Partial Purification and Antimycobacterial Screening of the Ethyl Acetate Extract of Alcaligenes faecalis BW1.

Aims: The focus of this study was to evaluate the antimycobacterial activity of Alcaligenes faecalis BW1 extract and to purify it partially. Study design: Partial purification of A. faecalis BW1 extract was performed by using thin layer chromatography and active substances responsible for the biological activity were localized. Place and Duration of Study: The study was carried out at laboratory of Microbial Biotechnology, Department of Biology, Faculty of Sciences and Technical, University Sidi Mohamed Ben Abdellah, BP 2202, Road of Immouzer, Fez, Morocco, during the period from January 2011 to July 2011. Methodology: Crude extract of A. faecalis BW1 was obtained by using ethyl acetate as an organic solvent and its antimycobacterial effect was investigated by agar discs diffusion method. The extract was then fractionated by thin layer chromatography and the bioactivity was assessed with a bioautography technique followed by spots elution tests. Results: The results showed that A. faecalis BW1 produced compounds with antimycobacterial activity. All the detected spots by thin layer chromatography inhibited the growth of M. smegamtis. Conclusion: Various metabolites of A. faecalis BW1 are responsible for the sought effect or they could act synergistically to inhibit mycobacterial growth. These compounds could be used after their total purification in further work against mycobacterial infections.

Antibacterial Effect of Pseudomonas aeruginosa Isolated from a Moroccan Hot Spring Discharge and Partial Purification of its Extract.

Aims: The focus of this study was to isolate and to identify microorganisms possessing an antibacterial activity followed by a partial purification of their extracts. Study Design: Screening and identification of bacteria with an antibacterial effect were performed and active substances responsible for the biological activity were localized and partially purified. Place and Duration of Study: The study was carried out at laboratory of Microbial Biotechnology, Department of Biology, Faculty of Sciences and Technical, University Sidi Mohamed Ben Abdellah, BP 2202, Road of Immouzer, Fez, Morocco, during the period from February 2010 to August 2010. Methodology: Samples of a hot spring discharge localized in the city Fez Morocco were explored to isolate compounds-producing microorganisms. The inhibitory spectrum of the isolate was evaluated against M. smegmatis, M. aurum, S. aureus, S. haemolyticus, B. subtilis, B. licheniformis, B. amyloliquefaciens, E. coli DH5α and Erwinia chrysanthemi by using agar well diffusion test and/or a modified spot-on-lawn assay. Identification of strain was executed on the basis of Gram stain, biochemical characteristics and PCR followed by DNA sequencing of 16S ribosomal RNA gene. Crude extract of the isolate was obtained by using ethyl acetate and was exposed to proteolytic enzymes (pepsin and proteinase K) and to heat treatment at 121°C (60 min), 100°C (20 min), 80°C (30 min), 37°C (3h) and kept at 4°C (six months). The antimycobacterial effect before and after every treatment was assessed by agar well diffusion method. Synthesis of antibacterial compounds was monitored during the isolate growth cycle. The extract was then fractionated by thin layer chromatography and the bioactivity was investigated with a bioautography technique followed by spots elution test. Results: One bacterium was isolated having a broad antagonistic effect against all the tested bacteria. Based on biochemical characterization and 16S rDNA sequence analysis, the strain was identified as Pseudomonas aeruginosa. The antibacterial compounds were synthesized during the exponential growth phase and were not affected following heat treatment and proteases that indicated the non-proteinaceous nature of the active agents. The crude extract developed in chloroform: acetone (9:1) showed metabolite (s) at Rf = 0.68 which it may be pyocyanin, inhibiting the growth of M. smegamtis. Conclusion: Metabolites of P. aeruginosa responsible for the sought effect were localized and characterized. These compounds might provide an alternative bio-resource for the bio-control of plant pathogens after their total purification in further investigation.

Tolerance Tests of Alcaligenes faecalis BW1 Extract.

Aims: To highlight whether metabolites of Alcaligenes faecalis BW1 extract can be administered orally for their possible antimycobacterial effects. Study Design: Study of the influence of certain parameters on the extract of Alcaligenes faecalis by using either discs or well diffusion methods against M. smegmatis. Place and duration of study: Laboratory of Microbial Biotechnology, Department of Biology, Faculty of Sciences and Technical, University Sidi Mohamed Ben Abdellah, BP 2202, Road of Immouzer, Fez, Morocco. From April to August, 2012. Methodology: The impact of acidic pH of gastric juice, bile, hydrogen peroxide, pancreatic enzymes and lysozyme on the antimycobacterial activity of Alcaligenes faecalis BW1 extract was evaluated by agar diffusion method. Detection whether or not antibacterial metabolites having a synergistic effect with rifampicin against M. smegmatis was also explored. Results: Antibacterial metabolites of Alcaligenes faecalis BW1 extract resist to the action of gastric pH, gallbladder bile and hydrogen peroxide. In addition, they are not affected by pancreatic enzymes and lysozyme. Moreover, they have a synergistic effect with rifampicin against M. smegmatis. Conclusion: Anti-mycobacterial metabolites of Alcaligenes faecalis BW1 extract are compatible with rifampicin and could be administered orally as antitubercular agents after their purification, identification in further work.

Antimycobacterial activity of a Brevibacillus Laterosporus strain isolated from a moroccan soil

The treatment of tuberculosis has become more difficult with the worldwide spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. Moreover, the prevalence of human disease caused by atypical mycobacteria has also increased in the past two decades and has further complicated the problem of the treatment of mycobacterial infections. It is therefore urgent to develop new highly active molecules against these bacteria. The present study reports the isolation from a Moroccan soil of a Bacillus strain that exhibits an important antimycobacterial activity. The strain was identified as Brevibacillus laterosporus using DNA sequencing of the 16S ribosomal RNA gene. The antimycobacterial activity was assigned to a substance with a protein nature. This nature was revealed using a liquid-liquid extraction with organic solvents, precipitation with ammonium sulfate and treatment with a protease. This study suggested the identification and the characterization of this active metabolite enabling therapeutic investigations further.