Sodium Azide induced enhancement of antimicrobial property of previously selected actinomycetes (SIMA)

A proposal on
Sodium Azide induced enhancement of antimicrobial property of previously selected actinomycetes (SIMA)

Principle Investigator: Pragya Sharma and Mandira Manandhar
Students from eighth semester
Universal Science College
Pokhara University
Maitidevi, Kathmandu 2009

Supervisor: Kiran Babu Tiwari
Universal Science College
Pokhara University
RLABBMaitidevi, Kathmandu

Research Lab
Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu

Introduction:
Actinomycetes, mostly known for their antibiotic activity, resemble both bacteria (prokaryotes) and fungi (eukaryotes). They have cell wall similar to gram positive bacteria with high G+C (>55%) content and also have 70S ribosome like that of bacteria. But their filamentous form, sporulating form and histone like protein (that are not found in prokaryotes) shows fungal characters. According to Bergey's Manual of Systematic Bacteriology, actinomycetes are divided into eight diverse families: Actinomycetaceae, Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae (Holt, 1989) and they comprise 63 genera (Nisbet and Fox, 1991). Characteristic biological aspects of actinomycetes is its ability to produce a wide variety of secondary metabolites including most of the antibiotics. Some examples of antibiotics produced actinomycetes are streptomycin, aureomycin, terramycin and chloromycetin.

Background:
In RLABB, Bhattarai and Tiwari (2006) developed a prototype methodology to explore mutagenic effects of sodium azide in Streptomyces spp., viz. both for loss of function (LOF) and gain of function (GOF) effects. The GOF is of particular importance and this type of work will be a good example of the foundation of applied research. The study should be extended in order to cover more parameters of actinomycetes, which helps to understand their physiology with respect to enhanced antibacterial properties among the mutants compared to respective wild types.

General objectives:
To study sodium azide induced enhancement of antimycrobial property of actionmycetes strain

Specific Objectives:
To revive and purify actinomycetes from preserved sample
To screen antibacterial activities of the strains by primary screening method
To verify the antibacterial activities of the strains by secondary screening method

Methodology Revival of the preserved actinomycetes: To revive them, they will be incubated at 28°C for one hour and then streaked on starch casein agar plates and further incubated at 28°C for 7 days.

Purification of actinomycetes : Streak plate method will be used to purify cultures of actinomycetes contaminated by bacteria and fungi (Williams and Cross, 1971, Singh and Agrawal 2002; Agrawal 2003). After isolation of the pure colonies based on their colonial morphology, colour of hyphae, color of aerial mycelium, they will be individually plated on another but the same agar medium.

Primary screening : Antibacterial activity of pure wild type and corresponding mutants will be determined by perpendicular streak method on Nutrient agar (NA). Test actinomycete strain will be streaked on the middle part of the NA plate and incubated for 5 - 7 days in room temparature. The test organisms to be used will be: Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Klebsiella species, Proteus species, Pseudomonas species, Salmonella typhi and Shigella species. Log phase culture (4hrs of NB at 37C) of the bacteria will be straeked perpendicular to the actinomycete streak line and incubated for overnight.

Secondary screening : Fresh and pure culture of each isolate selected from the primary screening will be inoculated in starch casein broth and incubated at 28°C for 7 days in water bath shaker . Growth of the organism in the flask will be confirmed by the visible pellets, clumps or aggregates and turbidity in the broth. Contents of flasks will be filtered through Whatman no.1 filter paper aseptically. The filtrate will be used for the determination of antibacterial activity against the standard test organisms by agar well-cut method.

Expected outcome: It has been found that azide induces gain of function and loss of function (Bhattarai and Tiwari, 2006). Enhanced antibacterial activity that they reported on primary screening (Bhattarai et al. 2007) is of particular importance; and hence, further elaboration of these applied aspects can be verified with secondary screening method. This study may explore some mutants with enhanced antibacterial properties compared to their respective wild types as a way of strain improvement among actinomycetes.

References:
Agrawal, V. P (2003) Biodiversity of Khumbu Region : Population Study of Actinomycetes, a Project Report Submitted to the Royal Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal "R"

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Enhanced antibacterial activity of sodium azide treated mutant Streptomyces strain. Journal of Nepal Association for Medical Laboratory Sciences, 8(1): 67-8.

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Loss-of-function (LOF) and gain-of-function (GOF) mutation of sodium azide in Streptomyces spp. Journal of Nepal Biotechnology Association. (accepted).

Holt, J.G., (1989) Bergey’s manual of systematic bacteriology, vol. 4, ed. S.T.Williams and M.E.Sharpe, Baltimore, Md : Williams and Williams.