ABSTRACT
A total of 106 actinomycetes isolated from the rhizosphere
of plants in abbatoir and refuse dumps in Awka and Onitsha were investigated
for the production of antimicrobial substances. Five of them were found to show
antimicrobial activity against Gram positive and Gram negative bacteria as well
as fungi on solid media. Three of the very active isolates designated MP-75, SP
-76 and QP-100 were further investigated in submerged medium in a shake-flask
experiment using glucose and soy bean as carbon and nitrogen sources
respectively. Isolates MP-75 and SP-76 were found to produce antimicrobial
substances. The antimicrobial substance produced by isolates SP-76 showed the
highest antimicrobial activity against Escherichia coli, Pseudomonas
aeruginosa, Klebsiella sp, Staphylococcus aureus and
Bacillus sp. The isolates were identified as Streptomyces
species based on their characteristic features. Activity of the antimicrobial
substance produced by Streptomyces SP-76 was maximum when 4% (w/v)
glucose and 2% (w/v) soybean were used in the fermentation process. Influence
of surfactants on accumulation of antimicrobial substance by Streptomyces
SP-76 in the fermentation broth showed that Tween 80, oleic acid, palmitic acid
and stearic acid enhanced antimicrobial activity. The effect of growth
promoters on antibiotic production by Streptomyces SP-76 indicated that
peptone, casein and yeast extract stimulated antimicrobial activity against the
test organisms. The effect of varying pH on antibiotic production by Streptomyces
SP-76 showed that there was maximum antimicrobial activity at pH 7. In a time
course for antibiotic production, maximum antimicrobial activity was obtained
at 120h. Paper chromatography of the culture filtrate of Streptomyces
SP-76 indicated that it contains more than one antimicrobial substance. From
this, it can be seen that the growth and subsequent production of bioactive
metabolites by Streptomyces SP-76 isolated from the soil.
CHAPTER ONE
INTRODUCTION
For centuries, preparations derived
from living matter were applied to wounds to destroy infection. The fact that a
microorganism is capable of destroying one another
was not established until the
latter half of the 19th century, when Pasteur noted the antagonistic effect of
other bacteria on the anthrax organism and pointed out that this action might
be put to therapeutic use. Meanwhile, the German Chemist, Paul Ehrlich
developed the idea of selective toxicity; that certain chemicals that would be
toxic to some organisms like infectious bacteria, would be harmless to other
organisms e.g humans (Limbird, 2004).
In 1928, Sir Alexander Fleming, a
Scottish biologist, observed that Penicillium notatum, a common mold, had
destroyed Staphylococcus bacteria in culture and in 1939, the American
microbiologist Rene Dubois demonstrated that a soil bacterium was capable of
decomposing the starchlike capsule of the Pneumococcus bacterium, without which
the Pneumococcus is harmless and does not cause pneumonia.
Dubois then found in the soil a
microbe, Bacillus brevis, from which he obtained a product, tyrothricin, that
was highly toxic to a wide range of bacteria (Limbird, 2004). Tyrothricin, a
mixture of the two peptides, gramicidin and tyrocidine, was also found to be
toxic to red blood and reproductive cells in humans but could be used to good
effect when applied as an ointment on body surfaces. Penicillin was finally isolated
in 1939, and in 1994 Selman Waksman and Albert Schatz, American
microbiologists, isolated streptomycin and a number of other antibiotics from Streptomyces
griseus (Calderon and Sabundayo, 2007).
Discovery of new antibiotics
produced by Streptomyces still continues. Today, due to the increasing
resistance of pathogenic bacteria to our current arsenal of antibiotics, a
great need exist for the isolation and discovery of new antibiotics and other
drug agents (Jarroff, 1994; Rice, 2003; Wenzel, 2004). Fifty years ago, it was
easy to discover new antibiotics by simply screening the fermentation broths of
actinomycetes and fungi. Today, it is much more challenging, but there are much
better tools to address this problem. Discovering new antibiotics,
pharmacophores is a long-term endeavor that requires deft orchestration and
support of many innovative sciences (Cuatrecasas, 2006). There are three
approaches that can be used to improve our chances of finding new antibiotic
substances: new test methods, new organisms, and variation of culture
conditions. None of these three options guarantees success alone and the
chances are best if the three are combined. There is need for long-term basic
microbiological research, which should cover the following areas: methods of
isolating and cultivating microorganisms that have not yet been accessed or
only with great difficulty, studies on the transportation of antibiotics into
the bacterial cell, comparative biochemistry of prokaryotes and eukaryotes,
mode of action of antibiotics and pathogenicity factors. In addition to search
for new antibiotics, long-term strategies to prevent the development and
spreading of resistant bacteria must be developed (Fiedler and Zanher, 1995).
Therefore, it is time to define natural product discovery from actinomycetes
and other microbes as a major priority for medical sciences and to engage the
most creative scientists in academia (Cuatrecasas, 2006) in close collaboration
with biotechnology and pharmaceutical companies, which would elevate the
science to a new level of achievement so as to be commensurate with past
successes and present demonstrated potential.
In this study, effort has been
made:
* To isolate Streptomyces from the
soil, capable of producing antimicrobial substances
* To study the cultural conditions
necessary for antibiotic production.
* To determine the time taken for the
optimum production of antimicrobial substance.
* To determine the type of
antimicrobial substance(s) present in the culture filtrate.
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