ISOLATION AND SCREENING OF SOIL MICROORGANISMS FROM CASSAVA FARMLAND IN UMUDIKE FOR ANTIBIOTIC PRODUCTION

ABSTRACT

The lack of new therapeutic options to replace many antimicrobial agents that are losing their efficacy against resistant bacteria is a growing concern. The search for new antimicrobials from rare and/or existing sources have thus become a worthwhile effort in recent years. Soil samples collected from Umudike were studied in an effort to screen for novel antibiotics produced by soil microbes. A total of 45 bacterial and 19 fungal isolates were successfully obtained from the soil samples. Antibiotic activity screening using the paper disc diffusion technique was used to evaluate the antagonistic potentials of the isolates against ATCC Strains of bacteria including Staphylococcus aureus (ATCC 25923), Salmonella typhi, Escherichia coli (ATCC 25922). Of the 64 isolates, 6.25% (4/64) were active against eight of the test organisms with diameter zones of clearing that ranged from 10.0mm to 17.0mm. Isolate VC7 exhibited the strongest antibacterial activity against S. typhi and E. coli with diameter zones of clearing of 17mm and 11.0mm respectively followed by VC29 which recorded a zone of inhibition of 14mm and 10mm against E. coli and S. aureus respectively. VC7 was tentatively identified as an Enterobacter species based on their colonial morphology and biochemical tests results. Among the isolates which demonstrated antagonism against the test bacteria, VC18, VC26, and VC7were identified as Bacillusspp., Penicillium spp and Enterobacter spp respectively.

TABLE OF CONTENTS

Title page                                                                                                                    i          

Certification                                                                                                               ii

Dedication                                                                                                                  iii        

Acknowledgements                                                                                                    iv        

Table of Contents                                                                                                       v         

Abstract                                                                                                                       vii     

CHAPTER ONE

1.0       Introduction                                                                                                    1

1.1       Aims and Objectives                                                                                      2         

CHAPTER TWO

2.0       Literature Review                                                                                           3

2.1       Isolation and Identification of Antibiotic Producing Microorganisms

            From Natural Habitats                                                                                                5

2.2       Screening for Antimicrobial Activities in Soil Microbes                               6

2.3       What Are Antibiotics                                                                                     8

2.5       Sources of Natural Occurring Antibiotics                                                      8

2.6       Antibiotic Producing Soil Microbes                                                               9

2.7       Biochemistry of Antibiotic Production                                                          10

2.8       Mechanism of Antimicrobial Action                                                              13

2.8.1    Inhibitors of Cell Wall Synthesis                                                                   13

2.8.2    Inhibitors of Cell Membrane Functions                                                         14

2.8.3    Inhibitors of Transcription and Replication of Genetic Material

            (Nucleic Acids)                                                                                               15

2.8.4    Inhibitors of Protein Synthesis                                                                       16

2.9       Review of Screening Methods                                                                       16

2.9.1    Primary Screening Methods                                                                           16

2.9.2    Primary Testing of Antibiotic Production                                                      17

2.10     Factors Affecting Antibiotic Production                                                        19

2.10.1  Carbon Source                                                                                                19

2.10.2  Nitrogen source                                                                                              19

2.10.3  Inorganic phosphate                                                                                       20

2.10.4  Trace metals                                                                                                   20

2.11     Biology and Pathogenicity of Some Test Organisms                                     20

2.11.1  Escherichia coli                                                                                              20

2.11.2  Salmonella typhi                                                                                             21

2.11.3  Staphylococcus aureus                                                                                   21

CHAPTER THREE

3.0       Materials and Methods                                                                                   22

3.1       Study Area                                                                                                      22

3.2       Collection of Samples                                                                                    22

3.3       Sterilization of Materials                                                                                22

3.4       Preparation of Culture Media                                                                         23

3.5       Inoculation of Soil Samples                                                                           23

3.6       Purification of Isolates                                                                                   23

3.7       Preparation of Inoculum of Test Organisms                                                  24

3.8       Gram Staining                                                                                                24

3.9       Morphological Identification of Bacteria Isolates                                          24

3.10     Biochemical Test                                                                                            25

3.10.1  Catalase Test                                                                                                   25

3.10.2  Indole Test                                                                                                      25

3.10.3  Citrate Utilization Test                                                                                   25

3.10.4  Hydrogen Sulphide (H₂S) Production Test                                                    25

3.10.5  Starch Hydrolysis                                                                                           26

3.11     Identificatin of Fungi Isolates                                                                        26

3.11.1  Microscopic Features                                                                                     26

3.12     Confirmation of Antibacterial Activity                                                          26

CHAPTER FOUR

4.0       Results                                                                                                            29

CHAPTER FIVE

5.0       Discussion and Conclusion                                                                            34

5.1       Discussion                                                                                                       34

5.2       Conclusion                                                                                                      37

LIST OF TABLES

CHAPTER ONE

1.0       INTRODUCTION

Antibiotics are substances used against infection caused by bacteria. Antibiotics act by inhibiting or destroying bacterial cells that cause certain disease. In nature, antibiotics are secondary metabolites produced by bacteria (Abdulkadir, and Waliyu, 2012) in order to maintain their niche and territory. Only limited groups of microorganisms are responsible for the sources of clinically useable antibiotics. According to Tortora et al. (2007), antibiotics can be found easily but only few are useful. Hence, concerted efforts have been carried out by many scientists in order to screen for novel antibiotic producing microbes. Through their efforts, many antibiotics have been successfully discovered in order to combat pathogenic bacteria that cause diseases. However, the emergence of new diseases and reemergence of multiple-antibiotic resistant pathogens have rendered the existence used antibiotics ineffectively. This problem has spurred the needs for the discovery of new antibiotics (Ahmed et al., 2013). Among the diverse sources of antibiotics, soil is the most important target for scientists in the discovery of novel antibiotics.

Antibiotics can be classified according to their mode of actions. Antibiotics are classified as broad-spectrum antibiotics when they have the ability to affect a wide range of Gram-positive and Gram-negative bacteria while antibiotics that are only effective towards certain group of bacteria are known as narrow-spectrum antibiotics. Several mechanisms of actions of antibiotics have been discovered by scientists. These actions include the inhibition of cell wall, protein and nucleic acids synthesis (Brooks et al., 2001; Tortora et al., 2007).

There are three important groups of microorganisms which are responsible for the production of antibiotics. These are the Gram-positive rod shape bacteria such as Bacillus, actinomycetes, and fungi such as Cephalosporium and Penicillium (Tortora et al., 2007).

Actinomycetes are the Gram-positive bacteria that contribute most of the clinically use antibiotics and as stated by Oskay et al., (2004), the discovery of new biological metabolites particularly useful antibiotics from actinomycetes need a vast amount of isolates. Majority of the antibiotics that have been identified and presently in use are isolated from the bacteria under the genus of Streptomycetes. Examples of these antibiotics are tetracycline and streptomycin.

As compared to antibacterial agents, the development of antifungal agents is not achieved at high advancement. This is because the lethal targets for infectious fungal species are hard to be identified due to the similarity of metabolic pathways possessed by them and their host, mammals since both are classified as eukaryotes. Griseofulvin and nystatin are examples of antifungal antibiotics that can be used to cure infections caused by Trichophyton and C. albicans, respectively.

1.1           AIMS AND OBJECTIVES

To isolate soil bacteria from MOUAU cassava farmland for potential production of antibiotics and their inhibitory activity against some pathogens.

The objectives are;

1.     To isolate and identify antibiotic producing bacteria from the cassava farmland of Michael Okpara University of Agriculture Umudike, Abia State.

2.     To determine the antimicrobial properties of soil microbes towards test microorganisms.

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