La finalidad de este trabajo fue comparar y evaluar los métodos fenotípicos que son utilizados de manera rutinaria en la identificación de estos microorganismos. Se estudiaron setenta y tres cepas de actinomicetos provenientes de tres laboratorios de microbiología de Venezuela. El comportamiento fisiológico y bioquímico de las cepas en estudio fue evaluado mediante pruebas de descomposición de diferentes sustratos. Los resultados obtenidos permitieron observar diferencias en la identificación preliminar de las cepas realizada por estos laboratorios, conduciendo algunas veces a una nueva identificación de las mismas, gracias a la utilización de estos sustratos. Se pudo observar que la metodología empleada permitió la reclasificación taxonómica de casi todas las cepas estudiadas, lo que sugiere que es necesario estandarizar la metodología de identificación para los actinomicetos.

Physiologic and biochemical tests constitute two of the main methodologies, mainly used in microbiology laboratories, for the identification and differentiation of actinomyces. The purpose of this work was to compare and evaluate the phenotypic methods used routinely for the identification of these microorganisms. The study included seventy three actinomyces strains from three microbiology laboratories in Venezuela. The physiologic and biochemical behavior of the strains was evaluated through decomposition tests using different substrates. The results obtained showed differences in the preliminary identification of the strains done in the various laboratories, leading occasionally to a new identification arisen from the use of these substrates. It was seen that with the methods used it was possible to taxonomically reclassify almost all the strains studied, suggesting that it is necessary to standardize the methods for the identification of actinomyces.

Comunidades de hongos actinomicetos en tres tipos de vegetación de la Amazonia colombiana: abundancia, morfotipos y el gen 16s ADNr

Communities of Actynomicetes fungy in three vegetation types of the Colombian Amazon: abundance, morphotypes and the 16s rDNA gene. Among soil microorganisms, Actinomycetes play an important role in the sustainability of natural and agricultural systems: decomposition of organic matter; degradation of recalcitrant compounds like lignin; nitrogen fixation; degradation of agricultural chemicals and biological control in plants and animals. We evaluated their diversity in soils under three different vegetation covers (pasture, tropical primary forest and stubble) at two depths in the Southern Colombian Amazon border. We collected five replicates per vegetation type (in each, three samples at 0-20cm and three at 20-30cm; for a total of 30 samples). Abundance and phenotypic diversity were determined by plate counting. Genomic DNA was extracted from the isolates: the 16s rDNA gene was amplified with specific primers, and its genetic diversity was estimated by means of an amplified restriction analysis (ARDRA). Actynomicetes abundance varied with vegetation and depth, possibly reflecting presence of earthworms, macro-fauna and physico-chemical characteristics associated to fertility, as well as organic matter, total bases, and optimal capacity to cationic interchange. Primary forests had the highest diversity. Sixteen morpho-types (six genera) were identified; Streptomyces was the most abundant everywhere. The heterogeneity of ARDRA patterns prevented species identification because of the intra-species variability in sequences of 16s rDNA operons. This community is a biological indicator of landscape alteration and could include new bio-active compounds of pharmaceutical interest. Rev. Biol. Trop. 57 (4): 1119-1139. Epub 2009 December 01.

Los actinomicetos son importantes en la sostenibilidad de sistemas naturales. Su diversidad fue evaluada en suelos de bosque, pastizal y rastrojo, y dos profundidades en el Sur del Trapecio Amazónico Colombiano. Se analizaron suelos de cinco repeticiones por cobertura para un total de 15 unidades. Se tomaron seis muestras en cada unidad y dos profundidades, para un total de 30. Los actinomicetos cultivables se determinaron por recuento en placa, se extrajo ADN, se amplificó el gen ADNr 16s y su diversidad genética se estimó por ARDRA. Hubo diferencias de abundancia entre coberturas y profundidades, relacionadas con la vegetación, presencia de lombrices, macrofauna, altos niveles de materia orgánica, y bases totales. Se obtuvieron valores de diversidad fenotípica similares para las tres coberturas, pero los bosques son más diversos. Se identificaron 16 morfotipos, agrupados en séis géneros, siendo Streptomyces el más abundante. La heterogeneidad de los patrones ARDRA no permitió la asignación de especies, reflejándose variaciones en las secuencias de diferentes operones ADNr 16s en un mismo organismo. Las perturbaciones en la cobertura influyen sobre los actinomicetos, generando cambios en su abundancia y diversidad. Su importancia ecológica permite proponerlos como indicadores biológicos de alteración del paisaje.

Comparación de la flora microbiana a partir de dos metodologías para el tratamiento de residuos sólidos domiciliarios en Garagoa, Boyacá / Comparing between microorganisms from two domiciliary solid remainder methodology, for treatment in Garagoa, Boyacá

El objetivo de esta investigación consistió en comparar la flora microbiana a partir de la metodología convencional implementada en Garagoa, Boyacá, para el tratamiento de residuos sólidos domiciliarios con una metodología experimental que consistió en humectar los residuos con agua en lugar de lixiviado, y no mezclar el material por tratar con otro de distintos tiempos de compostaje; para tal fin, se caracterizaron y determinaron los tamaños poblacionales de heterótrofas totales, pseudomonas, hongos y actinomicetos a partir de muestras tomadas en distintas etapas del proceso. De las distintas muestras analizadas se identificaron hongos de los géneros Cladosporium y Aspergillus, propios del proceso de compostaje, Fusarium, Scopulariopsis, Moniliella, Curvularia y levaduras del género Rhodothorula.En el agar ISP2 (para el aislamiento de actinomicetos) se identificaron diversos morfotipos: bacilos esporulados, bacilos gramnegativos, cocobacilos, cocos grampositivos y una bacteria del género Streptomyces. De acuerdo con los resultados de los tamaños poblacionales de los grupos de microorganismos estudiados no se presentaron diferencias significativas entre los tratamientos evaluados; sin embargo, el mayor promedio de los recuentos se presentó en el tratamiento experimental.

This research was aimed at comparing microorganisms from conventional household solid waste treatment in Garagoa, Boyacá, using an experimental methodology consisting of moistening waste with water instead of lixiviate and not mixing the material to be treated with other material having different composting times (i.e. organic fertilizer). Complete heterotopous, pseudomona, fungi and actinomycete population size were thus characterised from samples taken from different stages of the process. Fungi from the Cladosporium and Aspergillus genera (from composting) were identified from this analysis, as well as Fusarium, Scopulariopsis, Moniliella, Curvularia and yeasts from the Rhodothorula genera. Some morphotypes were identified in the ISP2 agar (for the actinomycete isolate): sporulated bacilli, gram-negative bacilli, cocobacilli, gram-positive coci and one bacterium from the Streptomyces genus. According to the results regarding the population sizes of the microorganism groups studied, no meaningful differences were presented between the treatments being tested; however, the experimental treatment presented the highest average count.

Streptomycetes antagonism against Cladosporium fulvum Cooke and Fusarium oxysporium f.sp. lycopersici / Antagonismo de estreptomicetos a Cladosporium fulvum Cooke e Fusarium oxysporium f.sp. lycopersici

This research aimed to evaluate the secondary effects of secondary metabolites produced by streptomycetes on spore germination and mycelial growth of the phytopathogenic fungi Cladosporium fulvum Cooke and Fusarium oxysporium f. sp. lycopersici from tomato plants. Metabolites produced by streptomycete isolates codified as AC-147 and AC-92 caused 94.1 percent inhibition of C. fulvum while AC-95 isolate caused 33.9 percent inhibition. AC-92 was the most efficient for F. oxysporum f. sp. lycopersici, causing 94.2 percent inhibition of spore germination. For mycelial growth, AC-26 and AC-92 were the most efficient in inhibiting C. fulvum growth by 46.6 percent and F. oxysporum f. sp. lycopersici by 29.9 percent. These streptomycetes are potential agents for biocontrol development methods of these tomato plant pathogenic fungi.

Este trabalho teve como objetivo avaliar o efeito de metabólitos secundários produzidos por estreptomicetos na germinação de esporos e no crescimento micelial dos fungos Cladosporium fulvum Cooke e Fusarium oxysporum sp. f. lycopersici da cultura do tomateiro. Metabólitos produzidos pelos isolados AC-147 e AC-92 causaram 94,1 por cento de inibição da germinação de esporos de C. fluvum, enquanto que o isolado AC-95 causou 33,9 por cento de inibição. O AC-92 foi o mais eficiente para F. oxysporum f. sp. lycopersici, causando 94,2 por cento de inibição na germinação de esporos. Para o crescimento micelial, AC-26 e AC-92 foram os mais eficientes na inibição dos fungos C. fulvum, em 46,6 por cento, e F. oxysporum f. sp. Lycopersici, em 29,9 por cento. Esses estreptomicetos são potenciais agentes para o desenvolvimento de métodos de controle biológico desses fungos fitopatogênicos do tomateiro.

Alkalithermophilic actinomycetes in a subtropical area of Jujuy, Argentina / Actinomicetos termoalcalófilos del área subtropical de Jujuy, Argentina

The objective of this study was to examine the alkalithermophilic actinomycete communities in the subtropical environment of Jujuy, Argentina, characterized by sugarcane crops. Laceyella putida, Laceyella sacchari, Thermoactinomyces intermedius, Thermoactinomyces vulgaris and Thermoflavimicrobium dichotomicum were isolated on the media with novobiocin, from sugar cane plants and renewal rhizospheres, and grass and wood soils. Soil pH was almost neutral or lightly alkaline, except for grass soil acidified by lactic liquor. A smaller number of actinomycetes was found on the living plants and bagasse (recently obtained or stored according to the Ritter method) with respect to decomposed leaves on the soil. Thermophilic species of Laceyella, Thermoactinomyces, Thermoflavimicrobium, Saccharomonospora, Streptomyces and Thermononospora were isolated on the media without novobiocin, from composted sugar cane residues. Air captured near composted bagasse piles, contained alkalithermophilic actinomycete spores.

El objetivo de este trabajo fue examinar los actinomicetos termoalcalófilos presentes en el área subtropical de Jujuy, Argentina, caracterizada por el cultivo de la caña de azúcar. Se aislaron en medio con novobiocina las especies Laceyella putida, Laceyella sacchari, Thermoactinomyces intermedius, Thermoactinomyces vulgaris y Thermoflavimicrobium dichotomicum a partir de la rizósfera de plantas y de renuevos de caña de azúcar, así como de suelos de pastura y de monte natural. El pH de los suelos era casi neutro a ligeramente alcalino, excepto en un solo caso en que el suelo estaba acidificado por licor láctico. El número de actinomicetos encontrados sobre los tejidos vivos y en el bagazo recién obtenido o almacenado según el método de Ritter fue pequeño en comparación con el observado sobre las hojas en descomposición. L. sacchari predominó respecto de T. vulgaris. Se aislaron especies termoalcalófilas de Laceyella, Thermoactinomyces, Thermoflavimicrobium, Saccharomonospora, Streptomyces y Thermononospora de los residuos compostados de caña de azúcar utilizando medio sin novobiocina. El aire capturado cerca de pilas de bagazo en compostaje contenía esporos de estos organismos.

Caracterização morfológica, fisiológica e infectividade em planta de estirpes de Frankia isoladas de nódulos de Casuarina / Morphological, physiological and plant infectivity characterization of Frankia strains isolated from Casuarina’s nodules

Frankia são microrganismos capazes de fixar N2 quando em simbiose com espécies arbóreas, denominadas plantas actinorrízicas. Este estudo foi conduzido para caracterizar morfologicamente, fisiologicamente e avaliar a nodulação de quatro estirpes referência de Frankia (HFPCcI3, JCT287, KB5 e F59) e 12 estirpes(IPRF) isoladas de nódulos de Casuarina. Todas as estirpes (Referência e IPRF) foram Gram-positivas e 50% foram “acid-fast”. As estirpes de Frankia alcalinizaram o meio de cultura, exceto IPRF006, IPRF008e IPRF010. As colônias das estirpes F59, IPRF002, IPRF004, IPRF005 e IPRF011, produziram melanina. Entre as estirpes referência, apenas a JCT287 cresceu no meio de cultura com pH 5,5, enquanto no meio com pH 6,0 as duas, JCT287 e KB5, apresentaram crescimento. A análise de regressão mostrou uma relação linear (Y = 67,56+ 3,88X e R2=0,5862, p<0,05) e coeficiente de correlação de Pearson (r=0,766,p<0,05) entre número de esporos e hifas (120 a 480 UFC mL-1) e proteína total (18 a 145 µg mL-1). Foi observado que a estirpe F59 apresentou uma maior proporção de proteína total (50%) do que a JCT287(7,0%) e, que as estirpes IPRF mostraram valores entre 17,5 e 29,3%. Todas as estirpes produziram compostos indólicos com valores variando de 5,9 a 98,8 µM.

Frankia are soil microorganisms that form symbiosis with roots of tree species called actinorhizal plants and are capable of fixing atmospheric N2. This study was carried out to characterize morphologically, physiologically and to assess the nodulation of four Frankia reference strains (HFPCcI3, JCT287, KB5and F59) and 12 (IPRF) isolated from root nodules of Casuarina plants. All strains (Reference and IPRF)were characterized as Gram-positive and 50% as acid-fast. The Frankia strains produced alkali in the culture medium, except the IPRF006, IPRF008 and IPRF010. The colonies of strains F59, IPRF002, IPRF004,IPRF005, and IPRF011 produced melanin. Among reference strains, only JCT287 grew in culture mediawith pH 5.5, while with pH 6.0 both strains JCT287 and KB5 presented growth. The regression analysis showed a linear relationship (Y = 67.56+ 3.88X and R2=0.5862, p<0.05) and Pearson’s correlation coefficient(r=0.766, p<0.05) between number of spores and hyphae (120 to 480 UFC mL-1) and total protein (18 to 145µg mL-1). It was observed that the strains F59 had a higher proportion of total protein 50.0% than JCT287with 7.0% and that the IPRF strains showed values between 17.5 and 29.3%. All strains presented ability to produce indolic compounds in growth media with values ranging from 5.9 to 98.8 µM.

Secretion of an alkaline protease from a salt- tolerant and alkaliphilic, Streptomyces clavuligerus strain Mit-1

An alkaliphilic and salt- tolerant actinomycete, Streptomyces clavuligerus strain Mit-1, was isolated from Mithapur, the western coast of India. The organism was Gram-positive, having filamentous, long thread like structure. The sporulation started after two days of growth and the optimum level of alkaline protease (130 U/ml) was produced during the early stationary phase. The strain could grow and produce protease with 0-10 percent NaCl (w/v), the optimum being 5 percent NaCl (w/v). Growth and protease production was optimum at pH 9 with substantial decline at neutral pH. Sucrose and gelatin were the best carbon and nitrogen sources respectively, whereas gelatin broth was the preferred medium for protease production. Mit-1 produced substantial protease with various amino acids, when employed as the sole nitrogen sources. Crude substrates, such as molasses, whey and wheat flour had significant effect on enzyme production. The results are quite valuable, as only few actinomycetes, particularly salt-tolerant alkaliphilic ones, have so far been explored for their enzymatic potential and process optimization.

Uma cepa halotolerante e alcalifílica de Streptomyces clavuligerus, Mit-1, foi isolada em Mithapur, na costa oeste da Índia. Esse microrganismo é Gram positivo e apresenta estrutura filamentosa na forma de longas cordas. A esporulação iniciou após dois dias de cultivo e o nível ótimo de produção de protease alcalina (130 U/ml) foi atingido no início da fase estacionária de crescimento. A cepa foi capaz de multiplicar com 0-10 por cento NaCl (w/v), com um ótimo de 5 por cento NaCl (w/v). O ótimo de crescimento e produção de protease foi atingido em pH 9, apresentando declínio substancial em pH neutro. Sacarose e gelatina foram as melhores fones de carbono e nitrogênio, respectivamente, enquanto o caldo gelatina foi o melhor meio para produção de protease. A cepa Mit-1 produziu bastante protease quando vários aminoácidos foram empregados como única fonte de nitrogênio. Substratos crus, como melaço, soro de leite e farinha de trigo, tiveram um efeito significativo na produção da enzima. Os resultados são bastante interessantes, considerando que somente poucos actinomicetos, especialmente os halotolerantes, já foram explorados por seu potencial de produção de enzimas e otimização de processos.

Actinomicetoma por Nocardia sp: informe de cinco casos tratados con imipenem solo o combinado con amikacina / Actinomycetoma and Nocardia sp: report of five cases treated with imipenem or imipenem plus amikacin

Antecedentes: El tratamiento estándar del actinomicetoma es dapsona con trimetoprim/sulfametoxazol. En casos seleccionados amikacina, estreptomicina, kanamicina, amoxicilina/ácido clavulánico o fosfomicina. El imipenem ha mostrado tener actividad in vitro e in vivo contra algunos actinomicetos; tiene un efecto sinérgico combinado con amikacina. Objetivos: Comunicar la respuesta al tratamiento con imipenem solo o combinado con amikacina en micetomas por Nocardia sp graves o multirresistentes. Material y métodos: Presentamos cinco pacientes con actinomicetoma que habían recibido múltiples tratamientos. Se hospitalizaron tres semanas para recibir imipenem (500 mg cada ocho horas) intravenoso por vía periférica por 21 días. En dos casos se combinó con amikacina. Resultados: Tres pacientes fueron hombres y dos mujeres. En tres casos la localización fue en el dorso, uno de ellos con afección ósea y pulmonar; en un caso hubo afección de la pared abdominal y en otro en la región cervical posterior. La evolución promedio fue de 7.4 años. En dos casos se logró curación clínica y bacteriológica a un año de seguimiento. En el paciente con afección pulmonar también hubo mejoría radiográfica. En el resto de los casos se logró cierre de la mayoría de las fístulas y una disminución importante de la inflamación, aunque hubo presencia de granos con cultivo negativo. Ningún tratamiento provocó efectos colaterales. Conclusiones: El imipenem es un antibiótico de amplio espectro y consideramos que es una buena alternativa para tratar actinomicetomas graves, resistentes al tratamiento habitual o con complicaciones viscerales.

INTRODUCTION: Dapsone with trimethoprim-sulfamethoxazol is currently the standard treatment for actinomycetoma. In select cases, amikacin, streptomycin, kanamycin, amoxicillin/clavulanic acid or phosphomycin may be also added. Imipenem has shown to be effective both in vitro and in vivo against some actinomycetes. Amikacin with Imipenem has a synergistic effect. OBJECTIVES: To report our preliminary findings using imipenem alone or with amikacin for severe or multi-resistant mycetomas due to Nocardia sp. MATERIAL AND METHODS: We present 5 cases of chronic mycetoma infection previously treated with anti-bacterial multidrug regimens. All patients were hospitalized and treated with imipenem 500 mg IV, three times a day for three weeks. Three patients received in addition amikacin. RESULTS: We included 3 male and 2 female patients. The average length of disease duration was 7.4 years. In 3 cases mycetoma was located on the back; one of them involved the rib and the lung. One case was localized in the abdominal wall, and another one involved the posterior side of the cervical region. Two patients achieved clinical and bacteriological cure one year after treatment with Imipenem, and the remaining three displayed clinical improvement, even though grains were observed, cultures where negative. None of the 5 patients studied showed clinical evidence of adverse reactions to Imipenem. CONCLUSIONS: Imipenem is a strong antibiotic and constitutes an important treatment alternative for severe or multi-resistant mycetoma especially for cases with bone and visceral involvement.