ISOLATION OF ANTIBIOTICS PRODUCING MICRO ORGANISMS FROM THE FARM LAND BEHIND CENTER FOR MOLECULAR BIOSCIENCES AND BIOTECHNOLOGY (CMBB) IN MICHAEL OKPARA UNIVERSITY OF AGRICULTURE, UMUDIKE

ABSTRACT

Antibiotics have substantially reduced the threat of infectious diseases. In the present study, twenty (20) soil samples were collected from the farm land behind Centre for Molecular Biosciences and Biotechnology (CMBB) in  Michael Okpara University of Agriculture Umudike (MOUAU). Streptomyces spp. and Bacillus spp. were isolated using Tryptone Soy Agar (T.S.A), Nutrient Agar (N.A), Sabouraud dextrose agar (S.D.A). A total of 50 isolates were gotten. These organisms were screened with regard to their antibacterial potential against different test organisms. The antibacterial activity of the isolates was tested using the disk diffusion method on a spread plate of the test organism (Staphylococcus aureus ATCC 2593TM, Pseudomonas aeruginosa ATCC 27853TM, Eschericha coli ATCC 25922TM, Enterococcus fecalis ATCC 7080TM). Five (5) isolates (10%) (A3, L2, C3, D1, J4) showed positive antibacterial activity. The positive isolates were further characterized based on their gram status and biochemical tests which include starch Hydrolysis, Hydrogen sulphide test, Catalase test. Some fungal spp which were isolated did not show any positive antibacterial activity and were not further characterized. The result obtained from this study, provides some information that would be useful to pharmaceutical industries that may be interested in the local production of antibiotics using locally isolated stains of microorganisms.

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TABLE OF CONTENTS

Title Page  i

Certification    ii

Dedication  iii

Acknowledgements  iv

Table of contents  v

List of Tables  vii

Abstract    x

CHAPTER ONE

1.0 INTRODUCTION 1

1.1 Aims and Objectives 3

CHAPTER TWO

2.0 LITERATURE REVIEW 4

2.1      Brief Overview of Antibiotic Isolation 8

2.2      Increasing Antibiotic Yield and Recovery 9

2.3      Natural Habitat of Antibiotic Producing Microorganisms 11

2.4      Factors Affecting Antibiotic Production and Activity 11

2.5      Isolation of Antibiotics from Microbial Biomass 12

CHAPTER THREE

MATERIALS AND METHODS

3.1      Collection of Soil Samples 14

3.2      Isolation and Enumeration of Microbes from Soil 14

3.3      Identification of Isolates 15

3.4      Gram Reaction 15

3.5      Biochemical Characterization 15

3.5.1 Starch Hydrolysis 15

3.5.2 Hydrogen Sulphide (H₂S) Production Test 16

3.5.3   Catalase Test 16

3.6      Test Microorganisms 16

3.7 Antibiotic Sensitivity Test 17

CHAPTER FOUR

4.1      RESULTS 18

CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion 23

5.2      Conclusion 25

5.3      Recommendations 25

REFERENCES

LIST OF TABLES

Table Title Page

1   Soil Samples with their total bacterial and fungal counts 19

2  Sample site and number of bacterial and fungal isolate  20

3 Colonial Morphology and biochemical test of isolate 21

4 Antibacterial Screening of isolates against test organisms with

their zone(s) of Inhibition in (mm) 22

CHAPTER ONE

1.0 INTRODUCTION

The world’s demand for antibiotics is steadily growing. Since their discovery in the 20th century, antibiotics have substantially reduced the threat of infectious diseases. The use of these miracle drugs, combined with improvements in sanitation, housing, food, and the advent of mass immunization programs, led to a dramatic drop in deaths from diseases that were once widespread and often fatal. Over the years, antibiotics have saved lives and eased the suffering of millions. By keeping many serious infectious diseases under control, these drugs also contributed to the increase in life expectancy during the latter part of the 20th century. There is a growing recognition of the pressing need for new antimicrobial agents for the treatment of infectious diseases (Dancer, 2004). As just one cogent example, new antibiotics are in high demand for the treatment of Staphylococcus aureus infections, particularly due to the emergence of methicillin-resistant S. aureus (MRSA) in communities and hospitals (Byarugaba, 2004). Further, providing effective and affordable antibiotics to people in the epidemic-prone Third World remains a major challenge. Historically, natural products have played a key role in the discovery and development of many antibiotics (Kavitha et al., 2010).

Due to the high resistance of microorganisms to the conventional antibiotics in the market, many antibiotics administered to people to combat bacterial and pathogenic disease have little or no effect to fulfilling their aim by eliminating the targeted pathogenic disease and this often result to dense prevalence of these ailments and in acute disease cases, death. This resistance however arises from factors such as abuse of the antibiotics and drug sensitivity problems. This either puts some antibiotics out of use due to intolerance or the microorganisms getting used to them.

Current drug development methods have been slow to produce effective new antibiotics as they have primarily focused on modifying existing classes of antibiotics or using genomics to identify new drug targets (Brooks et al., 2004). Unfortunately this approach is yielding results too slowly to keep up with the rapid pace of emergence of drug resistant bacteria. Hence, there is the need for new antibiotics from natural sources, and new classes of antibiotics. Exploring the possibility of discovering new antimicrobials from bacteria is reasonable because bacteria have been at war with each other for survival for many years. This would lead one to think that bacteria might be the best source for finding compounds to kill other bacteria.

There is a widespread acceptance that microorganisms are an unlimited source of new substances with many potential therapeutic applications. Microorganisms constitute an inexhaustible reservoir of compounds with pharmacological, physiological, medical or agricultural applications. The microbial products of secondary metabolism carry out an important role in human health, providing roadmaps for the biosynthesis of many synthetic and semi-synthetic drugs (Erving et al., 2009). In nature, microbial bioactive products are present as mycotoxins or bacteriocins derived from filamentous and non-filamentous bacteria and fungi (Abdulkadir and Waliyu, 2012). The soil-based actinomycetes have been the source of countless drugs, from streptomycin and actinomycin, to erythromycin and vancomycin. Natural soil harbors over 10⁹ microorganisms/gram and provides an ideal reservoir for bioactive microbiota, which springs virtually all clinical antibiotics used today. The nature of the active agents or the antibiotics produced by these organisms depends upon the species; frequently upon the strain; the composition of the medium in which it is grown, and the conditions of cultivation.

Today nearly 500 antibiotics are found each year and over 80% of antibiotics in clinical use are obtained from soil isolates (Dubey and Masheshwari, 2004). These bioactive microorganisms are most abundantly present at the top few inches of the soil, in soil containing straw and agricultural products (Usha et al., 2011). Studies have also suggested that soil from areas containing residential-derived materials, such as human fecal matter, contains 10-20 times more antibiotic resistant strains than recreational or industrial soils. Consequently and in addition to the elevated use of therapeutic drugs in urban or residential environments, residential soils contain exceptionally high bioactivity and are abundant reservoirs of antibiotic resistant microorganisms (Bush, 2004).

With the increasing number of drug-resistant pathogens, particularly the acquired multi-drug resistant strains, serious public health problems have arisen throughout the world. Therefore, the need for antimicrobial discovery and better treatments of these infections, particularly in hospitals where antibiotic resistance is immediately life threatening, is becoming a rapidly growing concern.

1.1 Aims and Objectives

This work is aimed at

1. Isolating microorganisms with antibiotic production potentials from the soil

2. Screening the isolated microorganism for antagonistic effect against known pathogens

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