ABSTRACT
Antibiotics are chemical substances that have the ability to inhibit or destroy microorganisms. Soil that was contaminated with abattoir effluents was investigated using general medium (nutrient agar) and selective media (glycerol nutrient agar and glycerol yeast agar). In the enumeration of total viable count, the result showed high bacteria counts, which ranged from 9.5 x 108 to 2.05 x 109 cfu/g soil. The soils were screened for antibiotic producing bacteria by agar sensitivity test. The bacterial isolated were identified by their colony features, microscopic structures, biochemical tests and by molecular characteristics. The bacterial isolates include Staphylococcus aureus, Bacillus subtilis, B. cereus, B.coagulans, Escherichia coli, Pseudomonas spp, Streptomyces globosus, S. hygroscopicus, S. oriantalis, and Micrococus spp. Among all the isolates, Bacillus subtilis , B. cereus, B.coagulans, Streptomyces globosus, S. hygroscopicus, and S. oriatalis exhibited antibiotics production by evidence of zone of inhibition round their colonies. The statistical analyses done on the antibacterial activity of the isolates revealed significant difference (P˂0.05) between the antibacterial agents produced by organisms and the chloramphenicol which is the standard. This study revealed that abattoir soils have the growth factors that can enhance the proliferation of antibiotic producing bacteria, which have the potency of secreting antibiotics that can be used in the now and in the future for the control of microbial growth.
TABLE OF
CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List
of Tables ix
Abstract x
CHAPTER 1: INTRODUCTION
1.1 Background
of the Study 1
1.2 Aim of the Study 2
1.3 Specific Objectives 2
1.4 Statement of the Problem 2
1.5 Justification
of the Study 3
1.5 Significance
of the Study 3
CHAPTER 2: LITERATURE REVIEW 4
2.1 Early Days of
Antibiotics Development 4
2.2 Classification and Nomenclature of Antibiotics 5
2.3 Biochemistry of Antibiotic Production 9
2.3.1 Mechanism of antimicrobial action 11
2.4 Mechanism of Antibiotic Resistance by Bacteria 14
2.5 Microorganisms Isolated from Abattoir Environment 19
2.6 Effect of Abattoir Effluents on Soil and
other Environment 20
2.6.1 Loss of land value 21
2.6.2 Soil contamination 22
2.6.3 Sources of heavy metals 23
2.6.4 Climate change and global warming 23
CHAPTER
3: MATERIALS AND METHODS 24
3.1 Collection of Samples 24
3.2 Sources
of Soil Samples and Sampling Designs 24
3.3 Preparation
of culture Media and Diluent 25
3.4 Isolation
of Bacteria from Abattoir Soil Samples 26
3.4.1 Enumeration
of total viable count 26
3.4.2 Isolation
of antibiotic producing bacteria 26
3.4.3 Isolation
of test bacteria 27
3.4.4 Determination
of occurrence of bacterial isolate 27
3.5 Characterization
and Identification of Isolates 28
3.5.1 Morphological
identification of isolates 28
3.5.2 Microscopic
structures 28
3.5.3 Biochemical reactions for identification of
bacterial isolates 28
3.5.4 Antibacterial sensitivity screening for the
isolates 31
3.5.5 Determination of relative antibiotic potency 32
3.6 Molecular Characterization of
Antibiotic Producing Isolates 32
3.7 Statistical
Analysis 33
CHAPTER 4: RESULTS
AND DISCUSSION 34
4.1 Results 34
4.2 Discussion 44
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS 47
5.1 Conclusion 47
5.2 Recommendations 47
References 49
Appendix 57
LIST OF TABLES
2.1 Groups of antibiotics named based on
their chemical structures 6
2.2: Antibiotics produced by various microorganisms 7
3.1 Coding of samples and point sources 25
4.1: Total
viable count (cfu/g) of bacteria in abattoir soil 36
4.2: Identification
of bacteria isolated from abattoir soils 37
4.3: Bacteria
isolated from the Abattoir Soil and their percentage
Occurrence 38
4.4: Characteristics of antibiotics producing
isolates (Bacillus and
Streptomyces species)
from abattoir soils 39
4.5 Antibacterial
sensitivity test for the isolates using S.
aureus and
E.
coli as test
organisms 41
4.6 Relative antibiotic potency of the test
bacteria on S. aureus and E.coli 42
4.7: Molecular
identification of antibiotics producing bacterium 43
4.8: ANOVA on the antibacterial activity of Bacillus and Streptomyces
isolates from abattoir soils 59
CHAPTER 1
INTRODUCTION
1.1
BACKGROUND OF THE STUDY
Soil is the major
reservoir of microorganisms that produce antibiotics. Those bacteria that are present in the soil
can be of different shapes such as rods, cocci, and spirilla. The number of Bacillus and Streptomyces present in the soil are said to be more than other
organisms, even as they are widely distributed (Brock and Madigam, 1991). An antibiotic produced by a bacterium, have
the potency of inhibiting other related organisms that are within for the
benefit of their bacterial genera (Jackie, 2011).
In the year 1929, Alexander Fleming
discovered the antibacterial effect of penicillin. In his work, he found that a
fungal colony had grown on an agar plate streaked with Staphylococcus aureus, and that the bacterial colonies around the
fungus were transparent, which shows that their cells have been lysed (Fleming,
1929).
Antibiotics are well produced by actinomycetes
which are Gram-positive bacteria. Actinomyces have DNA which is rich in guanine
plus cytosine (G+C) content (Gonzalz-Franco and Robles-Hernandez, 2009; Gurung et al., 2009; Ogunmwonyi et al., 2010). They are commonly
isolated from soil and also from other environmental niches such as marine
sediments. This genus Streptomyces are said to be the most
prolific antibiotic producers and they belong to a unique subgroup of bacteria called
the Actinomycetes. Streptomyces possess
high number of genes that encode for various enzymes, which are involved in the
synthesis of secondary metabolites (Tiwari and Gupta, 2013). However, previous information on the
isolation of antibiotic producing bacteria, from an abattoir soil, have shown
that re-isolation of strains has become a challenge when isolating cultures
that can yield new and vital secondary metabolites (Bushell, 1982). Abattoir
wastes that drains into the soil, pose threat to the organisms. These drained
wastes can cause oxygen to become less available, which will in turn encourage
denitrifying bacteria, to reduce available nitrate to gaseous nitrogen. When
this gaseous nitrogen enters the atmosphere, it has every tendency to cause
some negative effects (Edward, 1990). When an untreated wastewater is
introduced into the soil, certain elements like phosphorus, calcium, lead,
iron, and zinc are being released. Those released chemicals, can cause change
in the physicochemical status of the soil and some of these elements may be
harmful to the soil microbial flora and fauna (Tortora et al., 2007).
However, this work is expected to isolate
and characterize bacteria that have the potency of synthesizing antibiotics from
abattoir soil.
1.2 AIM
OF THE STUDY
The aim of this research is to isolate and characterize antibiotic
producing bacteria from abattoir soil at Ogbor-Hill market waterside. Aba, Abia
State, Nigeria.
1.3 SPECIFIC OBJECTIVES
i.
Screening and
identification of antibiotic producing bacteria from abattoir soil around
Ogbor-Hill Aba, Abia state, Nigeria.
ii.
Molecular
characterization of antibiotic producing isolates.
1.4 STATEMENT
OF THE PROBLEM
Antibiotics are chemical compounds
that have been used before being discovered as remedy for infections,
inflammations and diseases and these antibiotics are prevalent almost
everywhere. Abattoir is bio-diversified with various types of microorganisms
which include mainly bacteria, cyanobacteria, microfungi, microalgae and
protozoans. Some of these microorganisms found in abattoir are potential
reservoir for antimicrobial resistance genes. The secondary metabolites
produced by these antibiotic-producing bacteria are said to be of great
importance. Today almost all the known disease producing bacteria have
developed resistance to the existing drugs because of their extensive use.
Therefore, there is high need for new antimicrobial agents because of the
development of multidrug resistance microorganisms
1.5 JUSTIFICATION OF THE STUDY
The purpose for isolating and
identifying bacteria from abattoir soil is to see that new antibacterial agents
available in the abattoir soil are being harnessed in order to tackle the
development of multidrug resistance, microorganisms that are of great concern
to many. The identification of antibiotic-producing bacteria will make
subsequent analysis such as purification of the antibiotics to be easier.
1.6
SIGNIFICANCE OF THE STUDY
This work is all about prospecting
for antibiotic producing bacteria using abattoir soil as a case study. This
might lead to isolation of new antibiotic that may eventually form the basis
for development of other related drugs. The organisms could be deposited in
type culture collection centers for further studies.
The research study is of immense
importance because it will help discover specific antibiotic producing bacteria
that are needed to intensify the treatment of drug-resistant microorganisms.
To other researchers, this study will
help them to isolate antibiotics producing bacteria from abattoirs.
To other health professionals, the
study will help them to know that antibiotics producing bacteria are present in
an abattoir soil. The study will further help in the isolation of the bacteria
and their use, in the fight against infections and treatment of diseases.
The research will also help other
Researchers to know the efficacy of the antibiotics producing bacteria isolated
from abattoir soil.
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