ABSTRACT
Antibiotic production is a feature of several kinds of soil bacteria and fungi and may represent a survival mechanism where microorganisms can eliminate competition and colonize a niche. Soil samples were collected from 4 different mechanic workshops in Umuahia for the isolation and screening of microorganisms for antibiotic production. The media used for the isolation were Tryptone Soy agar, Sabouraud Dextrose agar and Nutrient agar. Sensitivity testing using Escherichia coli, Staphylococcus aureus, Samonella typhi, and Enterococcus feacalis as test organisms was employed to determine the ability of bacterial and fungal isolates to produce antimicrobial substance. Six out of 38 isolates obtained from the soil samples possessed antimicrobial activity against at least three of the test organisms. The six isolates included Bacillus subtilis, Bacillus spp. Micrococcus spp. Sphingomonas spp. Aspergillus spp. and Acremonium chrysogenium. Of all the isolates, Bacillus spp. and Bacillus subtilis showed the greatest antimicrobial activity. Bacillus spp. inhibited Salmonella typhi, Escherichia coli, Staphylococcus aureus with zones of inhibition 12mm, 10mm, 12mm, respectively and Bacillus subtilis inhibited Salmonella typhi, Escherichia coli, and Staphylococcus aureus with zones of inhibition 13mm, 10mm, and 9mm. The extracellular substances produced by Bacillus spp, Bacillus subtilis, Micrococcus spp, Sphingomonas spp, Acremonium chrysogenium and Aspergillus spp which inhibited the growth of some test organisms can be used to produce potent antibiotics if harnessed and purified as those of commercially available antibiotics.
TABLE
OF CONTENTS
Title
page i
Certification
ii
Dedication iii
Acknowledgements
iv
Table
of Contents v
List
of Tables
viii
List of Figures ix
Abstract
x
CHAPTER ONE
1.1
INTRODUCTION 1
1.2
Aims and Objectives
3
CHAPTER TWO
2.0
LITERATURE REVIEW
4
2.1 Soil
4
2.1.1 The Soil of
Mechanic Workshop 5
2.2.2 Soil
Bacteria
6
2.2 Antibiotics
6
2.3 Antibiotic
Producing Organism
7
2.4
Antibiotic Resistance
8
2.5
Previous Studies on Antibiotic Production
9
2.6
Mechanism of Antibiotics Resistance 10
2.7
The Need for New Antibiotics 11
2.8.
Production of Antibiotics
12
CHAPTER THREE
3.0
MATERIALS and METHODS 14
3.1
Sample Collection
14
3.2
Microbiological Methods
14
3.3
Media and Media Preparation
14
3.4
Isolation of Soil Microorganism
14
3.5
Subculturing / Purification of Isolates
15
3.6
Preparation of Inoculum of Test Organism
15
3.7
Screening for Antibacterial Activity
15
3.8
Characterization and Identification of Isolates
16
3.8.1
Gram Staining 16
3.8.2
Biochemical Testing
16
3.8.2.1
Indole Test
16
3.8.2.2
Citrate Utilization Test
16
3.8.2.3
Starch Hydrolysis 17
3.8.2.4 Catalase
Test
17
3.8.2.5 Hydrogen
Sulfide Production
17
CHAPTER FOUR
4.0
RESULTS 18
CHAPTER FIVE
5.1
DISCUSSION
25
5.2
Conclusion
26
5.3
Recommendation
27
References 28
LIST OF TABLES
Table Title Page
1 Sample sites and number of
isolates obtained 19
3 Colonial morphology and microscopic characteristics of fungal isolates 22
4 Antimicrobial
activity of the isolates 23
LIST OF PLATES
Plates Title page
1(a) & (b) Inhibition of the growth of the
test organisms by isolates 24
CHAPTER ONE
1.0
INTRODUCTION
The soil is an important reservoir of
several organisms. It is the largest source of microorganism. The number and
type of bacteria present in a particular soil is greatly influenced by
environmental factors such as soil temperature, soil type, soil pH, nutrient
availability and soil texture.
In Nigeria, it is common among mechanics
to dispense spent engine oil into gutters, water drains and soil (Okonokhua et al., 2007). Exploration and
exploitation of petroleum in Nigeria has led to the contamination of several
Nigerian ecosystem with petroleum hydrocarbon, especially in the Niger delta
region (Kadafa, 2012). Spent engine oil is defined as used lubricating oil
obtained after servicing or repair of automobile. Automobile (motor and motorcycle)
repairs and maintenance services by self-employed artesian mechanics generates
used petroleum products, comprising mainly used engine lubricating oil, diesel
and petrol used to wash out grease from machine parts (Orji et al., 2015). Spent engine oil causes
great damage to soil and soil micro flora. It creates unsatisfactory condition
for life in the soil due to poor aeration, immobilization of nutrients and
lowering of pH (Ugoh et al., 2011).
The trend of search for antibiotics in the
past and in recent times as a result of drug resistance by microbial species
has required combing the earth for various sources of antibiotics including the
soil (Ahmed et al., 2013). In the
soil of mechanic workshop, where most organism producing antimicrobial agent
are found, life is competitive. The inhabitant competes for carbon, nitrogen
and phosphate necessary for their growth. Successful competition may be ensured
by inhibition of the growth of other organism through the production and
secretion of substances interfering with their metabolism (Ogbonna et al., 2013). These microorganism in
the soil of mechanic workshop are capable of producing bioactive secondary
metabolite.
Antibiotics are chemical substances of
microbial origin that can prevent or destroy the growth of other
microorganisms. Antibiotics are one of the important commercially exploited
secondary metabolite produced by microorganisms and are been used to treat
human, animal and plant diseases. Historically, microorganisms have provided the
source of the majority of drugs in use today (Demain et al., 2009). For years, a pathogen’s ability to resist
antibiotics has kept them one step ahead of researchers when it comes to
developing new antibiotics that can truly overcome bacteria. Old antibiotics
lose their efficacy and are necessarily replaced with new ones for many species
of pathogenic bacteria (Hancock, 2007). Some antibiotics like Penicillin,
Erythromycin and Methicillin which used to be a one-time effective drug against
infectious disease (Raja et al.,
2010) are now less effective in treating infectious disease because bacteria
have become more resistant to such antibiotics.
Antibiotic resistant pathogens such as
Methicillin and Vancomycin resistant strain of Staphylococcus aureus cause huge threat to the treatment of serious
infections. In order to prevent this happening, quick replacement of the
existing antibiotic is important and the development of novel drugs against
drug resistant pathogens is significant for today.
Discovering new antibiotics for the
treatment of infection and disease as well as the practice of antibiotic
combination therapy can be able to delay the emergency of microbial resistance
and can also produce desirable synergistic effects in the treatment of infection
and disease caused by pathogenic microorganism.
Research in identifying newer antibiotics and
increasing productivity of antibiotics has been a very important activity
(Vengadesh et al., 2011; Retinowati,
2010). This is because some important drugs have side effects to the host or
some microbes are developing multidrug resistance. These factors have made the
need to discover new and effective antibiotics from microorganisms present in
different environment such as the soil to increase.
1.1
Aims and Objectives
1. To
isolate and screen soil microorganisms from mechanic workshops in Umuahia for
antibiotic production.
2. To
characterize the isolated microorganisms.
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