ABSTRACT
The genus Bacillus produces various classes of antibiotics, among which is bacitracin. Bacitracin is a mixture of related cyclic peptides produced by organisms of the licheniformis group of Bacillus subtilis var Tracy, first isolated in 1945. This study was carried out to screen and evaluate the antimicrobial activity of bacitracin produced by Bacillus subtilis at varying culture conditions. The crude bacitracin produced was tested at different dilutions against Escherichia coli and Staphylococcus aureus which are known to be susceptible to this antibiotic. The organism was isolated from the UGC farm of Michael Okpara University of Agriculture, Umudike and they were confirmed as Bacillus subtilis by using standard biochemical tests, gram and spore stains and other morphological characteristics. The efficiency of the bacitracin produced at different parameters were studied and determined by testing it against the test organisms through the agar well diffusion method. The susceptibility of the test organisms to the bacitracin extracts was determined by measuring the diameter of inhibition zones formed around agar wells in different agar plates prepared. From this analysis, the results showed that pH was the major contributing factor to increased activity of bacitracin, especially at pH 8, were it showed the highest inhibition of test organism. Glycerol and Calcium Carbonate also improved the effectiveness of the bacitracin produced, while Magnessium Sulphate greatly hindered its potency. Also, from the analysis, Staphylococcus aureus was more susceptible to the crude extracts of bacitracin compared to Escherichia coli which was less susceptible. From this work, it can be said that the effects of bacitracin produced by Bacillus subtilis cannot be optimized except with the variation of certain culture conditions. It can also be said that some cultural parameters decrease its effectiveness and thus should be avoided during industrial production of this antibiotic.
TABLE OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgements iv
Table of Contents v
List of Tables vii
List of figures viii
Abstract ix
CHAPTER
ONE
1.0 Introduction 1
1.1 Background of Study 1
1.2 Aims and Objectives of the Study 3
CHAPTER
TWO
2.0 Literature
Review 4
2.1 Bacillus subtilis 4
2.1.1 Description 4
2.1.2 Habitat 5
2.1.3 Reproduction 5
2.1.4 Uses 5
2.2 Bacillus Antibiotics 7
2.2.1 Non-Ribosomal
Biosynthesized Peptides 9
2.3 Bacitracin 10
2.3.1 Medical Use 11
2.3.2 Spectrum of activity and Susceptibility Data 12
2.3.3 Mechanism
of Action 12
2.3.4 History 12
2.3.5 Synthesis 13
2.3.6 Composition 13
CHAPTER THREE
3.0 Materials
and Methods 14
3.1 Materials 14
3.1.1 Source
of Material 14
3.1.2 Equipments
and Apparatus 14
3.1.3 Sample
and Sampling 14
3.2 Preparation
of Culture Media 14
3.2.1 Agar Preparation 14
3.2.2 Broth Preparation 15
3.2.3 Slant
Preparation 15
3.2.4 Inoculation
Procedures 15
3.3 Screening
for Bacillus subtilis 15
3.3.1 Gram Stain 16
3.3.2 Endospore Stain 17
3.3.3 Biochemical Tests 17
3.3.3.1 Catalase Test 17
3.3.3.2 Citrate
Utilization Test 17
3.3.3.3 Oxidase Test 18
3.3.3.4 Gelatin Hydrolysis Test 18
3.3.3.5 Methyl Red/Voges Proskauer Test 18
3.3.3.6 Sugar Fermentation Tests 18
3.3.3.7 Indole Test 19
3.3.3.8 Coagulase Test 19
3.3.3.9 Bacitracin Susceptibility Test 19
3.4 Varying
Culture Parameters 19
3.5 Extraction
of Crude Bacitracin 20
3.6 Preparation
of Test Organisms 20
3.6.1 Escherichia coli 20
3.6.2 Staphylococcus aureus 20
3.7 Effects of Crude bacitracin extract on
Test organisms 20
CHAPTER FOUR
4.0 Results 22
CHAPTER FIVE
5.0 Discussion,
Conclusion and Recommendations 31
5.0 Discussion 31
5.1 Recommendation 34
5.2 Conclusion 34
REFERENCES 36
APPENDIX 42
LIST OF TABLES
Table Title of Table
Table 4.1: Identification Tests for Bacillus subtilis 24
Table 4.2: Identification of the Test
Organisms 25
Table 4.3: Zones of Inhibition
measured in milimeters (mm) against the test organisms
from different
culture conditions 26
Table 4.4: Minimum Inhibition
Concentrations (MIC) for crude bacitracin against test
organisms 30
LIST OF FIGURES
Figure Title
of Figure
Figure 4.1: Comparism of the
inhibition zones of undiluted bacitracin from different carbon and nitrogen sources conditions 27
Figure 4.2: Comparism of the
inhibition zones of undiluted bacitracin from different pH levels, against the test organisms 28
Figure 4.3: Comparism of the
inhibition zones of undiluted bacitracin from other culture conditions 29
CHAPTER
ONE
1.0 INTRODUCTION
1.1 BACKGROUND OF STUDY
The
word “antibiotic” is derived from Greek term antibiosis, which literally means
“against life”. Antibiotics are low molecular-weight (non-protein) molecules
produced as secondary metabolites, mainly by microorganisms that live in the
soil. It can be purified from microbial fermentation and modified chemically or
enzymatically for either chemical use or for fundamental studies. The
antibiotics are widely distributed in the nature, where they play an important
role in regulating the microbial population of soil, water, sewage and compost.
Of the several hundred naturally produced antibiotics that have been purified,
only a few have been sufficiently non-toxic to be of use in medical practice.
Those that are currently of greatest use have been derived from a relatively
small group of microorganisms belonging to the genera Penicillium,
Streptomyces, Cephalosporium, Micromonospora and Bacillus. More than
5000 different antibiotics have been isolated from cultures of bacteria, fungi
and plant cells, 60% of them are contributed by the genus Streptomyces (Todar,
2002). In pharmaceutical industry, several peptide antibiotics of importance
are produced by Bacillus species such as bacitracin, polymyxin,
gramicidin, tyrocidine, subtilin, bacilysin etc. Bacilli are rod-shaped,
Gram-positive, sporulating, aerobes or facultative anaerobes. Most bacilli are
saprophytes. Each bacterium creates only one spore, which is resistant to heat,
cold, radiation, desiccation, and disinfectants. Bacilli exhibit an array of
physiologic abilities that allow them to live in a wide range of habitats,
including many extreme habitats such as desert sands, hot springs, and Arctic
soils. Species in the genus Bacillus can be thermophilic, psychrophilic,
acidophilic, alkaliphilic, halotolerant or halophilic and are capable at
growing at pH values, temperatures, and salt concentrations where few other organisms
can survive. A natural assumption is that soil microbes produce antibiotics in
their natural habitat and use them to gain advantage over their competitors;
that is, antibiotics are presumed to be involved in naturally occurring amensal
relationship in the soil. Most of the peptide antibiotics produced by bacilli
are active against gram-positive bacteria; however, compounds such as
polymyxin, colistin and circulin exhibit activity almost exclusively upon
gram-negative bacteria, whereas bacillomycin, mycobacillin and fungistatin are
effective against molds and yeasts (Katz and Demain, 1977). Berdy (1974)
reported the production of 167 peptide antibiotics from Bacillus subtilis and
Bacillus brevis. Of this total, 66 different peptide antibiotics are elaborated
by strains of Bacillus subtilis and 23 are products of Bacillus
brevis.
Bacitracin
is derived from cultures of Bacillus subtilis. It is a white to pale
buff, hygroscopic powder, odorless or having a slight odor. It is precipitated
from its solutions and inactivated by many of the heavy metals. Bacitracin
is a mixture of several polypeptides differing in their amino acid composition
(Ohki, 2003) and functions as an inhibitor of cell wall biosynthesis.
Bacitracin of other micro-organism is an antibiotic as well as non-ribosomally
produced by Bacillus licheniformis (Jyothi et al., 2010). Bacitracin affects protein synthesis, cell
wall synthesis and membrane functions.
Bacitracin
produced by B. subtilis is very effective topically and its action is
especially on Gram-positive cell walls. The biggest share of industrial enzymes
are produced by Bacillus, the laundry industry is consuming various
subtilisins, cellulases and amylases produced by B. subtilis (Jarnagin
and Ferrari, 1992). Other uses of enzymes isolated from B. subtilis include,
modification of milk proteins in dairy products by neutral proteases, starch
and maltose syrup production by the different amylases and pullulanases, high
fructose corn syrup production utilizing glucose isomerases and modification of
the barley cell wall in brewing processes by beta-glucanases (Zukowski, 1992).
1.2 AIMS AND OBJECTIVES OF THE STUDY
This study is aimed at understanding the effects
different culture conditions have on the bacitracin produced by Bacillus
subtilis isolated from a soil sample.
The objectives of this work are:
·
To
isolate bacitracin producing Bacillus subtilis from the soil.
·
To
vary the culture conditions needed for bacitracin production of the isolated Bacillus
subtilis.
·
To
determine the effects different cultural parameters have on the antimicrobial
activity of bacitracin produced by Bacillus subtilis.
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