ISOLATION OF BACILLUS SPECIE AND PRODUCTION OF BACITRACIN

ABSTRACT

Bacillus species was isolated from soil samples using spread plate method on nutrient agar while peptone water was used as the production medium. The production of bacitracin was authenticated by antimicrobial activity against Klebsiella pneumoniae, Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa. This was done using agar well diffusion method. After the production of bacitracin by Bacillus species, the minimum bactericidal concentration of the produced bacitracin was evaluated, using 70%, 60%, 50%, 30% and 20% each of the diluted bacitracin aliquot on Klebsiella pneumoniae, Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus and Pseudomonas aeruginosa. The zone diameter against the respective was calculated in duplicates and the mean diameter was obtained. Highest zone inhibition diameter was observed against Streptococcus pyogenes with a mean inhibition diameter of 10mm. Next in line was Staphylococcus aureus with a mean inhibition diameter of 9mm. After this came Escherichia coli with a mean inhibition diameter of 8mm, Klebsiella pneumoniae with a mean inhibition diameter of 7mm and lastly, Pseudomonas aeruginosa with a mean inhibition diameter of 6mm.  For Escherichia coli, the MIC was at 50% and minimum bactericidal concentration was at 50%. For Pseudomonas aeruginosa MIC was at 60% and minimum bactericidal concentration percentage was at 60%. For Staphylococcus aureus MIC was at 30% and MBC was at 30%. For Klebsiella pneumoniae, MIC was at 60% while MBC was at 60%. For Streptococcus pyogenes, MIC was at 30% while MBC was at 30%. Bacitracin is very effective against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes and Escherichia coli and as such can be used effectively in the treatment of diseases caused by these organisms.

TABLE OF CONTENTS

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          viii

Abstract                                                                                                                                 ix                                                                                                                                

Chapter One

1.0         Introduction…………………………………………………………………..1

1.1       Aim …………………………………………………………………………..2

1.2 Objectives………………………………………………………………….2

Chapter Two

2.0         Literature review……………………………………………………………3

2.1 Bacitracin…………………………………………………………………….3

2.1.1    History                                                                                                                        3

2.1.2    Synthesis                                                                                                                     3

2.1.3    Composition                                                                                                               4

2.1.4    Spectrum of activity and susceptibility data                                                              4

2.1.5    Mechanism of action                                                                                                  4

2.1.6    Clinical use                                                                                                                 5

2.2       Bacillus subtilis                                                                                                          9

2.2.1    Habitat                                                                                                                        9

2.2.2    Reproduction                                                                                                              10

2.2.3    Chromosomal replication                                                                                           10

2.2.4    Genome                                                                                                                      11

2.2.5    Transformation                                                                                                           12

2.2.6    Uses                                                                                                                            14

2.2.7    Safety                                                                                                                          15

2.2.7.1 In humans                                                                                                                   16

2.2.7.2 In animals                                                                                                                   17

Chapter Three

3.0       Materials and methods                                                                                                17

3.1       Source of material and collection                                                                               17

3.2       Preparation of culture media                                                                                      17

3.3       Sterilization of materials                                                                                            17

3.4.      Isolation of bacillus specie                                                                                         18

3.5.      Identification of the isolate                                                                                         18

3.5.1    Morphological appearance                                                                                         18

3.5.2    Gram reaction                                                                                                             18

3.5.3    Endospore stain                                                                                                          19

3.5.4    Biochemical characteristics                                                                                       20

3.5.5    Growth at 55⁰C                                                                                                           20

3.6.      Screening for crude bacitracin production by Bacillus specie                                    20

3.6.1.   Antimicrobial testing of bacitracin produced by Bacillus specie                                    20

3.7.      Minimum bactericidal concentration                                                                         21

Chapter Four

4.0.      Results                                                                                                                        30

Chapter Five

5.1.      Discussion                                                                                                                   31

5.2.      Conclusion                                                                                                                  32

References                                                                                                                              34

Appendix

LIST OF FIGURES

TABLE                         TITLE                                              PAGE

TABLE 1     Shows the Morphological And Biochemical Tests Carried Out On The Environmental Isolates To Authenticate Their Identity As Being Of Bacillus Species.

TABLE 2    Displays The Zone Inhibition Produced Against The Test Organisms.         27

TABLE 3     Depicts the Minimum Bactericidal Concentration Of The Bacitracin As Tested On Escherichia coli                                        28

TABLE 4     Shows The Minimum Bactericidal Concentration Of The Bacitracin As Tested On Pseudomonas aeruginosa.                                                      29

TABLE 5   Depicts The Minimum Bactericidal Concentration Of The Bacitracin As Tested On Staphylococcus aureus.                                    30

TABLE 6    Shows The Minimum Bactericidal Concentration Of The Bacitracin As Tested On Klebsiella pneumoniae.                                          31

TABLE 7   Depicts The Minimum Bactericidal Concentration Of The Bacitracin As Tested On Streptococcus pyogenes.                                         32

                                              CHAPTER ONE

 1.0     INTRODUCTION

The word antibiotic ‘is derived from Greek term antibiosis, which literally means ‘Against life’. It can be purified from microbial fermentation and modified chemically or enzymatically for basic research (Satpal, 2011). The discovery and use of antibiotics, which has been produced by several microorganisms through secondary metabolic pathways has been one of the major scientific achievements in the earliest of 20th century and these compounds can fight against various diseases. Generally antibiotic is a chemical substance, possessing a molecular weight lesser than 2 kilo Dalton and used to kill or prevent growth of any other type of microorganisms at a lower Dosage (Satpal, 2011). Antibiotics are molecules that kill, or stop the growth of, microorganisms, including both bacteria and fungi. Antibiotics have many classes which include;

1.         β-Lactam antibiotics examples: penicillins (e.g. amoxicillin), cephalosporins,  carbapenems.

2.         Tetracyclines example: tetracycline                                                 

3.         Macrolide antibiotics example: erythromycin

4.         Aminoglycosides examples: Gentamicin, Tobramycin, Amikacin

5.         Quinolones example: Ciprofloxacin (a fluoroquinolone)

6.         Cyclic peptides examples: Vancomycin, Streptogramins, Polymyxins

7.         Lincosamides example: clindamycin

8.         Oxazolidinoes example: Linezolid (Zyvox)

9.         Sulfa antibiotics example: sulfisoxazole

Most of the peptide antibiotics are produced by bacilli that are active against gram-positive bacteria; however, compounds such as polymyxin, colistin and circulin exhibit activity almost exclusively upon gram-negative bacteria, whereas bacillomycin, mycobacillin and fungistatin are effective against molds and yeasts.

Bacillus species are the predominant soil bacteria, because of their resistant endo-spore formation and production of essential antibiotics such as bacitracin. This bacillus species are gram-positive, endospore-forming chemoheterotrophic rod-shape bacteria which are usually motile with peritrichous flagella; they are aerobic or facultative anaerobic and catalase positive (Waites, 2008). Members of the bacillus genus are generally found in the soil. Many bacillus species are of remarkable importance because they construct antibiotics, such as bacitracin, polymyxin, gramicidin, tyrocidine, subtilin, bacilysin etc (Trookman, 2011).    

1.1       AIM

The aim of the study was: Isolation of bacillus specie and production of an antibiotic bacitracin.

1.2       OBJECTIVES

The objectives of the study were:

·       To isolate and characterize Bacillus specie

·       To  produce bacitracin

·   To determine the susceptibility of the test organisms to the produced bacitracin.

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