Resumen P. azurea es una especie de rana bien distribuida geográficamente en América del Sur, que incluye biomas brasileños como Pantanal y Cerrado. En comparación con otros anuros de Phyllomedusinae, existen pocos informes sobre el potencial bioactivo de las moléculas derivadas de la piel de esta especie. En esta perspectiva, el objetivo del presente estudio fue evaluar la actividad antibacteriana in vitro de la secreción de la piel de P. azurea mediante la detección de la Concentración Inhibitoria Mínima (CIM) del crecimiento de cepas indicadoras bacterianas y determinar si ocurre un cambio en la permeabilidad de la envoltura celular bacteriana. La determinación de MIC se llevó a cabo mediante el método de la placa de microdilución. La absorbancia se midió y se analizó estadísticamente mediante la prueba t para comparar dos grupos (0.05 de significancia). El impacto del extracto crudo sobre la permeabilidad de la envoltura celular de Staphylococcus aureus ATCC 25923 se realizó mediante el ensayo de cristal violeta, y se midió la absorbancia mediante espectrofotometría seguida del cálculo del porcentaje de absorción de violeta cristal. La CIM específica para S. aureus ATCC 25923 y Escherichia coli ATCC 25922 fue 31.25 μg / ml, mientras que para Bacillus subtilis ATCC 6633 de 125 μg / ml y Pseudomonas aeruginosa ATCC 27853 de 250 μg / ml. El tratamiento con concentraciones crecientes de secreción de piel de rana aumentó la absorción de violeta cristal por las células de S. aureus ATCC 25923, sugiriendo una acción sobre la membrana plasmática de la célula. Los resultados demostraron que la secreción de la piel de P. azurea presenta actividad antibacteriana y amerita más investigaciones para caracterizar las moléculas bioactivas.

Abstract Phyllomedusa azurea is a frog species well distributed geographically in South America, including Brazilian biomes as Pantanal and Cerrado. Compared with other anurans from the Phyllomedusinae family, there are few reports on the bioactive potential of skin-derived molecules from this species. In this perspective, the aim of the present study was to evaluate the in vitro antibacterial activity of skin secretion of P. azurea by detection of Minimum Inhibitory Concentration (MIC) of the growth of bacterial indicator strains and to determine if occurs a changing in the bacterial cell envelope permeability. The MIC determination was carried out by the microdilution plate method. The absorbance was measured and analyzed statistically using the t-test to compare two groups (0.05 % of significance). The impact of the crude extract on cell envelope permeability of Staphylococcus aureus ATCC 25923 was conducted by the crystal violet assay, and the absorbance was measured spectrophotometry followed by the calculation of the crystal violet uptake percentage. The specific MIC for S. aureus ATCC 25923 and Escherichia coli ATCC 25922 was 31.25 µg/mL, while for Bacillus subtilis ATCC 6633 was 125 µg/mL and Pseudomonas aeruginosa ATCC 27853 was 250 µg/mL. The treatment with crescent concentrations of frog skin secretion increased the crystal violet uptake by S. aureus ATCC 25923 cells, suggesting an action on the cell plasma membrane. The results demonstrated that the skin secretion of P. azurea presents antibacterial activity and merit further investigations to characterize the bioactive molecules.

Resumo Os venenos de serpentes são reconhecidos como uma fonte promissora de substâncias farmacologicamente ativas e potencialmente úteis para o desenvolvimento de novas drogas antimicrobianas. Esse trabalho teve como objetivo investigar a atividade antimicrobiana do veneno de Crotalus durissus terrificus contra várias bactérias. A determinação da atividade antibacteriana foi realizada pelo método de microdiluição em placas e a ação na estrutura do envelope bacteriano pelo ensaio violeta de cristal. As proteínas do extrato bruto foram separadas por eletroforese e caracterizadas quanto à sua atividade proteolítica. O veneno de C. d. terrificus apresentou ação antimicrobiana frente bactérias gram-positivas e gram-negativas. Os valores de MIC foram definidos para Pseudomonas aeruginosa ATCC 27853 (62.5 µg/ mL), Staphylococcus aureus ATCC 25923 (125 µg/mL) e Micrococcus luteus ATCC 9341 (≤ 500 µg/mL). Para Salmonella enterica serovar typhimurium ATCC 14028 e Corynebacterium glutamicum ATCC 13032, o decréscimo no crescimento bacteriano não foi detectado visualmente, mas foi estatisticamente significante. O teste do cristal violeta demonstrou que o veneno bruto aumentou a permeabilidade das células bacterianas e o perfil de proteína secretada está em consonância com relatos anteriores. Os resultados sugerem que as proteínas com atividade lítica contra bactérias no veneno de C. d. terrificus merecem atenção para uma melhor caracterização, uma vez que podem trazer reforços para o escasso arsenal terapêutico empregado para combater a multirresistência microbiana.

Abstract Snake venoms are recognized as a promising source of pharmacologically active substances and are potentially useful for the development of new antimicrobial drugs. This study aimed to investigate the antimicrobial activity of the venom from the rattlesnake Crotalus durissus terrificus against several bacteria. Antibacterial activity was determined by using the plate microdilution method and the activity on the bacterial envelope structure was screened by using the crystal violet assay. The proteins in crude venom were separated by electrophoresis and characterized regarding their proteolytic activity. C. d. terrificus venom exhibited antimicrobial action against gram-positive and gram-negative bacteria. MIC values were defined for Pseudomonas aeruginosa ATCC 27853 (62.5 µg/mL), Staphylococcus aureus ATCC 25923 (125 µg/mL), and Micrococcus luteus ATCC 9341 (≤500 µg/mL). For Salmonella enterica serovar typhimurium ATCC 14028 and Corynebacterium glutamicum ATCC 13032, the decrease in bacterial growth was not detected visually, but was statistically significant. The crystal violet assay demonstrated that the crude venom increased bacterial cell permeability and the secreted protein profile agreed with previous reports. The results suggest that the proteins with lytic activity against bacteria in C. d. terrificus venom deserve further characterization as they may offer reinforcements to the weak therapeutic arsenal used to fight microbial multidrug resistance.