ISOLATION AND IDENTIFICATION OF ANTIBIOTIC PRODUCING BACTERIA FROM THE SOIL SAMPLES IN MICHAEL OKPARA UNIVERSITY OF AGRICULTURE, UMUDIKE, ABIA STATE.

ABSTRACT

Isolation and identification of antibiotic producing bacteria from soil were carried out. Ten (10) soil samples were collected from different sites around the institution and screened for potential antibiotic producing bacteria. The samples collected were analyzed for total heterotrophic bacteria and coliform count. The result showed that total heterotrophic count ranged from 2.0 × 104 cfu/g – 12 × 102 cfu/g and coliform count ranged from 1.4 × 102 cfu/g – 8.5 × 102 cfu/g. The bacteria isolated were Bacillus species, Micrococcus species, Pseudomonas species Staphylococcus species Streptococcus species Proteus species and Escherichia coli. Staphylococcus aureus, Bacillus species, Proteus species and Micrococcus species showed heavy growth as all the organisms grow in the media at different rates.  Bacillus species showed evidence of antibiotic production by inhibiting the growth of the test organisms such as Staphylococcus aureus (zone of inhibition 10mm), Escherichia coli (zone of inhibition 11mm), Pseudomonas species (zone of inhibition 3mm) and Streptococcus species (zone of inhibition 5mm). Moreover, the production of potential antibiotics as seen in this preliminary work was not recovered in large quantity around the university.

TABLE OF CONTENTS

Title Page  i

Certificate  ii

Dedication                                                                                                   iii

Acknowledge    iv

Table of Contents  v

List of Tables              viii

Abstract  ix CHAPTER ONE: INTRODUCTION 1      1.1      STATEMENT OF PROBLEM 3  1.2 JUSTIFICATION                                                3         1.3       AIMS AND OBJECTIVES 5  

CHAPTER TWO: LITERATURE REVIEW 6

2.1       MICROORGANISMS AS SOURCES OF NATURAL PRODUCTS 7

2.1.1 ENZYME PRODUCTION 7

2.1.2 ANTIBIOTIC PRODUCTION 8

2.2 SOURCES OF ANTIBIOTIC PRODUCING MICROORGANISMS 9

2.3 CLASSIFICATION AND NOMENCLATURE OF ANTIBIOTICS 9

2.4 ANTIBIOTIC DIVERSITY 15

2.5 CONCEPT OF INDUSTRIAL RESEARCH IN ANTIBIOTIC PRODUCTION 15

2.6 PROGRESS OF TRENDS IN ANTIBIOTIC RESEARCH 17

2.7 GOALS OF ANTIBIOTIC RESEARCH 17

2.8 FACTORS CONSIDERED IN SELECTION OF ANTIBIOTIC 18

2.9 FACTORS AFFECTING ANTIBIOTIC PRODUCTION 19

2.10 BIOCHEMISTRY OF ANTIBIOTIC PRODUCTION 20

2.11 MECHANISMS OF ANTIMICROBIAL ACTION 22

2.12 SPECTRA OF ANTIMICROBIAL ACTIVITY 26

2.13 SCREENING METHODS 26

2.14 PRIMARY TESTING OF ANTIBIOTIC PRODUCTION 28

2.15 MICROBIAL DIVERSITY IN SOIL 29

CHAPTER THREE: MATERIALS AND METHODS

3.1 COLLECTION OF SOIL SAMPLE 31

3.2 PREPARATION AND STERILIZATION 31

3.3 SAMPLE PREPARATION AND INOCULATION 32

3.4 COLONIAL MORPHOLOGY 32

3.4.1 SUBCULTURING 32

3.4.2 GRAM STAINING 32

3.5 BIOCHEMICAL TESTS 33

3.5.1 CATALASE TEST 33

3.5.2 COAGULASE TEST 33

3.5.3 CITRATE UTILIZATION TEST 34

3.5.4 INDOLE TEST 34

3.5.5 OXIDASE TEST 34

3.5.6 UREASE TEST 35

3.5.7 STARCH HYDROLYSIS 35

CHAPTER FOUR: RESULTS 36

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 DISCUSSION 40

5.2 CONCLUSION 41

             REFERENCES

LIST OF TABLES

Table         Title    Pages

1        The thirteen named groups of antibiotics based chemical structure        12

2        Microorganisms producing various antibiotics        13

3         Mean Colony Count (cfu/g)        37

4                Bacteria isolates and their percentage occurrence                   38

5                Zone of inhibition diameter (mm) of bacterial isolates against test organisms                  39

 CHAPTER ONE

1.0 INTRODUCTION

The term soil refers to the outer loose material of the earth crust. It may be regarded as a three phase system composed of solids, liquids and gases dispersed to form a heterogeneous matrix. The whole soil is composed of five major components, which include, mineral matter (contain less than 20% organic carbon), water, organic matter (contains humic substances, non-humic substances, humic acid, fulvic acid and humin), air and living organisms. The various components of the soil environment constantly changed and the quantity of these constituents are not the same in all soil but vary with locality. However, the living portion of the soil body includes small animals and microorganisms that play the most important role in the release of nutrient and carbon dioxide for plant growth (Bhagabati et al., 2004). That soil is rich in microorganisms capable of antibiotic synthesis is well accepted, but the frequency with which synthesis occurs at ecological significant levels in nature has been much less clear (Brun et al., 2000). Soil provides optimum conditions such as moisture, temperature, pH, and organic matter which are favorable for the growth of microorganisms. These microorganisms produce primary metabolites in the latter stages of their growth, which are needed for their defense and survival (Abdulkadir et al., 2012).

The bacteria are the most abundant group usually more numerous than the four combined. Predominantly, Bacillus and Actinomycetes are present (Abdulkadir et al., 2012). Soil bacteria can be rod (bacilli), cocci (spherical) and spirilla (spirals) (Bhagabati et al., 2004). The term antibiotic means against life. In our every day usage however, we use the word to describe a set of chemical that inhibit or kill bacteria (Nester et al., 2009).

Antibiotic can be defined technically as a chemical heterogeneous group of small organic molecules of microbial origin that at low concentrations, are deleterious to the growth or metabolic activities of other microorganisms (Brun et al., 2000). Antibiotics are one of the most important commercial exploited secondary metabolites produced by bacteria and employed in a wide range. Most of the antibiotic producers used today are the soil microbes (Arpigny et al., 1999). Over the past decades however, genetic and molecular techniques, have been applied to demonstrate conclusively that microorganisms synthesize a variety of antibiotics, even under field conditions, in Rhizosphere i.e. that portion of the soil enriched in carbon and energy resources released by the plant roots (Brun et al., 2000). There are numbers of bacteria having potential to produce antibiotic example of which is Actinomycetes which are best known for their ability to produce antibiotics and are Gram-positive bacteria which comprise a group of branching unicellular microorganisms. Among Actinomycetes, Streptomycetes are the dominant. Streptomyces species produce antibiotic like tetracycline, chloramphenicol, vancomycin, gentamycin, Bacillus species which produce antibiotic like bacitracin, pumulin and gramicidin which are active against Gram-positive bacteria such as Staphylococcus, Streptococcus, Corynebacter species, and Lactobacillus species (Lactobacillus lactis) which produces antibiotic nisin (Waites et al., 2008). They have been widely utilized over the decades to fight several bacterial infections.

The diversity of soil microorganisms has been exploited for many years based on the cultivation and isolation of microbial species (Rolf, 2004). Various unconventional culture media such as low nutrient media have been used in the recent past to isolate rarely isolated groups of bacteria in soil. Such media prevent growth of fast growing bacteria from low nutrients habitats (Postgate et al., 1964), a situation that is common with high nutrient standard cultivation media (Zengler et al., 2002). Janssen et al., 2002 cultivated novel bacteria using Dilute Nutrient Broth (DNB) plus agar or gellan media and extended cultivation of slow growing bacteria at an incubation temperature of 25⁰C. Temperature is an important factor in regulating microbial activity and shaping the soil microbial community (Pietikainen et al., 2005). However, little is known on how temperature affects microbes found in termite gut, mound and soil.

1.1 Statement of Problem

Bacteria are known producers of antimicrobials and enzymes used in pharmaceuticals and industries respectively. However, the emergence of multi-drug resistance pathogens has rekindled the need to discover new antimicrobials from the environment. Low discovery rate of these antimicrobial is attributed to standard cultivation methods that use nutrient rich media (Strohl, 2000). Low nutrient media has therefore been used in the recent past to cultivate uncultured group of bacteria from soil. However, little is known on how temperature affects bacteria cultured from soil while using low nutrient agar medium to isolate antibiotic producing bacteria from soil samples.

1.2 Justification

There still exist large discrepancy between the numbers of bacterial colonies that form on solid media and actual total number of bacterial cells present in that same soil (Rappe et al., 2003). The discrepancy has limited our understanding of species diversity of soil bacterial communities and it has been associated with the inadequacy of standard cultivation methods (Joseph et al., 2004). These unrecovered microorganisms represent an unexplored reservoir of novel strains that may produce novel natural products (Rolf, 2004).

The problem of resistance to drugs currently available in the market by pathogens coupled with toxicity of most of them has called for urgent development of newer and more effective substances that could improve the fight against infectious diseases (Keller et al., 2004).

Most pathogens have evolved mechanisms that always keep them one step ahead of us, and therefore there is need to try and also stay one or two steps ahead of them. This can only be achieved by discovering and developing new substances capable of interfering with certain key processes inherent in them (Leeb, 2004). Soil type dictates the soil texture hence influencing the general microorganism’s survival (England et al., 1993). For instance, soil texture determines the water content holding capacities. Consequently, soils that are poor at retaining water films, thus restricting the movements of grazing protozoa and improving the survival potential of prey cells (Heijnen et al., 1991). Soil texture through the amalgamation of soil aggregates and organic matter also acts as a provider of microhabitats that may affect the survival of microorganisms in the soil. Hence, the presence of clay enhances retention of microorganisms and increases the provision of protective niches (England et al., 1993). Soil type also dictates the soil structure and physical make up of the soil pore network. The habitable pore space that arises through the given soil structure means that organisms of different diameters may only inhabit pores to which they can gain physical access. Cells that inhabit smaller pores become less susceptible to predation by larger microorganisms which cannot access the narrow pore networks (Young et al., 2000).

1.3 Aims and Objectives

· To isolate, identify, and characterize antibiotic-producing bacteria from soil samples obtained from Michael Okpara University of Agriculture, Umudike, Abia State.

· To investigate the production of antibiotics from bacteria isolated from soil and evaluate its antimicrobial activities on other microorganisms.  

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