Resumen Introducción: La actividad antimicrobiana realizada por las bacterias asociadas con los corales, además de promover la salud de su huésped, representa una fuente para obtener nuevos compuestos bioactivos. Objetivo: Analizar la actividad antimicrobiana de las bacterias asociadas con el zoantario Palythoa caribaeorum de los arrecifes de Carapibus, Paraíba, Brasil. Metodología: El análisis filogenético de la bacterias se realizó con base en secuencias parciales del gen RNAr 16S utilizando herramientas moleculares y de bioinformática. La actividad antimicrobiana de las cepas se probó contra cuatro cepas bacterianas y una cepa de levadura: Bacillus cereus (CCT0198), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa y Candida albicans (ATCC 10231), utilizando ensayos antibiosis y antibiograma, y la concentración inhibitoria mínima (CIM) que se determinó por el método de microdilución. Resultados: Las cepas bacterianas pertenecían a Firmicutes (84 %) y Gammaproteobacteria (16 %). Entre 49 cepas se encontraron cinco géneros de bacterias: Bacillus, Vibrio, Pseudomonas, Staphylococcus y Alteromonas. Un total de 19 cepas exhibieron actividad antimicrobiana, siendo el género Bacillus el responsable del mayor número de bacterias antagonistas, con 12 cepas positivas en el ensayo de antibiosis y cuatro en la prueba de antibiograma. El mayor número de bacterias antagonistas se encontró en Bacillus (12 aislamientos), seguido por Vibrio (tres aislamientos) y Pseudomonas (un aisladmiento). El NC8, clasificado como Bacillus subtilis, inhibió todas las cepas estándar en el ensayo de antibiosis y las cepas de B. cereus, S. aureus y C. albicans en la prueba de antibiograma. El material liofilizado del B. subtilis NC8 mostró acción bacteriostática contra B. cereus, con un valor de CIM de 125 μg/μl. En la prueba de citotoxicidad, el grado de hemólisis fue del 4.8 % para el material liofilizado a las concentraciones probadas, lo que indica su baja citotoxicidad. Conclusión: Los resultados muestran el potencial antimicrobiano de algunos aislamientos bacterianos asociados al P. caribaeourum, especialmente los pertenecientes al género Bacillus.

Abstract Introduction: The coral-associated bacteria with antimicrobial activity may be important to promote the health of their host through various interactions, and may be explored as a source of new bioactive compounds. Objective: To analyze the antimicrobial activity of bacteria associated with the zoanthid Palythoa caribaeorum from the coral reefs of Carapibus, Paraiba state, Brazil. Methods: The phylogenetic analysis of the bacteria was conducted based on partial sequences of the 16S rRNA gene using molecular and bioinformatics tools. The antimicrobial activity of the 49 isolates was tested against four bacterial strains and one yeast strain: Bacillus cereus (CCT0198), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa and Candida albicans (ATCC 10231). The antibiosis and antibiogram assays were conducted and the Minimal Inhibitory Concentration (MIC) was determined by the microdilution method. Results: The bacterial isolates belonged to Firmicutes phylum (84 % of the isolates) and the Proteobacteria phylum (16 % of the isolates). Among the 49 isolates five genera were found, with the Bacillus genus being the most abundant (82 % of the isolates), followed by Vibrio (10 %), Pseudomonas (4 %), Staphylococcus (2 %) and Alteromonas (2 %). Antibiosis test revealed that 16 isolates (33 %) showed antimicrobial activity against one or more of five tested reference strains. The highest number of antagonistic bacteria were found in the Bacillus genus (12 isolates), followed by Vibrio (three isolates) and Pseudomonas (one isolate) genera. The B. subtilis NC8 was the only isolate that inhibited all tested strains in the antibiosis assay. However, antibiogram test with post-culture cell-free supernatant of NC8 isolate showed the inhibition of only B. cereus, S. aureus and C. albicans, and the lyophilized and dialyzed material of this isolate inhibited only B. cereus. The lyophilized material showed bacteriostatic activity against B. cereus, with a MIC value of 125 μg/μl, and in the cytotoxicity assay, the hemolysis value was of 4.8 %, indicating its low cytotoxicity. Conclusions: The results show the antimicrobial potential of some bacterial isolates associated with the P. caribaeourum tissue, especially those belonged to Bacillus genus.

ResumenLas cianobacterias se encuentran ampliamente distribuidas en ecosistemas terrestres, de agua dulce y marinos, y en las últimas décadas han sido reconocidas como una poderosa fuente de compuestos bioactivos. En este estudio, las cepas de cianobacterias fueron aisladas a partir de agua de mar, agua salobre y muestras de tejidos de invertebrados bentónicos de arrecifes (zoanthid Protopalythoa variabilis, las esponjas Cynachrella sp. y Haliclona sp., el coral Siderastrea stellata y ascidias) recogidas en los estados de Paraíba y Rio Grande do Norte, en el noreste de Brasil, en el período comprendido entre julio 2010 y febrero 2014. La mayoría de las dieciocho cepas de cianobacterias pertenecían a la Familia Synechococcaceae. Los géneros: Synechocystis, Synechococcus y Romeria estuvieron representados por diez, seis y dos cepas, respectivamente. Las cepas fueron analizadas para la producción de microcistina por ELISA y para la actividad antibacteriana contra Staphylococcus aureus y Pseudomonas aeruginosa por el método de difusión en agar. La detección del gen mcyB, uno de los genes relacionados con la síntesis de microcistina, se realizó mediante la técnica de reacción en cadena de la polimerasa (PCR). El cultivo de las cepas se realizó en cámara de cultivo aclimatada (25 ° C) bajo aireación constante durante 15 días con un fotoperíodo de 12 horas utilizando los medios Conway y BG11 elaborados con agua de mar filtrada. Se observó la producción de microcistina en cinco cepas pertenecientes al género Synechocystis. La presencia del gen mcyB fue detectada en doce cepas de cianobactérias: Synechocystis (tres cepas), Synechococcus (seis cepas) y Romeria (dos cepas). Sólo una cepa (Synechocystis aquatilis) mostró tanto la producción de microcistina como la presencia del gen mcyB. Se observó la actividad antibacteriana de una cepa de Romeria gracilis, de una cepa de Synechocystis aquatilis y dos cepas de Synechococcus sp. Los extractos etanólicos de las cepas de R. gracilis y Synechococcus sp. inhibieron el crecimiento de P. aeruginosa. Entre los extractos metanólicos de cianobacterias solamente S. aquatilis mostró actividad contra S. aureus y R. gracilis contra P. aeruginosa. Varias cepas de cianobacterias estudiadas en este trabajo fueron positivas para la producción de microcistina y actividad antibacteriana frente a bacterias patógenas de S. aureus y P. aeuruginosa, y pueden ser explotadas para aplicaciones biotecnológicas.

AbstractCyanobacteria are widely distributed in terrestrial, freshwater and marine environments, and over the past decades have been recognized as a powerful source of bioactive compounds. In this study, some cyanobacterial strains were isolated from samples of seawater, brackish water and tissue of reef benthic invertebrates (zoanthid Protopalythoa variabilis, the sponges Cynachrella sp. and Haliclona sp., the coral Siderastrea stellata, and ascidians), collected at the states of Paraíba and Rio Grande do Norte (Northeast of Brazil), during the period between July 2010 and February 2014. After standard isolation methods, the cultivation of the strains was carried out in acclimatized culture chamber (25 °C) under constant aeration, for 15 days at 12-hour photoperiod, using Conway and BG11 media made with filtered seawater. The cyanobacterial cells were analysed for the microcystin production by the ELISA technique and their ethanolic and methanolic extracts for the antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa by the agar well diffusion method. The detection of the mcyB gene, one of the genes related to the microcystin synthesis, was done by the Polymerase Chain Reaction (PCR) technique. The majority of the eighteen cyanobacterial strains belonged to Synechococcaceae Family. The genera of Synechocystis, Synechococcus and Romeria were represented by ten, six and two strains, respectively. The production of microcystins was observed in five strains belonging to the genus Synechocystis. The presence of mcyB gene was detected in 12 strains of cyanobacteria: Synechocystis (three strains), Synechococcus (six strains) and Romeria (two strains). Only one strain (Synechocystis aquatilis) showed both the microcystin production and the mcyB gene presence. The antibacterial activity was observed for one strain of Romeria gracilis, one strain of Synechocystis aquatilis and two strains of Synechococcus sp. The ethanolic extracts of R. gracilis strain and two Synechococcus spp. strains inhibited the growth of P. aeruginosa. Among methanolic extracts of cyanobacteria, only one strain of S. aquatilis showed activity against S. aureus, and one R. gracilis strain against P. aeruginosa. Some cyanobacterial strains studied were positive for the microcystin production and antibacterial activity against pathogenic bacteria S. aureus and P. aeuruginosa, and may be further explored for additional biotechnological applications. Rev. Biol. Trop. 65 (3): 890-899. Epub 2017 September 01.

We report here the occurrence of seasonal changes in the pigmentation of colonies of Siderastrea spp. during bleaching events on coastal reefs in northeastern Brazil. Bleached affected almost 50% of coral colonies analyzed in Cabo Branco reefs (Paraiba state) in the summer of 2005. The same phenomenon was detected along various coastal reefs in northeastern Brazil during the summer of 2007 and 2008. These events were seasonal, and began with the emergence of pale colonies that became pale-pink and then purple during the rainy months. The patterns and intensity of colonies pigmentation changes varied with the studied sites. The decrease in zooxanthellae density and chlorophyll-a content was quantified in the colonies with their pigmentation pattern altered (bleaching). Microbiological analyses revealed higher densities of bacteria in pink colonies (bleached) as compared to brown colonies (normal). Environmental disturbances may lead to the pigmentation changes in Siderastrea spp., but the immediate causes of this phenomenon are not clear and require further investigations.

Se relata aquí la aparición de cambios estacionales en la pigmentación de colonias de Siderastrea spp. durante los eventos de blanqueamiento en arrecifes costeros del noreste de Brasil. En verano de 2005, existían casi 50% de colonias blanqueadas en los corales analizados en Cabo Branco, Estado de Paraíba. El mismo fenómeno se detectó a lo largo de varios arrecifes costeros en el noreste del país durante el verano de 2007 y 2008. Estos eventos son asociados a la variación estacional, que comenzaron con la aparición de colonias pálidas que luego se convirtieron en rosa pálido y después a morado, durante los meses de lluvias. Los patrones e intensidad de los cambios en la pigmentación de las colonias fueron diferentes en los sitios estudiados. La disminución de la densidad de zooxantelas y del contenido de clorofila-a fue cuantificada en las colonias y comparados con el patrón de pigmentación alterada (blanqueo). Los análisis microbiológicos revelaron densidades más altas de bacterias en colonias rosas (blanqueada) en comparación con las colonias marrones (considerados normales). Las alteraciones ambientales pueden conducir a estos cambios de pigmentación en Siderastrea spp., pero las causas inmediatas de este fenómeno no están claras y requieren más investigación.

Atividade proteolítica total e biomassa de uma linhagem de Bacillus sp. termofílico foram analisados em vários níveis de pH inicial e temperatura utilizando a metodologia de superfície de resposta. O meio mineral com extrato de levedura (0.01%) e amido (1%) foi utilizado no experimento. Os resultados de análise estatística da metodologia de resposta de superfície definiram um modelo polinomial para a biomassa e atividade proteolítica da linhagem de Bacillus sp. com alto coeficiente de determinação (R² = 0.8), mostrando um ajuste satisfatório do modelo estatístico obtido com os dados experimentais. Os valores de p mostraram que a temperatura e pH tiveram efeito significante em biomassa e atividade total (P <0.05) da linhagem testada. A atividade proteolítica mais alta (2.368 U/ml/h) da linhagem de Bacilus sp. foi prevista pelas curvas de superfície de resposta em temperatura a 41º C e pH igual a 4.8. Os valores de biomassa altos foram previstos para ampla faixa de temperatura e pH.

Proteolytic activity and cell biomass of thermophilic Bacillus sp. strain were evaluated at various levels of initial pH and temperature by applying response surface methodology. The mineral medium containing yeast extract (0.01%) and starch (1%) was used throughout the experiment. The results of statistical analysis revealed the polynomial model with high coefficient of determination (R² = 0.8) for the biomass and total proteolytic activity of the strain studied. This model showed a satisfactory adjustment of the statistic model with the experimental data. The p values showed that the temperature and pH had significant effect on biomass and proteolytic activity (P<0.05) of strain tested. The highest proteolytic activity (2.333 U/ml/h) of the Bacillus sp. was predicted at 41º C and pH 4.8. The high biomass values were observed at broad range of temperature and pH.

O estudo sobre água e sedimento do mangue do rio Paraíba do Norte (Nordeste do Brasil) e carne de caranguejo-uçá (Ucides cordatus) foi conduzido para investigar a relação entre a abundância de bactérias heterotróficas totais e patogênicas no ambiente e na carne de caranguejo. As amostras de água, sedimento e caranguejos U. cordatus foram coletadas simultaneamente em duas áreas do manguezal que diferiam em influencia da maré e do esgoto lançado no mangue. O número de bactérias heterotróficas totais nas amostras de água do mangue variaram de 0,04 a 6 log UFC 100mL-1, e no sedimento de 5 a 6 log UFC g-1. Em local mais próximo a desembocadura do esgoto evidenciou-se maior incidência de bactérias heterotróficas totais, coliformes, Staphylococcus aureus e Salmonella spp. As contagens de bactérias totais nas amostras da carne de caranguejo variaram de 1,3 a 4,4 log UFC g-1, não diferindo estatisticamente entre as áreas estudadas. Nenhuma amostra da carne de caranguejo apresentou coliformes, S. aureus e Salmonella spp.

Study on mangrove water and sediment of Paraiba do Norte river (Northeastern Brazil) and crabmeat of Ucides cordatus was carried to investigate the relation between the abundance of bacteria in the mangrove habitat and in the crabmeat. Simultaneous collection of samples of water, sediment and crabs (U. cordatus) was done during the rainy and dry seasons at two areas of mangroves that differed in influence of tide and sewage discharge. The numbers of total heterotrophic bacteria in the mangrove water were between 0.04 and 6 log CFU 100mL-1, and in the sediment between 5 and 6 log CFU g-1. The location closer to waste discharge showed higher incidence of total heterotrophic bacteria, coliforms, Staphylococcus aureus and Salmonella spp. The counts of total heterotrophic bacteria in the crabmeat varied from 1.3 to 4.4 log CFU g-1, and did not differ significantly between the locations analysed. No crabmeat sample showed the presence of coliforms, S. aureus and Salmonella spp.

A dinâmica das populações bacterianas meso e termofílicas foi estudada no solo sem raízes e rizosférico associado à gramínea Aristida adscensionis L. na caatinga da zona semi árida do nordeste brasileiro. As bactérias totais heterotróficas, as bactérias esporuladoras e actinomicetos mesofílicos foram significativamente mais abundantes que os termofílicos, suas contagens variando na ordem de 10(6) - 10(7) UFC g-1 de solo seco, apresentando flutuações discretas ao longo de um período de um ano. Microrganismos termófilos foram encontrados numa ordem de 10(6) UFC g-1 de solo seco e demonstraram, em geral, flutuações mais evidentes durante o mesmo período de estudo. Entre os termófilos, os actinomicetos apresentaram um maior declínio durante a estação seca. Nenhuma diferença significativa no número de microrganismos foi observada entre o solo rizosférico e o solo não rizosférico. As bactérias mesofílicas mais observadas foram as filamentosas Gram positivas (actinomicetos) e os bastonetes esporulantes Gram positivos. Todos os isolados termofílicos foram bactérias esporuladoras Gram positivas em forma de bacilo. A maioria dos isolados mesofílicos produziu amilases e proteases, enquanto que 38% das linhagens produziram todas as enzimas extracelulares estudadas: amilases, proteases, celulases e quitinases. Entre as bactérias termofílicas, todos os isolados apresentaram atividade proteolítica e 77% produziram amilases, mas nenhum produziu quitinases ou celulases.

The dynamics of mesophilic and thermophilic bacterial population was studied in the bulk soil and rhizosphere of the grass Aristida adscensionis L. in a caatinga of the semi-arid Brazilian Northeast. Mesophilic heterotrophic bacteria, sporeforming bacteria and actinomycetes were significantly more abundant than thermophiles, and their counts were 10(6) - 10(7) CFU g-1 dry soil, showing weak fluctuations over the one-year study period. Thermophiles were below 10(6) CFU g-1 dry soil and, in general, demonstrated stronger fluctuations over the studied period. Among them, thermophilic actinomycetes showed the greatest decline during the dry season. No significant differences in microbial number were observed between bulk and rhizosphere soil. Mesophilic bacteria were represented mainly by Gram positive, filamentous organisms (actinomycetes) and by Gram positive, endospore-forming, rod-shaped organisms. All isolates of thermophilic bacteria were Gram positive, endospore-forming rods. Most of mesophilic isolates produced amylases and proteases, and 38% of isolates produced all studied extracellular enzymes: amylases, proteases, cellulases and chitinases. Among thermophilic bacteria, all isolates showed proteolytic activity and 77% produced amylases, but no one produced cellulases or chitinases.

O objetivo deste estudo foi avaliar o potencial de inibição de nisina e o pH elevado em relação à multiplicação de Staphylococcus aureus e Salmonella sp. em culturas puras e inoculadas na carne de caranguejo-uçá. Em culturas puras, a multiplicação de S. aureus foi fortemente inibida por nisina e a de Salmonella sp. por nisina-EDTA (20 mM). A multiplicação de S. aureus foi inibida até 8h de incubação, enquanto que a de Salmonella sp. foi inibida durante todo o experimento (24h). O pH elevado (tampão NaHCO3-NaOH, pH 10 e 11) mostrou-se efetivo na inibição da multiplicação de S. aureus e Salmonella sp. Nisina e o pH elevado aplicados na carne contaminada não apresentaram o mesmo efeito. A nisina não mostrou eficiência na inibição dos patógenos quando inoculados na carne de caranguejo, enquanto que o tampão em pH 10 demonstrou inibição significante sobre a multiplicação de Salmonella sp. Estes resultados sugerem que o pH elevado apresenta um potencial como agente antibacteriano, podendo ser útil na preservação química da carne de caranguejo.

The aim of this study was to evaluate the potential of nisin and high pH to inhibit the growth of Staphylococcus aureus and Salmonella sp. in broth culture and when inoculated into meat of land crab. In pure cultures, the growth of S. aureus was bly inhibited by nisin and the growth of Salmonella sp. was inhibited by nisin-EDTA (20 mM). The inhibition of S. aureus lasted for eight hours and Salmonella sp. growth was inhibited throughout the experiment (24 h). The high pH (pH 10.0 and 11.0 with NaHCO3-NaOH buffer) was very effective for in vitro inhibition of S. aureus and Salmonella sp. Nisin and high pH, when applied to the contaminated meat, did not yield the same effect. Nisin was not effective in preventing growth of both pathogens in the crab meat, while pH 10.0 showed significant inhibitory effect on Salmonella sp. The results suggest that high pH has a potential as antibacterial agent, and may be useful in chemical preservation of crab meat.