ANTIBIOTIC PRODUCTIONS POTENTIAL OF SOIL BACTERIAL ISOLATES

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 Strains of bacteria including Staphylococcus aureus, Salmonella typhi, Escherichia coli. of the 64 isolates, 6.25% (4/64) were active against at least one of the test organisms with diameter zones of clearing that ranged from 10.0mm to 17.0mm. Isolate Enterobacter species exhibited the strongest antibacterial activity against S. typhi and E. coli with diameter zones of clearing of 17mm and 11.0mm respectively followed by bacillus species which recorded a zone of inhibition of 14mm and 10mm against E. coli and S. aureus respectively. Enterobacter species 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, bacillus species, Penicillium species, and Enterobacter species were identified as Bacillus spp., Penicillium spp and Enterobacter spp respectively

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          viii

Abstract                                                                                                                                  ix

CHAPTER ONE

INTRODUCTION

1.1       Background of the Study                                                                                            1

1.2       Purpose of Study                                                                                                         4

1.3       Scope of the Study                                                                                                      5

1.4       Justification of the Study                                                                                            5

CHAPTER TWO

LITERATURE REVIEW

2.1       Introduction                                                                                                                6

2.2       What are Antibiotics                                                                                                   8

2.3       Classification and Nomenclature of Antibiotics                                                        9

2.4       Antibiotics from Microorganisms                                                                              12

2.5       Biochemistry of Antibiotic Production                                                                      13

2.6       Mechanism of Antimicrobial Action                                                                          16

2.7       Spectra of antimicrobial activity                                                                                19

2.8       Screening methods                                                                                                     21

2.8.1    Primary Screening Methods                                                                                       21

2.8.2    Primary Testing of Antibiotic Production                                                                  22

2.8.3    Factors affecting antibiotic production                                                                      23

2.8.4    Extraction and purification methods                                                                          25

2.9       Some suggested newer methods of searching for antibiotic-producing microorganisms 25

2.9.1    The use of super sensitive mutants                                                                             26

2.9.2    The application of criteria other than death or inhibition                                          26

2.9.3    The use of chemical assays                                                                                         26

2.9.4    Search in novel environments                                                                                    27

2.9.5    Changed culture conditions                                                                                        27

2.9.6    Strategy based on multiple antibiotic resistance                                                        28

CHAPTER THREE

MATERIALS AND METHODS

3.1       Collection and Preparation of Soil Sample                                                                29

3.2       Isolation of microorganisms from samples                                                                29

3.3       Taxonomic characterization                                                                                       29

3.4.      Biochemical Analysis                                                                                                 29

3.4.1    Gram staining                                                                                                             29

3.4.2    Motility test                                                                                                                30

3.4.3    Indole test                                                                                                                   30

3.4.4    Catalase test                                                                                                                31

3.4.5    Coagulase test                                                                                                             31

3.4.6    Citrate test                                                                                                                   31

3.4.7    Voges –Proskauer                                                                                                       32

3.4.8    Oxidase test                                                                                                                32

3.4.9    Triple Sugar Iron Agar Test (TSI)                                                                              33

3.5       Challenge bacteria                                                                                                      33

3.6       Preliminary Screening for Antibiotic Production by the Bacterial Isolates                        33

3.7       Antibiotic Productivity Test by Agar Diffusion Method                                           34

3.7.1    Inoculation of Mueller hinton with test organism                                                      34

CHAPTER FOUR

4.0       Results                                                                                                                        36

CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1       Discussion                                                                                                                   41

5.2       Conclusion                                                                                                                  44

            References                                                                                                                  45

LIST OF TABLES

CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

The term ‘antibiotic’ literally means ‘against life’. An antibiotic was originally defined as a substance, produced by one microorganism, or of biological origin which at low concentrations can inhibit the growth of other microorganisms or infectious organisms. Antibiotics include a chemically heterogeneous group of small organic molecules of microbial origin that, at low concentrations, are deleterious to the growth or metabolic activities of other microorganisms. According to Gottlieb and Shaw (2007) antibiotics are substances produced by natural metabolic processes of some microorganisms that can inhibit or destroy other microorganism. Berdy (2010) defined antibiotics as the miracle drugs of modern times that act as magic bullets shooting down the infective organisms that have invaded the human body and caused infections.

 Originally, the term antibiotics referred only to organic compounds, produced by bacteria and fungi, which are toxic to other microorganisms. Antibiotics represent the single contribution of drug therapy for the health care of increasing population of the world, and provide effective control of many microbial pathogens that have been the cause of death of human and animals. The advent of synthetic methods has, however, resulted in a modification of this definition and an antibiotic now refers to a substance produced by a microorganism, or to a similar substance (produced wholly or partly by chemical synthesis), which in low concentrations inhibits the growth of other microorganisms

Antibiotic, generally, refers to antibacterial. However, because the term is loosely defined, it is preferable to specify compounds as being antibacterials, antifungals and antivirals.

It was not until 1940 with the discovery of penicillin, the first, best-known and most widely used antibiotic in 1928 by an English Bacteriologist, late Sir Alexander Fleming that the first clinical trials of penicillin were tried on humans. This antibiotic was obtained from a blue green mould of the soil called Penicillium notatum. Penicillin was discovered accidentally in 1928 by Fleming, who showed its efficacy in laboratory cultures against many disease producing bacteria. This discovery marked the beginning of the development of antibacterial compounds produced by living organisms.

 Another antibiotic, streptomycin was isolated in 1944 by Waksman, a Microbiologist, from a species of soil bacteria, called Streptomyces griseus, particularly tubercle bacilli, and has proved to be very valuable against tuberculosis. A vigorous search for more antibiotics was on at this time and in 1947, another antibiotic, chloromycetin was discovered by Burkholder. It was isolated from S. venezuelae. It has a powerful action on a wide range of infectious bacteria both Gram positive and Gram negative.

 The ability to produce antibiotics has been found mainly in fungi of the group Aspergillales, and in a few other bacteria. The streptomycetes are remarkable for the chemical diversity of antibiotics that they produce. Altogether about 2,000 antibiotics have been characterized so far; but only 50 are used therapeutically.

 As more antibiotics were discovered, designed and studied, scientists found that they had different properties. Some of these properties include their source, range of activity and their kinds. These were used to classify them.

 Perhaps one of the few most important discoveries regarding the beneficial use of fungi for humans was the identification in 1928 by Sir Alexander Fleming, that an isolate of Penicillium notatum produced a substance capable of killing Gram positive bacteria. This compound was subsequently identified as penicillin and was the first member of the β-lactam class of antibiotics to be discovered. These compounds function by inhibiting peptidoglycan synthesis in bacteria and their use has reduced the importance of the Gram positive as a cause of disease.

Subsequent to the identification of penicillin production by P. notatum, screening experiments revealed that P. chrysogenum was a superior producer of penicillin. A typical fermentation yields three types of Penicillin, namely, Penicillin F, Penicillin G and Penicillin V. 

 Antibiotics produced by fungi, are widely used in current chemotherapy especially the penicillin, cephalosporin and fusidic acid, which have antibacterial and antifungal activity. A number of antibiotic drugs have been discovered from soil-inhabiting microorganisms which include fungi (20% of isolated antibiotics), actinomycetes (70%) and eubacteria (10%). 

With advances in organic chemistry many antibiotics are now also obtained by chemical synthesis, such as the sulfa drugs. Drugs used in the chemotherapy of infectious diseases are classified into two groups. Drugs that have been synthesized by chemical procedures in the laboratory are called synthetic drugs while those produced by bacteria and fungi are called antibiotics. 

The antibiotics are widely distributed in the nature, where they play an important role in regulating the microbial population of soil, water, sewage, and compost. Of the several hundred naturally produced antibiotics that have been purified, only a few have been sufficiently non-toxic to be of use in medical practice. 

Those that are currently of greatest use have derived from a relatively small group of microorganisms belonging to the genera Penicillium, Streptomyces, Cephalosporium, Micomonospora and Bacillus.  

Treatment of infectious diseases caused by pathogenic bacterial and fungal strains was one of the most traditional problems in the clinical field. This necessity encouraged the investigators to synthesize novel and more potent inhibitory compounds (like azoles and quinolones derivatives) to fight them. However, the adverse effects and also appearance of bacterial or fungal resistances persuaded the investigators to study on natural products from microorganisms or herbal extracts to discover novel and safe lead compounds. 

Most of the peptide antibiotics produced by Bacillus are active against Gram positive bacteria. However, compounds such as polymyxin, colistin, and circulin exhibit activity almost exclusively upon Gram-negative forms, whereas bacillomycin, mycobacillin, and fungistatin are effective agents against molds and yeasts. As more antibiotics were discovered, designed and studied, scientists found that they had different properties. Some of these properties include their source, range of activity and their kinds. These were used to classify those.

Some antibiotics like penicillin, erythromycin, and methicillin which used to be one-time effective treatment against infectious diseases, are now less effective because bacteria have become more resistant to such antibiotics. Antibiotic resistant pathogens such as methicillin and vancomycin resistant strains of Staphylococcus aureus (S. aureus) and others cause an enormous threat to the treatment of serious infections. Moreover, some important drugs are expensive and/or have side effect to the host, some microbes have no successful antibiotics. These situation requires more attention to find solutions by searching and producing new and effective antibiotics from microbes. However, there is little scientific report on antibiotic producing microbes from soil samples collected in MOUAU.

1.2 Purpose of Study

The main purpose of the present study is to isolate and determine antibiotic production potential of soil samples collected around MOUAU.

Specific objectives include;

      i.         Isolation of bacteria and fungi from soil samples collected from MOUAU

     ii.         Determination of the antibiotic production potentials of the isolated microbes against Staphylococcus aureus, Pseudomonas aeruginosa, Micrococus sp, Candida utilis and Escherichia coli

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