EVALUATION OF ANTIMICROBIAL ACTIVITY OF PRODIGIOSIN PRODUCED FROM SERRATIA MARCESCENS AGAINST SOME PATHOGENIC ORGANISMS

ABSTRACT

Serratia marcescens was isolated from soil samples using spread plate method on Mac.Conkey agar while Mac.Conkey broth was used for fermentation and production of prodigiosin. The production of secondary metabolite (prodigiosin) was authenticated by antimicrobial activity against Streptococcus pyogenes, Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa. Highest zone of inhibition was observed with Staphylococcus aureus with a mean inhibition diameter of 22.33mm. Next was Streptococcus pyogenes with a mean inhibition diameter of 20.67mm. This was closely followed by Klebsiella  pneumoniae, with a mean inhibition diameter of 16.67mm. Lastly was Pseudomonas aeruginosa with a mean inhibition diameter of 13.67mm. The Minimum inhibitory concentration of prodigiosin against Staphylococcus aureus was tested. The various dilution concentrations of the antibiotics were obtained by diluting the corresponding decimal percentage mil of the supernatant from the production broth into 10ml peptone water, already inoculated with the corresponding test organism. At concentration percentages of 70, 65, 50 and 45, prodigiosin was inhibitory to Staphylococcus aureus. At concentration percentages of 30, prodigiosin was not effective on Staphylococcus aureus. The Minimum inhibitory concentration of prodigiosin against Klebsiella pneumonia was tested. At concentration percentages of 70 and 65, prodigiosin was inhibitory to Klebsiella pneumonia. At concentration percentages of 50, 45 and 30, prodigiosin was not effective on Klebsiella pneumonia. The Minimum inhibitory concentration of prodigiosin against Streptococcus pyogenes was also acertained. At concentration percentages of 70, 65 and 50, prodigiosin was inhibitory to Streptococcus pyogenes. At concentration percentages of 45 and 30, prodigiosin was not effective on Streptococcus pyogenes. The Minimum inhibitory concentration of prodigiosin against Pseudomonas aeruginosa was also deciphered. At concentration percentages of 70 and 65, prodigiosin was inhibitory to Pseudomonas aeruginosa. At concentration percentages of 50, 45 and 30, prodigiosin was not effective on Pseudomonas aeruginosa. Finally, since prodigiosin produced marked inhibitory effects on these pathogenic organisms, that means prodigiosin can be used to treat diseases caused by this organisms.

TABLE OF CONTENT

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          viii

List of Figures                                                                                                                         ix

Abstract                                                                                                                                  x

CHAPTER ONE

1.0.INTRODUCTION……………................……………………………………….2

1.1. AIM…………………………………………………………….............…………2

1.2. OBJECTIVES…………………………………………………............…………2

CHAPTER TWO

LITERATURE REVIEW

2.1. PRODIGIOSIN………………………………………………..........…………….3

2.2.      Serratia marcescens…………………………………………........……………….4

2.3.      IDENTIFICATION OF Serratia marcescens………......……………………….7

2.4.      EPIDEMIOLOGY AND PATHOGENECITY……………………......……….7

2.5.      CLINICAL MANIFESTATION…………………………………….....……….8

2.6. PATHOGENESIS……………………………………………….........………..9

2.7.      USES AND APPLICATION OF PRODIGIOSIN PIGMENT….......……..10

2.8.      ANTIMICROBAL SCREENING METHODS ……………………......…..12

CHAPTER THREE

MATERIAL AND METHOD

3.1.      COLLECTION OF SAMPLES…........…………………………………….13

3.2.      COLLECTION OF TEST ORGANISMS........……………………………13

3.2.1    BIOCHEMICAL TESTS RUN ON THE TEST ORGANISMS TO       

           AUTHENTICATE THEIR IDENTITY……………......…………………..14

3.2.      PREPARATION OF MEDIA………………………………………........…15

3.3.      ISOLATION OF Serratia marcescens………………………………......….16

3.3.1.CONFIRMATORY TESTS RUN ON THE ISOLATES TO AUTHENTICATE THEIR IDENTITY AS SERRATIA MARCESCENS………………………...…..16

3.4.      PRESUMPTIVE TEST FOR THE PRODUCTION OF PRODIGIOSIN..18

3.5.      PRODUCTION OF PRODIGIOSIN……………………………………….18

3.6.      ANTIMICROBIAL ASSAY ON THE PRODUCTION OF PRODIGIOSIN

            (AGAR WELL DIFFUSION METHOD)………………………………..19

3.7 MINIMUM INHIBITORY CONCENTRATION DETERMINATION…….19

CHAPTER FOUR

4.0      RESULTS…………………………………………………………………..27

CHAPTER FIVE

DISCUSSION AND CONCLUSION……………………………………………..28

5.1. DISCUSSION………………………………………………………………..28

5.2. CONCLUSION………………………………………………………………29

REFERENCE……………………………………………………………………..32

LIST OF TABLES

TABLE                    TITLE                                       PAGE

Table 1: Morphological and biochemical test used to confirm the isolates as                  22

Serratia marcescens.

Table 2: The zone inhibition diameter produced on Staphylococcus aureus,

              Klebsiella pneumoniae, Streptococcus pyogenes and Pseudomonas

aeruginosa by prodigiosin.                                                                                    23

Table 3 depicts the Minimum inhibitory concentration of prodigiosin against                

Staphylococcus aureus.                                                                                             24

Table 4: Minimum inhibitory concentration of prodigiosin against                                 

Klebsiella pneumoniae.                                                                                         25

Table 5: The Minimum inhibitory concentration of prodigiosin against                         

Streptococcus pyogenes.                                                                                            26

Table 6: The Minimum inhibitory concentration of prodigiosin against                                

Klebsiella pneumoniae.                                                                                             27

                                                     LIST OF FIGURES

FIGURE               TITLE                                                                PAGE

Figure 1           Shows the structure of Prodigiosin highlighting the pyrrole rings.      3

CHAPTER ONE

1.0   INTRODUCTION

Microorganisms are found in every sphere and biome of life. If these organisms are left to keep proliferating without any control measures, they would one day outgrow the human population. Any compound that inhibits or kills microorganism, is said to have an antimicrobial activity. Antibiotics are produced by only a few genera of microorganisms. These antibiotics are an example of secondary metabolites and are produced at the stationary and death phases of growth.

Prodigiosin is a red pigment produced by many strains of the bacterium Serratia marcescens and other Gram negative gamma proteobacteria, collectively called prodiginines.

Serratiaspecies are opportunistic gram negative bacteria classified in the group Klebsielleae and the large family Enterobacteriaceae. Serratiaspecies are wide spread in the environment but are not a common component of the human faecal flora (Elsevier, 2010). Serratiamarcescens is the primary pathogenic specie of Serratia. Rare reports have described disease resulting from infection with Serratia plymuthica, Serratia oclorifera, Serratia liquefaciens and Serratia fonticola. Some strains of S. marcescens are capable of producing a pigment called prodigiosin, which ranges in colour from dark red to pale pink depending on the age of the colonies. The chemical structure of prodigiosin has been unicited. Serratiaare capable of surviving in diverse environment including water, soil and the digestive tract of various animals. S. marcescens has a predilection for growth on starchy foodstuff where the pigmented colonies are easily mistaken for drops of blood (Eleanor et al., 2014).

In 1819, Bartholomeo Bizio, a pharmacist from Italy, discovered and named an organism as S. marcescens which He discovered the organism to be the cause of a miraculous bloody discolouration in a corn meal mush called polenta. Bizio named the organism as Serratia, in honour of an Italian physicist called Serrati, who invented the steam boat and Bizio chose the name marcescens from the Italian word for decay, because of the bloody pigment which was observed to deteriorate quickly.

Since 1906, physicians have used S. marcescens as a biological marker for studying the transmission of microorganisms because until the1950’s, this bacterium was generally considered a harmless saprophyte. Only since the 1960s has S. marcescens been recognized as an opportunistic pathogen in humans.

Derivatives of prodigiosin have recently been found to have immunosuppressive properties and anti-tumor activity in vivo and are also currently being considered as candidate treatment for chagas disease (Genes, 2011). Optimum growth of all strains of S. marcescens have been observed at pH 9 and at temperature of 20-37^OC.

S. marcescens can be identified in the laboratory by the use of methyl red test, of which they are negative. Their negation is due to their ability to produce 2, 3-butanediol and ethane. S. marcescens is positive to Voges-Proskaeur which shows their ability to convert pyruvate to acetoin.

1.1.      AIM

·       To evaluate the antimicrobial activity of prodigiosin produced from S. marcescens against pathogenic microorganisms.

1.2.      OBJECTIVES

·       To isolate S. marcescens from environmental samples.

·       To produce prodigiosin from S. marcescens.

·       To test the efficacy of the produced prodigiosin on pathogenic organisms.

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