Abstract. Tuberculosis is considered one of the most important epidemiological diseases worldwide. The current treatment for tuberculosis is a combination of antituberculosis drugs for at least 9 months. Nowadays, the search for new pharmacological agents for the treatment of tuberculosis is an urgent endeavor. Microalgae produce a wide variety of molecules that possess biological activities of pharmaceutical interest. Therefore, the objective of this work was to evaluate the antimycobacterial activity of hexane crude extracts of 6 microalgae: Chlamydomonas mexicana, Porphyridium cruentum, Isochrysis galbana, Rhodomonas sp., Aphanocapsa marina, and Nitzschia palea. All of the hexane extracts inhibited at least 90% of the growth of Mycobacterium tuberculosis H37Rv at a concentration of 100 μg ml-1. The hexane crude extract of I. galbana had the highest antimycobacterial activity displaying a percentage inhibition that was equal to that of the antituberculosis drug isoniazid and having a minimum inhibitory concentration of 50 μg ml-1. These findings demonstrate that microalgae are an excellent source for the search of novel antimycobacterial compounds.

Resumen. La tuberculosis es considerada una de las enfermedades epidemiológicas más importantes a nivel mundial. El tratamiento actual para la tuberculosis consiste en la combinación y uso de fármacos antituberculoso por al menos 9 meses. En la actualidad, la búsqueda de nuevos agentes farmacológicos para el tratamiento de la tuberculosis es una tarea urgente. Las microalgas producen una amplia variedad de moléculas que poseen actividades biológicas de interés farmacéutico. Por lo tanto, el objetivo de este trabajo fue evaluar la actividad antimicobacteriana de los extractos crudos hexánicos obtenidos de 6 microalgas: Chlamydomonas mexicana, Porphyridium cruentum, Isochrysis galbana, Rhodomonas sp., Aphanocapsa marina y Nitzschia palea. Todos los extractos crudos inhibieron al menos el 90% del crecimiento de Mycobacterium tuberculosis H37Rv a una concentración de 100 μg ml-1. El extracto crudo obtenido de I. galbana presentó la mayor actividad antimicobacteriana con un porcentaje de inhibición igual al del fármaco antituberculoso isoniazida y con una concentración inhibitoria mínima de 50 μg ml-1. Estos hallazgos demuestran que las microalgas son una excelente fuente para la búsqueda de nuevos compuestos antimicobacterianos.

Abstract Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 μg), goat (HU50 = 13.2 ± 0.3 μg), rabbit (HU50 = 34.7 ± 0.5 μg), and human (HU50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.

The axenic microalgae cultures are a difficult task and they are hard to maintain. Microalgae cultures with reduced bacterial load can be an option to axenic microalgae cultures to produce compounds with biotechnological and pharmaceutical potential. Also they can be used for cryopreservation and in biochemical, physiology, ecology and genetic studies. The aim of this study was to develop a protocol to decrease the bacterial load in an Isochrysis galbana culture, through washes by centrifugation and the administration of various antibiotics (ampicillin, neomycin, kanamycin, chloramphenicol, sulphate G418, streptomycin, and carbencillin), at several doses and combinations. The concentrations of heterotrophic bacteria and I. galbana cell densities were monitored daily. Maximum non-lethal concentration and lethal concentration 50% (LC50) were calculated. Individually, antibiotics and washes by centrifugation failed to reduce bacterial load, but their combination removed bacteria from the cultures. Peak survival (84.6 ± 1.4%) and reduction of bacterial load in I. galbana cultures were effected with the combination of 5 washes by centrifugation and administration of a cocktail, comprising ampicillin, kanamycin, neomycin, and streptomycin at 48 h. Values of maximum non-lethal concentration varied from 75 to 106 µg mL-1 and LC50 between 194 and 332 µg mL-1, thus, our protocol is an effective and rapid method of producing I. galbana cultures with reduced bacterial load.

La obtención de cultivos axénicos es una tarea laboriosa y resultan difíciles de mantener. Los cultivos de microalgas con carga bacteriana reducida pueden ser una alternativa a los cultivos axénicos de microalgas para producir compuestos con potencial biotecnológico y farmaceútico. También éstos pueden ser usados para la criopreservación, y en estudios bioquímicos, fisiológicos, ecológicos y genéticos. El propósito de este estudio fue desarrollar un protocolo para reducir la carga bacteriana en cultivos de Isochrysis galbana por medio de lavados por centrifugación y la administración de varios antibióticos (ampicilina, neomicina, kanamicina, cloranfenicol, sulfato G418, estreptomicina y carbencilina), adicionados en varias dosis y combinaciones. Todos los tratamientos fueron realizados por triplicado. La concentración de bacterias heterotróficas y la densidad de células de I. galbana fue evaluada diariamente. Se calculó la concentración no letal máxima y la concentración letal (LC50). De forma individual, los antibióticos y los lavados por centrifugación no fueron efectivos para reducir la carga bacteriana, pero su combinación removió las bacterias de los cultivos. El máximo de sobrevivencia (84,6 ± 1,4%) y la reducción de la carga bacteriana en I. galbana fue efectiva con la combinación de 5 lavados por centrifugación y la administración de un coctel de ampicilina, kanamicina, neomicina, y estreptomicina por 48 h. Los valores de concentración no letal máxima varían entre 75 a 106 µg mL-1 y LC50 se encontró entre 194 y 332 µg mL-1, por lo anterior, este protocolo resulta ser un método efectivo y rápido para obtener cultivos de I. galbana con carga bacteriana baja.

The antibiotic activity of organic extracts from cultures of six species of phytoplankton, Synechococcus elongatus, Synechocystis sp., Amphiprora paludosa, Porphyridium cruentum, Chaetoceros muelleri and Dunaliella tertiolecta was examined. The bacteria used were Gram(-) strains, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, and the Gram(+) strains were Bacillus subtilis, Staphylococcus aureus, and Microccocus luteus. All extracts inhibited the growth of B. subtilis and effected the generation of inhibition halos, ranging in diameter from 7.06 to 15.23 mm, for several bacteria species. Wide-spectrum antibacterial activity was observed in extracts from S. elongatus, Synechocystis sp., A. paludosa, P. cruentum, and C. muelleri. Thus, this five phytoplankton species can be used to control bacteria.