ABSTRACT
This research work was conducted to access the antibacterial activity of Bacillus species isolated from agricultural sites in two different communities (Umudike and Ndoru in Ikwuano local government area) in Abia State of Nigeria. The antibacterial activities of the isolated Bacillus spp was tested against three different bacterial strains (Staphylococcus aureus, micrococcus spp, Escherichia coli) by agar well diffusion method and incubated for 24hrs at 370C. The Research Results shows that most strains possess significant antibacterial potentials mostly against S. aureus and micrococcus sp but less activity against the Gram negative E. coli. Thereafter, Further investigations was carried out to determine the optimum growth conditions for these Bacillus species for maximum antibiotic production. Optimum activity was achieved using nutrient broth culture at different optimization conditions (temperatures; 300C., 370C. and 450C. incubation time; 24hrs,48hrs and 72hrs and pH; 6, 7, 8), The medium was then centrifuged at 10,000rpm for 20minutes to get the cell free supernatant and a Bacitracin assay done on nutrient agar plates by measuring the diameter zones of inhibition in millimeters(mm) against the test organisms being used. And the highest zone of inhibition for incubation temperature was observed, (24mm) against Micrococcus spp at 370C, (27mm) at 72hrs incubation period and (24mm) at pH8, antibacterial activity was also produced maximally against S. aureus at optimized temperature, incubation time and pH as (18mm, 20mm and 14mm) respectively and zero effect was seen on E. coli. Therefore, antimicrobial compounds from these Bacillus species can be good candidates for future antibiotics production. Further screening for antimicrobial agents should be carried out in search of novel therapeutic compounds.
TABLE OF CONTENTS
Title page I
Certification II
Dedication III
Acknowledgement IV
Table of content V
List of Tables VIII
Abstract IX
CHAPTER ONE
1.1 Introduction 1
1.2 Objectives of this Study 3 CHAPTER TWO
2.0 Literature Review 4
2.1 Bacillus and its Taxanomy 4
2.1.1 Bacillus Cereus Group 4
2.1.2 Bacillus Subtilis Group 7
2.2 The Nature Of Bacillus Endospore 8
2.3 Bacillus Endospore Formation Process 8
2.4 Structure, Biosynthesis And Mechanism Of Action Of Bacitracin 10
2.4.1 Structure Of Bacitracin 10
2.4.2 Biosynthesis Of Bacitracin 11
2.4.3 Mechanism Of Action Of Bacitracin 11
2.5 Antimicrobial Spectrum Of Bacitracin 11
2.6 Uses Of Bacitracin 12
CHAPTER THREE
3.0 Materials And Methods 13
3.1 The Study Area 13
3.2 Soil Sample Collection 13
3.3 Sample Preparation 14
3.4 Sterility 14
3.5 Preparation Of Culture Media 14
3.7 Collection And Confirmation Of Test Isolates 15
3.7.1 Colonial Morphology 15
3.7.2 Gram Staining 15
3.7.3 Motility Test 15
3.7.4 Biochemical Test 16
3.8 Preliminary Screening For Bacitracin Producing Bacillus Spp 18
3.9 Optimization Of Conditions For Bacitracin Production 18
3.9.1 Optimization For Incubation Temperature 19
3.9.2 Optimization For Time Of Incubation 19
3.9.3 Optimization For pH Of Incubation 19
3.10 Presentation And Analysis Of Data 19
CHAPTER FOUR
4.0 Results 20
4.1 Results Presentation 20
CHAPTER FIVE
5.0 Discussion, Conclusion And Recommendation 28
5.1 Discussion 28
5.2 Conclusion 29
5.3 Recommendations 30
REFERENCES
LIST OF TABLES
Table 1: Sample collection sites 22 Table 2: Morphological and biochemical characteristics of isolates 23
Table 3: Preliminary screening for bacitracin 24
Table 4: Antimicrobial effect of crude extracts of Bacitracin against E. coli,
S. aureus and Micrococcus spp at optimized incubation temperature 25
Table 5: Antimicrobial effect of crude extracts of Bacitracin against E. coli,
S. aureus and Micrococcus spp at various incubation periods 26
Table 6: Antimicrobial effect of crude extracts of Bacitracin against E. coli,
S. aureus and Micrococcus spp at various optimized pH 27
CHAPTER ONE
1.1 INTRODUCTION
Bacillus bacteria belong to the bacillaceae family, they are Gram positive bacteria and can be found in diverse habitats but majorly in soil ( Splepecky and Hemphil, 2006; Graumann, 2012).
Their cells are straight, rod shaped and can be either found in pairs or singles , chains, or even as long filaments as well as peritrichous flagella in some motile species ( Baruzzi et al., 2011). Moreover for each of the cells, an endospore forms to resist adverse and harsh conditions such as radiations, heat and chemicals i.e. disinfectants (Tan and Ramamustii, 2014). Although most species are facultative aerobes, some can be anaerobic (Prieto et al., 2014). Several studies have revealed that some species such as Bacillus subtilis, Bacillus brevis, Bacillus circulans, Bacillus polymyxa, Bacillus cereus and Bacillus lichenformis are capable of producing antibiotics ( Hirad et al., 2013; Sawale et al., 2013; Ral et al., 2017).
Some of them were extracted and used for many medical treatments for example: Tyrothricidin for sore throat infections and polymyxin for ear inflammations (Mondol et al., 2013) antibiotics.
Antibiotics are low molecular-weight (non-protein) molecules produced as secondary metabolites, mainly by microorganisms that live in the soil i.e. Bacillus species (Bushra et al., 2007). A natural assumption is that soil microbes produce antibiotics in their natural habitats and use them to gain advantage over their competitors; that is antibiotics are presumed to be involved in naturally occurring commensal relationship in the soil. Regardless of the toxicity of some antibiotics produced by bacteria from Bacillus genus to the cells of mammals (eg. Polymyxins, bacitracin, etc). They and continued to be in the focus of scientists. The amount of antibiotics produced by these bacillus is approaching 167. From that more than 66 derived from B. subtilis and about 23 originated from B.brevis (Katz and Demain, 1977; Holt et al., 1994). As is generally recognized these antibiotics are mostly polypeptides. Most of the peptides antibiotics produced by Bacillus are most active against Gram positive organisms (Ming and Epperson, 2002). Examples of peptide antibiotics includes some well-known or commonly used drugs such as bacitracin, polymyxin etc.
In the case of Bacillus species antibiotics are produced during the early stages of spore formation which suggests that this phenomenon is considered as defense mechanism of these bacterial species against other microorganism (Sumi et al., 2015). In addition the antibiotics synthesized by Bacillus species can be classified into four categories according to their mechanism of action Cyclic oligopeptides (For Example, Bacitracin ) that hinder cell wall synthesis; Linear oligopeptides ( for example gramicidins and polymyxins )that interferes with cell membrane functions; Basic peptides ( for example, edeines ) that restrains the initiation complex formation on the small ribosome subunit; Aminoglycosides antibiotics ,that distresses ribosomes function ( Graumann, 2012).
Various scientific literatures have revealed that most Bacillus species isolated from soil samples especially soil samples from marine sites and agricultural sites showed more antagonistic activities against Gram positive bacteria such as Staphylococcus aureus and Staphylococcus epidermidis, but however less toward Gram negative bacteria such as Pseudomonas aeruginosa ( Kannahi and Eshwari, 2016 ). This phenomenon is supported by recent studies in India and Arabian Gulf ( Pavarthi et al., 2009; Hirad et al., 2013; Sawale et al., 2013). In addition, this case could also be explained due to differences in cell wall composition. Unlike Gram positive bacteria which has only one thick layer peptidoglycan, Gram negative bacteria has far more complex components, in which the antimicrobial components of the must cross to interfere with its molecules required during cell wall biosynthesis ( Malanovic and Lohner, 2016 ).
Moreover, antibiotics inhibit growth of the Gram positive bacterium S.aureus via the obstruction of a carrier of peptidoglycan monomers, “lipid II”, across the cytoplasm membrane to the exterior side. As a consequence, the incorporation of the growing peptidoglycan network by lipid II is inhibited during the cell wall synthesis (Baruzzi et al., 2011; Sumi et al., 2015). Another possible explanation for the antimicrobial peptides’ action is that these disrupt the cell cytoplasm by binding to and aggregating with the membrane. As a result, these forms pores through which ions can pass and lead to cell components leakage and eventually, cell death ( kuta et al.,2015 ).
1.2 OBJECTIVES OF THIS STUDY
· The main aim of this study is to isolate Bacillus species that exhibit antibacterial properties against Gram positive and Gram negative bacteria as well as to investigate the production of these antibiotics at different optimal conditions
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