ABSTRACT Endophytic microorganisms have proven to be potential sources of antimicrobials against recalcitrant pathogens. Extracts of an endophytic fungus, Emericella nidulans exhibited antibacterial activity against selected pathogens. Emericella nidulans was cultivated in broth and fermented at 28 ± 2°C for 7days and 21 days to obtain bioactive components. Solvents extraction of bioactive compounds from Emericella nidulans in the order of their polarity – n-Hexane, ethyl acetate and ethanol – revealed significant susceptibilities of the test organisms to the extracts viz: Staphylococcus aureus ATCC 6538, Salmonella enterica subspecies enterica serovar typhimurium ATCC 14028, Escherichia coli ATCC 35218, and Enterococcus faecalis ATCC 51299 using the agar well diffusion method. Standard antibiotics were used as positive control while dimethyl sulfoxide (DMSO) was used as negative control. The n-hexane and ethyl acetate extracts inhibited the growth of the test organisms at 8.03 ± 0.35mm – 28.00 ± 0.56mm and 9.77 ± 0.66 – 24.50 ± 0.60 with minimum inhibitory concentration of 16mg/ml – 256mg/ml and 32mg/ml – 256mg/ml respectively. n-hexane extracts were bactericidal to the test organisms except Escherichia coli ATCC 35218; while ethyl acetate was bactericidal against Staphylococcus aureus ATCC 6538 and Enterococcus faecalis ATCC 51299 only. However in this study, ethanolic extracts had no activity against the test organisms. Thus, extracts of Emericella nidulans have purported activity against selected pathogens.
TABLE OF CONTENTS
Content Page
Title page………………………………………………………………………………i Certification…………………………………………………………………………...ii Dedication…………………………………………………………………………….iii Acknowledgement……………………………………………………………………iv
Table of content………………………………………………………………………vi
List of tables………………………………………………………………………….ix
List of plates………………………………………………………………………….x Abstract………………………………………………………………………………xi
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1 Antimicrobial agents: Brief history and development……………….….……1
1.2 Antimicrobial resistance…………………………………………….….……..2
1.2.1 Causes of antimicrobial resistance………………………………….….……..3
1.2.2 Mechanism of resistance………………………………………….….……….4
1.3 Medical importance of test organisms…………………………….………….5
1.3.1 Staphylococcus aureus………………………………………………….……………5
1.3.2 Escherichia coli………………………………………………….……………6
1.3.3 Salmonella enterica…………………………………………….……………..7
1.3.4 Enterococcus faecalis………………………………………….……………...8
1.4 Plants as antimicrobial agents……………………………….………………..9
1.4.1 Ethno pharmacology……………………………………….………………..10
1.4.2 Endophytes in plants……………………………………….………………..11
1.4.2.1 Fungi…………………………………………………….……………….....13
1.4.2.2 Bacteria………………………………………………….………………….14
1.4.2.3 Actinomycetes………………………………………….…………………..14
1.5 Moringa oleifera…………………………………………….……………………...15
1.5.1 Morphology of Moringa oleifera………………………….……………………...15
1.5.2 Etymology of Moringa oleifera……………………….…………………….16
1.5.3 Geographical source of Moringa oleifera…………….……………………..16
1.5.4 Taxonomic classification of Moringa oleifera……….………………………….16
1.5.5 Chemical composition of Moringa oleifera………….…………………………..17
1.5.6 Pharmacology……………………………………….……………………….17
1.6 The endophyte Emericella nidulans……………….………………………...18
1.6.1 Morphology of Emericella nidulans……………….………………………...18
1.6.2 Habitat of Emericella nidulans……………………….…………………………….20
1.6.3 Applications of Emericella nidulans………………..……………………………..20
1.7 Endophytes of Moringa oleifera…………………………………………………..20
1.8 Justification…………………………………………………………………..21
1.9 Aim of the study……………………………………………………………..21
1.10 Specific objectives…………………………………………………………...22
CHAPTER TWO: MATERIALS AND METHODS
2.1 Sterilization of materials……………………………………………………..26
2.2 Preparation of culture media.……………………………..…………………26
2.3 Source of test organisms…...…………………………………………………27
2.4 Maintenance of test organisms……………………………………………….27
2.5 Source of the endophytic fungus Emericella nidulans.......………………….27
2.6 Cultivation and Extraction…………………………………………………..27
2.7 Reconstitution of extracts…………………………………………………...28
2.8 Antimicrobial susceptibility tests…………………………………………...28
2.8.1 Determination of antimicrobial activity of the extract……………………...28
2.8.2 Determination of minimum inhibitory concentration………………………29
2.8.3 Determination of minimum bactericidal concentration…………………….30
2.8.4 Antimicrobial susceptibility test using standard antibiotics………………..30
2.9 Statistical analysis…………………………………………………………..31
CHAPTER THREE: RESULTS
3.1 Antimicrobial susceptibility of test organisms to extracts…………………32
3.2 Minimum inhibitory concentration………………………………………...32
3.3 Minimum bactericidal concentration………………………………………32
3.4 Susceptibility of test organisms to standard antibiotics……………………37
CHAPTER FOUR: DISCUSSION……………………………………………….39 Recommendations…………………………………………………………………..46 Conclusion…………………………………………………………………………..47 References…………………………………………………………………………..48
Appendix…………………………………………………………………….……...65
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LIST OF TABLES
Table Title
Page 1. Antimicrobial susceptibilities to extracts of Emericella nidulans…... 33
2. Minimum inhibitory concentration…………………………………...35
3. Minimum bactericidal concentration…………………………………36
4. Susceptibility of test organisms to standard antibiotics………………38
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LIST OF PLATES Table Title Page 1a. Seeds of Moringa oleifera Lam. ……………………………………..23 1b. Leaves of Moringa oleifera Lam. ……………………………………24 1c. Flowers of Moringa oleifera Lam. …………………………………..25 2. Pure culture of Emericella nidulans……………………………Appendix I
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