Time-kill study and morphological changes of Proteus mirabilis cells exposed to ethyl acetate crude extract of Lasiodiplodia pseudotheobromae IBRL OS-64

@#Aims: To investigate time-kill curve and morphological changes of Proteus mirabilis cells exposed to ethyl acetate crude extract of endophytic fungus, Lasiodiplodia pseudotheobromae IBRL OS-64, isolated from Ocimum sanctum. Methodology and results: Inhibitory effect of the fungal extract against the test bacteria via disc diffusion assay showed a fair antibacterial activity with diameter of inhibition zone was 12.0 ± 0.4 mm. The Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC) values of the ethyl acetate extract against P. mirabilis was 250 and 500 µg/mL, respectively. The value of MBC which is two-fold higher than MIC value indicated that the fungal extract exerted bactericidal effect on bacterial cells of P. mirabilis. Time-kill curve study revealed that the bactericidal effect of the crude extract towards test bacteria was both dose and time dependent. Scanning electron microscope (SEM) observation revealed that the bacterial cells of P. mirabilis exposed to fungal crude extract resulted in formation of pits, irregular shape of the bacterial cell and ultimately cell death beyond repair. Conclusion, significance and impact of the study: The time-kill curve study, and cell morphological changes suggested the potential of ethyl acetate extract of L. pseudotheobromae IBRL OS-64 against P. mirabilis infection by formation of cavities, irregular bacterial cell that leads to ultimate cell death and the extract may have pharmaceutical potential to be develop as antibacterial agent.

Antimicrobial activities of stembark and wood extracts from Nauclea subdita against pathogenic microorganisms

Aims: The aim of the study was to investigate the antimicrobial activity of Nauclea subdita (Korth) Steud against six pathogenic microorganisms. Methodology and results: Young and matured trees of N. subdita were cut and separated into bark and wood parts, respectively, prior to extraction process. Phytochemical screening tests, antimicrobial activity, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were determined. Preliminary screening for phytochemical components showed that both young and matured tree had similar constituents. Extracts from matured tree showed more potency in terms of the zones of inhibition sizes than the young tree. Extract of N. subdita was more potent to both marine bacteria, Vibrio parahaemoliticus and V. alginolyticus, while Candida albican and Aspergillus niger were resistant to it. The sensitivity test showed that 500 µg/mL is the optimum concentration for extract of bottom sapwood of mature tree to act as bactericidal. Conclusion, significance and impact study: The results from this study suggest that N. subdita bark and wood extracts may serve as potential source of antimicrobial agents for future development in medicine applications.

Analysis of the composition of Brazilian propolis extracts by chromatography and evaluation of their in vitro activity against gram-positive bacteria

Brazilian propolis from São Paulo state was submitted to extraction using several solvents, resulting in extracts with different composition. These extracts were submitted to Thin Layer Chromatography (TLC). Bioauthographic analysis of the TLC plates identified fractions with inhibitory activity, which were then analysed by High Performance Liquid Chromatography (HPLC). In vitro assays, commonly used to evaluate the activity of propolis against Gram-positive bacteria, were compared to determine which rendered the most consistent results. The bactericidal activity of these extracts were analysed by Serial Dilution in Tubes and Agar Plate Diffusion. Serial Dilution in Tubes obtained the most consistent results, with the Minimal Bactericidal Concentration of the extracts ranging between 2.5 and 20.0 mg/mL, for the species of Gram-positive bacteria tested. The results of the Agar Plate Diffusion were directly proportional to the hydro-solubility of the extracts and did not evaluate their bactericidal activity correctly. The bactericidal activity of this sample of propolis was due to the combined effect of several components that were identified by HPLC and were best extracted using 50% ethanol as a solvent.

Própolis brasileira, proveniente do estado de São Paulo, foi submetida à extração usando vários solventes, resultando em extratos com diferentes composições. Estes extratos foram submetidos à Cromatografia em Camada Delgada (CCD). Análise bioautográfica das placas de CCD permitiu identificar as frações com atividade antimicrobiana, que foram então analisadas por Cromatografia Líquida de Alta Eficiência (CLAE). Ensaios in vitro freqüentemente utilizados para avaliar a atividade de própolis frente a bactérias Gram-positivas foram comparados para determinar qual renderia os resultados mais consistentes. A atividade bactericida destes extratos foi analisada por Diluição Seriada em Tubos e por testes de Difusão em Agar. O método de Diluição em Tubos permitiu obter os resultados mais consistentes e a Concentração Bactericida Mínima dos extratos variou entre 2,5 e 20,0 mg/mL, para as espécies de bactérias Gram-positivas testadas. Os resultados do método de Difusão em Agar foram diretamente proporcionais à hidrossolubilidade dos extratos, e não avaliaram a atividade bactericida corretamente. A atividade bactericida desta amostra resultou da combinação de vários componentes, identificados por CLAE, que foram extraídos preferencialmente usando etanol 50% como solvente.