Thesis Abstract and Dissertation Abstracts

Actinomycetes are one of the most attractive sources of antibiotics and other biologically active substances of high commercial value. It accounts roughly for two-thirds of the total known antibiotics isolated from these gram positive, branched spore bearing filamentous group of Eubacteria with controversial bacto-fungoid morphology. Among the Actinomycetes, Streptomyces spp. has been the most abundant sources of all types of antibiotics. Screening and isolation of promising strains of Actinomycetes with potential antibiotics is still a thrust area of research and it is suggested that the explorations of materials from new areas and habitats have a pivotal role to play in the search for new microbes and novel metabolites and is urgent to counter the threats posed by the fast emerging phenomenon of antibiotic resistance.
The tropical forests of Western Ghats are rich in flora and fauna and so are naturally rich in microbial-diversity. The Neyyar wild life sanctuary is located in the western slop of the Southern Western Ghats of Kerala having a vegetation of tropical evergreen, moist deciduous forests to grasslands and is harboring a good number of endemic and endangered flora. The present study deals with the isolation and screening of the antibiotic producing Actinomycetes from Neyyar wild life sanctuary of Kerala state.

120 soil samples were analysed for antibiotic producing Actinomycetes. The soil samples showed an average of 18.265 x 106 number of Actinomycetes per gram of soil. Among the soil samples from different habitats, evergreen forest showed the highest number of Actinomycetes (24.57x106/gram soil), followed by 17.89x106 in deciduous forest and 12.32x106 in grassland. 412 Actinomycetes isolates of were obtained as pure cultures from evergreen forest soils, 374 from deciduous forest soils and 241 from grassland soil samples. The results showed that the soils of Evergreen forests of Neyyar wild life sanctuary are rich in Actinomycetes flora than that of deciduous forests and grasslands.

Generic level identification of the isolates showed the presence of genera like, Streptomyces, Streptoverticillium, Actinoplanes, Streptosporangium, Micromonospora and others. Among these Streptomyces spp. are most abundant in occurrence.

The evaluation of the antagonistic properties of the isolates showed 58.9% as antagonists. The habitat wise comparison of the per cent occurrence of antagonists showed that 47.33% are from evergreen forest soils, deciduous forest soils showed 75.13% and grasslands 53.526%. These results proved that the deciduous forest soils of Neyyar Wild Life sanctuary are more potential sources of antagonistic Actinomycetes than that of evergreen and grasslands.

Four Actinomycetes isolates with strong antimicrobial activity (broad spectrum) were selected for further studies. These isolates include three Streptomyces spp. and one Streptosporangium sp.. Among these the Streptomyces sp. TBG-19NRA1 showed high antibiotic activity compared to others, producing an average inhibition zone of 26.23mm diameter against the test organisms tried. This isolate was selected for detailed studies.

On biochemical analysis of the cell wall contents of the isolate 19NRA1showed the presence of LL-Diaminopimilic acid (LL-DAP) as the major cell wall peptidoglycan. There are no diagnostic sugars detected in cell wall. The Mol% of G+C of the isolate 19NRA1 is calculated as 73.44%. The isolate also shows a typical Streptomyces type morphology. These characters suggested to place the isolate 19NRA1 as a representative of the genus Streptomyces Waksman & Henrici.

The isolate 19NRA1 is having a spore mass colour of predominantly white. Grey spores were also produced occasionally. The reverse colony colour is yellow brown and is not producing any diffusible pigments. The microscopic examination shows that the conidial chains are Rectus-flexibilis (RF) and with more than 50 smooth walled spores which are oblong in shape (0.86 ~ 1.03 x 0.6 ìm). It shows the physiological characters like nitrate reduction and production of H2S. It do not produce melanin pigment and are not utilizing Adonitol, L-Arabinose, meso-Inositol, D-Melibiose, Raffinose, Salicin, Sucrose, Xylitol and D-Xylose as the carbon source. Based on these characters and using standard keys and by comparison to published descriptions, the isolate is identified as Streptomyces setonii (Millard & Burr, 1926) Waksman 1953.

The partial 16s rRNA sequence analysis of genomic DNA of the isolate 19NRA1 using the primers, 8-27 F and 1500 R is done for the confirmation of the identity of the strain. A 16S database search was performed in BLAST (NCBI 2.0) to find the Actinomycetes strains with the most similar 16S sequences to the 19NRA1 isolate and it showed 99.525% similarity with Streptomyces setonii. The identified isolate was deposited to Microbial Type Culture Collection & Gene Bank (MTCC), Chandigarh, with an accession number MTCC 3756.

In this study 12 different media were tried for maximum antibiotic accumulation. Among these the antibiotic assay broth gave the maximum growth and antibiotic activity. The nutrient sources of the medium was modified by supplementing different carbon and nitrogen compounds, inorganic phosphate, NaCl, trace elements etc. at different concentrations. Based on the results a modified fermentation broth was formulated having 1% Starch and 0.3%glycerol as the major carbon sources and 0.5% Tryptone as nitrogen source, for improved antibiotic production using Streptomyces setonii strain 19NRA1.

The physiological parameters of antibiotic production in batch fermentation was standardised for optimum production. An inoculam prepared in Tryptone-Yeast-extract broth at a seed concentration of 5ml and at an incubation period of 48hrs is found ideal for initiation fermentation. Batch fermentations at an incubation period of 5 days (120hrs) at room temperature (28+1oC), shaking at a speed of 120 rpm is found ideal for growth and antibiotic accumulation at laboratory conditions using Streptomyces setonii19NRA1.

The antibiotic compound was isolated and purified. The purified compound is having a mass of 473 Daltons and a melting point of 132-135oC. The compound is white powder and soluble in most organic solvents but insoluble in water and DMSO and show positive reaction with alcoholic Ferric Chloride and Dragen dorft reagent. This compound is named as TBGA-1. It is showing activity against gram positive bacteria and also against some gram negative bacteria like Escherichia coli, Pseudomonas fluorescens and Salmonella typhi. The biological and chemical properties of the compound were compared with that of the early reported antibiotic compounds from the Streptomyces setonii and as the characters of the active principle differ from the reported ones this compound may be a different one.

Streptomyces setonii 19NRA1 strain is a potential source of antibiotic with antibacterial properties. The active principle may be a new compound. Further elaborate chemical, biochemical and pharmacological studies are necessary to develop this active principle to the level of a drug (antibiotic).

This Thesis Abstract may be cited as follows:
S. Shiburaj (2003) Screening, isolation and characterization of an antibiotic producing Actinomycete, Streptomyces setonii 19NRA1 (Ph.D thesis), University of Kerala

Submission Details: Thesis Abstract submitted by S. Shibu Raj from India on 18-Oct-2005 18:25.
Abstract has been viewed 6135 times (since 7 Mar 2010).

S. Shibu Raj Contact Details: Email: drshiburaj@gmail.com