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

Abstract

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.