Caracterización bioinformática y producción de L-asparaginasa de Bacillus sp. M62 aislado de las salinas de Maras, Cusco, Perú

El objetivo del estudio fue realizar la caracterización bioinformática, así como optimizar la producción de L-asparaginasa extracelular de Bacillus sp. M62 aislada de las salinas de Maras (Cusco). Para ello, se verificó la producción de L-asparaginasa mediante el viraje del medio M9 modificado con azul de bromofenol 0.0075%, pH 7.4 a 37 °C por 72 h. A la vez, se extrajo el ADN genómico para amplificar los genes ribosómicos 16S y el gen ansA3. La secuencia aminoacídica codificada por el gen ansA3 se predijo mediante análisis bioinformático. La producción de L-asparaginasa intracelular y extracelular se evaluó a diferentes niveles de glucosa, L-asparagina, NaCl y pH en el medio M9 modificado. Adicionalmente, las actividades enzimáticas de L-asparaginasa y L-glutaminasa se determinaron mediante cuantificación del amonio liberado por el método de Nessler. Así, Bacillus sp. M62 produjo el viraje del medio M9 modificado, obtuvo alta similitud y cercanía evolutiva con Bacillus licheniformis, se encontró que el gen ansA3 amplificado codificaba para 319 aa, dentro de la cual se predijo una secuencia patrón del sitio activo (GFVITHGTDTM ) y 15 sitios inmunogénicos. La producción de L-asparaginasa extracelular fue superior a la intracelular, la que se optimizó de 0.37 U/mL (0.24 U/mg) a 2.15 ± 0.39 U/mL (0.63 U/mg). Finalmente, se encontró que Bacillus sp. M62 presenta L-asparaginasa extracelular con mínima actividad de L-glutaminasa.

The aim of this study was to perform bioinformatics characterization and optimize the production of extracellular L-asparaginase from Bacillus sp. M62, isolated from the Maras salt ponds (Cusco). To achieve this, the production of L-asparaginase was verified by the change in color of modified M9 medium, containing 0.0075% bromophenol blue, at pH 7.4 and 37°C for 72 hours. Genomic DNA was extracted to amplify the 16S ribosomal genes and the ansA3 gene. The amino acid sequence encoded by the ansA3 gene was predicted using bioinformatic analysis. The production of intracellular and extracellular L-asparaginase was evaluated at different levels of glucose, L-asparagine, NaCl, and pH in modified M9 medium. Additionally, the enzymatic activities of L-asparaginase and L-glutaminase were determined by quantifying the released ammonium using the Nessler method. Bacillus sp. M62 showed the change in color of the modified M9 medium, high similarity, and evolutionary closeness to Bacillus licheniformis. The amplified ansA3 gene was found to encode for 319 amino acids, with a predicted active site pattern (GFVITHGTDTM) and 15 immunogenic sites. The production of extracellular L-asparaginase was found to be higher than intracellular L-asparaginase and was optimized from 0.37 U/mL (0.24 U/mg) to 2.15 ± 0.39 U/mL (0.63 U/mg). Finally, it was found that Bacillus sp. M62 presents extracellular L-asparaginase with minimal L-glutaminase activity.

Registro fotográfico del gato andino (Leopardus jacobita), en la Reserva Nacional Salinas y Aguada Blanca, Perú

El gato andino es uno de los felinos más raros de América del Sur, habita en los altos Andes de Argentina, Bolivia, Chile y Perú; se encuentra la categoría de En Peligro según la UICN. En Perú, se ha reportado en nueve departamentos y en cuatro áreas naturales protegidas; presentamos dos registros para la Reserva Nacional Salinas y Aguada Blanca, de principios de 2015; la primera cerca del pueblo de Pillones y la segunda cerca de Viscachani. Siendo las primeras imágenes para la Reserva Nacional Salinas y Aguada Blanca, registros anteriores fueron de pieles y ADN fecal. Ambos registros son los más recientes para Perú y las primeras imágenes completas de gato andino.

The Andean cat is a rarest wildcat of South America, categorized as Endangered by IUCN. Its habitat is in the high Andes of Argentina, Bolivia, Chile and Peru. In Peru, it has been reported in nine departments, and in four natural protected areas. We present two records from Salinas y Aguada Blanca National Reserve from early 2015, in two different locations. The first was near the Pillones village and the second near to Viscachani village; those are the first image record for Salinas y Aguada Blanca National Reserve; previous records were from skins and fecal DNA. Both records are the most recent from Peru and the very firsts complete images of full Andean cats live in the county.

Tolerance of Coffea arabica L. seeds to sub zero temperatures / Tolerância de sementes de Coffea arabica L. à temperaturas sub zero

ABSTRACT Preservation of the quality of coffee seeds is hindered by their intermediate behavior in storage. However, long-term storage at sub zero temperatures may be achieved by adjusting the water content of the seeds. The aim of this study was to evaluate the tolerance of coffee seeds to freezing, in relation to physiological and enzymatic modifications. Coffee seeds were dried in two manners, rapid and slow, to water contents of interest, 0.67, 0.43, 0.25, 0.18, 0.11, and 0.05 g H2O g-¹ dw (dry basis). After drying, the seeds were stored at a temperature of -20 ºC and of 86 ºC for 24 hours and for 12 months, and then compared to seeds in cold storage at 10 ºC. The seeds were evaluated through calculation of percentage of normal seedlings, percentage of seedlings with expanded cotyledonary leaves, dry matter of roots and of hypocotyls, and viability of embryos in the tetrazolium test. Expression of the enzymes superoxide dismutase, catalase, and peroxidase were evaluated by means of electrophoretic analysis. Only seeds dried more slowly to 0.18 g H2O g-1 dw present relative tolerance to storing at -20 °C for 12 months. Coffee seeds do not tolerate storage at a temperature of -86 ºC for 12 months. Water contents below 0.11g H2O g-¹ dw and above 0.43 g H2O g-¹ dw hurt the physiological quality of coffee seeds, regardless of the type of drying, temperature, and storage period. Coffee seed embryos are more tolerant to desiccation and to freezing compared to whole seeds, especially when the seeds are dried to 0.05 g H2O g-¹ dw. The catalase enzyme can be used as a biochemical marker to study tolerance to freezing in coffee seeds.

RESUMO A preservação da qualidade de sementes de café é dificultada pelo comportamento intermediário no armazenamento. Porém, a conservação a longo prazo em temperaturas subzero pode ser conseguido com o ajuste do teor de água das sementes. Objetivou-se neste trabalho avaliar a tolerância de sementes de café ao congelamento, com relação às modificações fisiológicas e enzimáticas. As sementes foram submetidas a dois tipos de secagem, rápida e lenta, até os teores de água de interesse, de 0,67, 0,43, 0,25, 0,18, 0,11, 0,05 g H2O g-¹ dw (base seca). Após secagem, as sementes foram armazenadas em temperatura de -20 e de -86 ºC, por 24 horas e por 12 meses, sendo comparadas às sementes armazenadas em câmara fria a 10 ºC. As sementes foram avaliadas pela porcentagem de plântulas normais, plântulas com folhas cotiledonares expandidas, matéria seca de raízes e de hipocótilos e viabilidade dos embriões no teste de tetrazólio. A expressão das enzimas superóxido dismutase, catalase e peroxidase foi avaliada por meio de análise eletroforética. Apenas as sementes secadas lentamente até 0,18 g H2O g-¹ dw apresentam relativa tolerância ao armazenamento a -20 °C por 12 meses. Sementes de café não toleram o armazenamento à temperatura de -86 ºC por 12 meses. Umidades abaixo de 0,11g H2O g-¹ dw e acima de 0,43 g H2O g-¹ dw são prejudiciais à qualidade fisiológica das sementes de café, independentemente do tipo de secagem, temperatura e período de armazenamento. Embriões de sementes de café são mais tolerantes à dessecação e ao congelamento quando comparado às sementes inteiras, principalmente quando as sementes são secadas até 0,05 g H2O g-¹ dw. A enzima catalase pode ser usada como um marcador bioquímico para estudar a tolerância de sementes de café ao congelamento.

Diagnóstico e fatores de risco do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais / Diagnosis and risk factors of bovine taeniasis-cysticercosis complex in Salinas, Minas Gerais, Brazil

Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais, foram coletadas amostras de sangue de 355 bovinos distribuídos em 18 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também um levantamento epidemiológico dos casos de teníase diagnosticados nos laboratórios credenciados pela Secretaria Municipal de Saúde de Salinas, no período de 2007 a 2010. A prevalência de cisticercose bovina foi de 4,70% enquanto as prevalências de teníase, encontradas durante os quatro períodos avaliados, foram de 0,29%, 0,36%, 0,24% e 0,24%. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Foi concluído que a cisticercose bovina está presente no município de Salinas, Minas Gerais, sendo o tratamento térmico ineficiente da carne bovina o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária.(AU)

In order to diagnose the situation of bovine taeniasis-cysticercosis complex in the municipality of Salinas, Minas Gerais, Brazil, blood samples were collected from 355 cattle in 18 randomly selected farms. A socioeconomic questionnaire was filled in each farm for the analysis of factors which favor the maintenance of the taeniasis-cysticercosis complex. An epidemiological survey of human taeniasis was performed through analyses of the Municipal Health Department in the 2007-2010 period. A prevalence of 4.7% for bovine cysticercosis and the frequency of 0.29, 0.36, 0.24 and 0.24% for human taeniasis, during the evaluated period, was found. Among the risk factors, a statistically significant correlation was found between the occurrence of bovine cysticercosis and the ingestion of undercooked meat. It was concluded that bovine cysticercosis is present in the municipality of Salinas, due to inefficient heat treatment of the meat as the main risk factor for maintenance of the taeniasis-cysticercosis complex, reinforcing the need to adopt control measures with continuous epidemiological and health surveillance.(AU)

Análisis microbiológicodel pulpo rojo en puertos pesqueros de Campeche, México / Microbiological analysis of red octopus in fishing ports of Campeche, Mexico

Resumen: Objetivo: Estudiar la calidad microbiológica del pulpo rojo dado su importante impacto económico y social en la región sur-sureste de México. Material y métodos: Se tomaron muestras en diversas zonas de captura de la especie y se analizaron con pruebas bioquímicas descritas en las normas oficiales mexicanas. Se identificaron cepas pertenecientes al género Vibrio, Salmonella, coliformes fecales y E. coli O157:H7. Con el empleo del Sistema BAx, se logró la identificación de microorganismos a través de su ADN bacteriano. Los resultados obtenidos en los métodos bioquímicos y moleculares fueron contrastados. Resultados: El método estadístico de Bland-Altman indicó que ambas técnicas pueden usarse indistintamente. La prueba de McNemar demostró que ambos métodos cuentan con la misma eficacia para la identificación de patógenos (valor X2=0.5 ρ=0.4795). Conclusión: La calidad microbiológica del pulpo en la región sur-sureste de México es deficiente debido a la presencia de flora bacteriana patógena que podría representar un riesgo epidemiológico. Los índices establecidos por las normas sugieren la necesidad de aplicar técnicas de identificación eficaces y rápidas como el Sistema BAx. Este método alternativo de análisis puede coadyuvar a la implementación de estrategias efectivas que permitan cumplir con especificaciones mínimas sanitarias durante el procesamiento de los productos pesqueros, y así elevar los sistemas de control para disminuir los riesgos de brotes epidemiológicos en la región.

Abstract: Objective: In this work we studied the microbiological quality of the red octopus given its important economic and social impact on the region South-Southeast of Mexico. Materials and methods: Samples were taken in different areas of capture of the species and analyzed with biochemical tests described in the Mexican official standards, identifying strains belonging to the genus Vibrio, Salmonella and faecal coliforms, and E. coli O157: H7. We used the BAx System for the identification of microorganisms through their bacterial DNA. The results obtained in biochemical and molecular methods were confirmed. Results: Bland-Altman statistical method pointed out that both techniques can be used interchangeably. McNemar test showed that both methods have the same efficacy for the identification of pathogens (value X2=0.5 ρ=0.4795). Conclusion: The microbiological quality of the octopus in the South-Southeast region of Mexico is deficient due to the presence of pathogenic intestinal flora that might represent an epidemiological risk. The indexes established by the regulations suggest the need to apply effective and rapid identification technologies, such as the BAx System.This alternative method of analysis can contribute to the implementation of effective strategies that allow compliance with the minimal sanitary specifications during the processing of fishing products, thus strengthening the control systems to decrease the risks of epidemiological outbreaks in the region.

Caracterización de bacterias halófilas productoras de amilasas aisladas de las Salinas de San Blas en Junín / Characterization of halophilic bacteria producing amylase isolated from San Blas Salterns in Junin

El objetivo de este estudio fue caracterizar bacterias halófilas con actividad amilolítica provenientes de las Salinas de San Blas-Junín, ubicadas en los Andes peruanos aproximadamente a 4100 m de altitud. Este estudio se realizó con 34 bacterias aisladas de muestras de suelos las cuales se cultivaron en agar agua de sales (SW) 5 % conteniendo extracto de levadura 0,5 % y almidón 1 %. El 41 % de bacterias mostró la capacidad de hidrolizar almidón, éstas fueron caracterizadas mediante pruebas fisiológicas y bioquímicas convencionales. Tres bacterias fueron Gram-negativas y once Gram-positivas. El 21 % (3/14) creció en un amplio rango de concentración de sales, entre 5 y 20 %. El 14 % (2/14) de las bacterias presentó actividad lipolítica, proteolítica y nucleolítica, y el 29 % (4/14), presentó actividad proteolítica y nucleolítica. Las bacterias se identificaron mediante los perfiles de restricción de los genes ribosómicos 16S amplificados, las enzimas usadas fueron Hae III, BstU I, Hinf I y Cfo I. Los genes ribosómicos 16S de siete bacterias que presentaron perfiles de ADN diferentes se amplificaron, secuenciaron y analizaron mediante programas bioinformáticos. Del análisis fenotípico y molecular de las 14 bacterias amilolíticas se obtuvieron dos grupos, uno perteneciente al género Halomonas (3) y el otro, al género Bacillus (11). Las bacterias amilolíticas caracterizadas podrían ser de potencial uso a nivel industrial.

The aim of this study was to characterize halophilic amylolytic bacteria from San Blas Salterns-Junin, located in the Peruvian Andes at approximately 4 100 m of altitude. This study was conducted with 34 bacteria isolated from soil samples which were cultured in salt water medium (SW) 5 % containing 0,5 % yeast extract and 1 % starch. It was found that 41 % were starch-degrading bacteria, which were further characterized with conventional physiological and biochemical tests. Three bacteria were Gram-negative and eleven Gram-positive. Also, 21 % (3/14) was able to grow in a wide range of saltconcentration from 5 to 20 %. We reported that 14 % (2/14) of bacteria had all lipolytic, proteolytic and nucleolytic activity, and 29 % (4/14) had both proteolytic and nucleolytic activity. Bacteria were identified by restriction 16S ribosomal genes profiles, enzymes used were Hae III, BstU I, Hinf I and Cfo I. 16S ribosomal genes of seven isolated wich showed different DNA profiles were amplified, partial sequenced and analyzed using bioinformatic programs. By both phenotypic and molecular analysis of 14 amylolytic bacteria two groups were obtained, one belonged to the genus Halomonas (3) and the other, to the genus Bacillus (11). The characterized amylolytic bacteria could have a potential industrial use.

New species of Tetramorium Mayr (Hymenoptera: Formicidae) from Puebla State, Mexico

Tetramorium notomelanum sp. n. is described from the Tehuacán Valley, state of Puebla, México. Its distribution and relation with other species of the tortuosum-group is discussed. The new species of Tetramonium is described from workers, and distinguished from others of the group by several characters: i) black coloration of the body; ii) size: T. notomelanum sp. n. is smaller than T. hispidum (Wheeler), T. mexicanum Bolton and T.spinosum (Pergande), but larger than T. bicolorum Vásquez-Bolaños and T. placidum Bolton; iii) length of the hairs of the dorsal of the head are equal to the diameter of eye; iv) the length of the hairs on the scape and tibiae less than the width of the appendage where they are located. This is the second species of the tortuosum group of Tetramorium found in the State of Puebla, and the fourth recorded in Mexico.