ISOLATION OF ANTIBIOTIC PRODUCING MICROORGANISMS FROM SOIL SAMPLES FROM OHOKOBE NDUME

ABSTRACT

This study was aimed at investigating and exploring indigenous soil microflora for antimicrobial production. 12 soil samples were collected, serially diluted and spread plated on Tryptone soy agar and Sabouraud dextrose agar plates. 18 isolates were recovered from the soil samples. Agar disc diffusion method was used to check the antagonistic activity of the resulting isolates against four test bacteria (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 7080, and Salmonella typhi). Inhibition zones obtained from the isolates showed that Isolate A2 was active against Staphylococcus aureus (14mm) while another isolate C2 produced a zone of 10mm against Salmonella typhi. Among Gram negative bacteria E. coli was least susceptible as none of the isolates could inhibit its growth. The isolated organisms were in the genera Micrococcus and Bacillus. Development of new antibacterial agents with activity against multi-drug resistant bacteria is therefore perceived as a critical public health need.

TABLE OF CONTENTS

Title page                                                                                                                                 i

Certification                                                                                                                              ii

Dedication                                                                                                                                iii

Acknowledgements                                                                                                                   iv

Table of contents                                                                                                                       v

List of Tables                                                                                                                            vii

Abstract                                                                                                                                    viii

CHAPTER ONE

1.1       Introduction                                                                                                                1

1.2       Aims and Objectives                                                                                                  4

CHAPTER TWO

2.1       LITERATURE REVIEW                                                                                           5

2.1.1    Soil Bacteria                                                                                                               5

2.1.2    Soil Actinomycete                                                                                                      6

2.1.3    Soil Fungi                                                                                                                   8

2.1.4    Soil Antibiotic                                                                                                            9

2.1.5    Soil pH and Alkalinity                                                                                               10

CHAPTER THREE

Materials and Methods

3.1       Sample Collection                                                                                                      14

3.2       Isolation of Soil Microorganisms                                                                               14

3.3       Purification of isolates                                                                                                15

3.4       Preparation of inoculum of test organisms                                                                 15

3.5       Preparation of inoculum of each isolates                                                                   15

3.6       Characterization and Identification of isolates                                                           16

3.6.1    Gram staining                                                                                                             16

3.7       Biochemical tests                                                                                                        16

3.7.1    Catalase test

3.7.2    Indole test                                                                                                                   16

3.7.3    Citrate utilization test                                                                                                 17

3.7.4    Hydrogen sulphide (H₂S) Production test                                                                  17       

3.7.5    Starch Hydrolysis                                                                                                       17

CHPATER FOUR

4.0       RESULTS                                                                                                                   18

CHAPTER FIVE

5.1       Discussion                                                                                                                   22

5.2       Conclusion                                                                                                                  24

            References                                                                                                                  26

LIST OF TABLES

CHAPTER ONE

1.1       INTRODUCTION   

Antibiotics are the most important bioactive compounds for the treatment of infectious diseases. But now, because of the emergencies of multi-drug resistant pathogens, there are basic challenges for effective treatment of infectious diseases (Hopwood, 2007). Thus, due to the burden for high frequency of multi-drug resistant pathogens in the world, there has been increasing interest for searching effective antibiotics from soil micro-organism of diversified ecological niches. In the present study, the randomly selected soil samples were taken from Ohokobe Ndume farmland for isolation of microorganisms. The successful isolation of microorganism from environmental samples requires an understanding of the potential soil sample areas and environmental factors affecting their growth. Previous studies showed that selection of different potentials areas such as farm soil samples were an important activity for isolation of different types of potent antibiotics producing soil microorganism (Gurung et al., 2009).

The antibacterial effect of penicillin was discovered by Alexander Fleming in 1929. He noted that a fungi colony had grown as a contaminant on an agar plate streaked with the bacterium Staphylococcus aureus, and that the bacterial colonies around the fungus were transparent, because their cells were lysing (Demain et al., 2009). Fleming had devoted much his career to finding methods for treating wound infections and immediately recognized the importance of a fungal metabolite that might be used to control bacteria. The substance was named penicillin because the fungal contaminant was identified as Penicillum notanum (Tiwari, 2013). Fleming found out that it was effective against many Gran positive bacteria in laboratory conditions, and he even used locally applied crude preparations of this substance, from culture filtrates, to control eye infections. However, he could not purify this compound because of its instability and it was not until the period of the second world war (1939-1945) that two other British scientist, Florey and Chain, working in the USA, managed to produce the antibiotic on an industrial scale for wide spread use. All three scientists shared the Nobel Prize for this work, and rightly so penicillin rapidly became the “wonder drug” which saved literally millions of lives. It is still a “front line” antibiotic, in common use for bacterial infections although the development of penicillin-resistance in several pathogenic bacteria now limits its effectiveness.

The term antibiotics literally means against life. An antibiotic was originally defined as a substance, produced by the/one microorganism or of biological origin which at low concentrations can inhibit the growth of other microorganisms or infectious organisms (Baltz 2009).

Penicillin has an interesting mode of action: it prevents the cross-linking of small peptide chains in peptidoglycan, the main wall polymer of bacteria. Pre-existing cells are unaffected, but all newly produced grow abnormally, unable to maintain their wall rigidity and they are susceptible to osmotic lysis. This morphogenetic effect of penicillin can be demonstrated by growing either Gram-positive or Gram-negative bacteria in the presence of sub-lethal concentrations of penicillin. Ampicillin is the Gram-stained cells of Bacillus cereus that has been cultured in the absence of penicillin or in the presence of a low concentration of penicillin derivative termed. By affecting the cross-linking of the bacteria cell-wall (Brotze-Oesterhelt et al., 2008).

Penicillin has caused the bacterium to grow as larger cells with less frequent cell divisions. The two natural penicillins obtained from culture filtrates of Penicillium notatum or the closely related species Penicillium chrysogenum are penicillin G and the more acid resistant penicillin V. (Alekshun et al., 2007). They are active only against Gram-positive bacteria (which have a thick layer of peptidoglycan in the wall) and not against Gram-negative species, including many serious pathogens like Mycobacterium tuberculosis (the cause of tuberculosis).

Nevertheless, the natural penicillins were extremely valuable for treating wound pathogens such as staphylococcus in wartime Europe. An expanded role for the penicillins came from the discovery that natural penicillins can be modified chemically by removing the acyl group to leave 6 aminopenicillanic acid and then adding acyl group that confer new properties. These modern semi-synthetic penicillin such as Ampicillin, Carbenicillin and Oxacillin have various specific properties such as: resistance to stomach acids so that they can be taken orally, a degree of resistance to penicillinase (a penicillin-destroying enzyme produced by some bacteria) extended range of activity against some Gram-negative bacteria. Although the penicillin are still used clinically, their value has been diminished by the wide spread development of resistance against target microorganisms and also by some peoples allergic reaction to penicillin.

Microorganism are frequently present in soil, manure and decaying plant tissues which are able to degrade wastes that are correlated with the substrate organic matter(Allen et al., 2009). Agriculture soil is a dynamic medium in which a large number of pathogenic and non-pathogenic bacterial and fungal flora live in close association. Microbes in the soil are the key to carbon and nitrogen recycling. Microorganisms produce some useful com-pounds that are beneficial to soil health, plant growth and play an important role in nutritional chains that are important part of the biological balance in the life in our planet (Handelsman. 2010).farm lands are basically naturally ventilated climate controlled. Farmland cultivation has been evolved to create favorable micro-climates, which favors the crop production and could be possible all through the year or part of the year as required. Wherein, off season crops are also grown under a favorable controlled environment and other conditions viz. temperature, humidity, light intensity, ventilation, soil media, irrigation, and other agronomical practices throughout the season. The present study aimed to isolate effective microorganisms that are pre-sent in farm land soil and find out the optimize culture condition (Gillings et al., 2008).

1.2       AIMS AND OBJECTIVES

The objectives of this study include:

a.     To isolate and identify indigenous microorganisms present in the soil

b.     To screen the isolates for production of antimicrobials against selected test bacteria.

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