ISOLATION OF SERRATIA MARCESCENS FROM THE SOIL AND INVITRO PRODIGIOSIN PRODUCTION AS SOURCE OF ANTIBIOTIC, ACTIVE AGAINST OXACILLIN RESISTANT STAPHYLOCOCCUS AUREUS

 

ABSTRACT

This research work involve the isolation of two isolates titled RMN1 and RMN2, belonging to the Genus Serratia  from two soil samples collected from the following two strategic locations in university of Abuja, permanent site: Termite hill besides Girls hostel dump ground and from Faculty of Agricultural Science farm land. The isolates were morphologically distinct on the basis of spore colour, aerial and substrate mycelium formation and production of diffusible pigment. Isolates were observed under a microscope by using Gram staining technique, they were all Gram negative. Biochemical tests revealed that the two isolates were positive to catalase test and citrate test, and was negative to oxidase test. One of the two isolates was observed to have significant antibiotic producing potential, and the antibacterial activity of the produced antibiotics (red pigment extracted from cultural supernatants of the isolates grown on Peptone glycerol agar) was assessed using the well-in-agar diffusion method and streaking agar method. The results indicated that the extracted pigment had varying antibacterial activity at different concentration levels of 500 mg/ml, 250 mg/ml 125 mg/ml and 62.5 mg/ml against four strains of the test organism (Staphylococcus aureus) titled S1 to S4, the extracted pigment was more effective at concentration 500 mg/ml against the antibiotic resistant Staphylococcus aureus with the MIC at 125 mg/ml. The results indicated that the soil of this region could be a good source of prodigiocin having antibacterial activity and thus enable the use of micro-organisms as biological control agents.

 

 

 

 

 

 

TABLE OF CONTENT

TITTLE PAGE

DEDICATION..................................................................................................ii

DECLERATION................................................................................................iii

CERTIFICATION...............................................................................................iv

ACKNOWLEDGEMENT...................................................................................v

ABSTRACT........................................................................................................vi

TABLE OF CONTENT .....................................................................................vii

LIST OF TABLES ..............................................................................................xi

 

CHAPTER ONE

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

1.1 Justification…………..………………………………………………….…..2

1.2 Aim………………………………………………………………………….3

1.3 Objectives..,…………………………………………………………...….....3

 

CHAPTER TWO

2.0 Literature review……………………………………………………..……...4

2.1 Antibiotics…………………………………………………………………...4

2.1.1 Antibiotics Resistance …………………………………………………….5

2.1.2 Antibiotics resistances to staphylococcus aureus…………………………8

2.1.3 Antibiotics production from microorganism…………………………….10

2.1.4 Historical background……………………………………………………10

2.1.5 Why an effort for antibiotics search……………………………………..10

2.1.6 Concrete objectives of antibiotics research……………………………...11

2.1.7 The Mechanism of action of antibiotics…………………………………12

2.1.8 Functions of soil microorganisms………………………………………..12

2.1.9 Some antibiotic producing microorganisms……………………………..14

2.2.0 Serratia marcescens……………………………………………………..15

2.2.1 Prodigiosin……………………………………………………………….16

 

CHAPTER THREE

3.0 Materials and methods……………………………………………………..18

3.1 Material ……………………………………………………………………18

3.1.1 Soil sample collection …………………………………………………..18

3.1.2 Chemicals and re-agents ………………………………………………..18

3.1.3 Equipment and glass wares ……………………………………………..18

3.2 Methods …………………………………………………………………...19

3.2.1 Sample preparation ……………………………………………………..19

3.2.2 Sterilization of glss wares ……………………………………………….19

3.2.3 Antibiotic sensitivity assay ……………………………………………..19

3.2.4 Preparation of isolation media  …………………………………………20

3.2.5 Plating …………………………………………………………………...20

3.2.6 Identification of growth …………………………………………………20

3.2.6.1 Gram staining …………………………………………………………21

3.2.6.2 Motility test …………………………………………………………...21

3.2.7 Biochemical identification ………………………………………………22

3.2.7.1 Oxidase test  …………………………………………………………...22

3.2.7.2 Catalase test  …………………………………………………………..22

3.2.7.3 Citrate utilization test…………………………………………………..22

3.2.8  Invitro prodigiosin production ………………………………………….23

3.2.9 Anti-microbial activity of extracted pigment on clinical isolate ………..24

3.3  Determination of minimum inhibitory…………………………………….24

concentration (MIC) of extracted pigment

3.3.1 Statistical analysis ……………………………………………………….25

 

CHAPTER FOUR

4.0 Results ……………………………………………………………………..26

4.1  Staphylococcus aureus antibiotic resistance pattern ……………………...26

4.2 Physical characteristics of soil samples collected …………………………27

4.3 Morphological characteristics ……………………………………………..27

4.4 Biochemical characterisation of RM1 isolate ……………………………..28

4.5 Antimicrobial Assay ………………………………………………………32

4.6 Minimum inhibitory concentration of the red……………………………...32

      pigment produced by serratia marcescens

 

 

CHAPTER FIVE

5.0     Discussion and conclusion.......................................................................35

5.1     Discussion.................................................................................................35

5.2     Conclusion................................................................................................37

 

REFERENCE.....................................................................................................38

APPENDIX 1.....................................................................................................xii

APPENDIX 2.....................................................................................................xvi

APPENDIX 3..................................................................................................xviii

APPENDIX 4......................................................................................................xx

STATISTICAL ANALYSIS

Data Analysis......................................................................................................xii

Introduction .......................................................................................................xii

Analysis..............................................................................................................xii

Analysis of Variance (ANOVA)........................................................................xii

To test the effect of extract on S.aureus ............................................................xii

Descriptive Statistics.........................................................................................xiii

GRAPHICAL REPRESENTATION

Figure 1      Graphical Representation of Antimicrobial Activity ...................xvi

                   of oxacilin Antibiotic against Isolated S.aureus

 

Figure 2      Graphical Representation of Antimicrobial Activity...................xvii

                   of prodigiocin extract  against Isolated S.aureus

 

LIST OF TABLES

Table 1 Antibiotic sensitivity and resistance pattern…………………………27

          of four strains of S.aureus against Oxacilin

Table 2   Characterization of soil samples collect …………………………….29

Table 3  Cultural characteristics of soil isolates ………………………………30

Table 4  Biochemical characterization of RMN1……………………………...31

Table 5  Minimum inhibitory concentration (MIC)…………………..............33

          of Red pigment produced by S.marcescens  

Table 6 Antimicrobial activities of red pigment extracted ……………………34

Table 7 Analysis Of Variance (ANOVA)……………………………………..xii

Table 8 Descriptive  Statistics………………………………………………...xiv

Table 9 Summary  Statistics…………………………………………………...xv

 

PICTORIAL REPRESENTATION OF PLATES AND MICROSCOPY

Isolated sarretia marsceines on Pepton glyceron Agar.................................xviii

                   media plates

Microscopy view of stained isolate after Gram stain.....................................xix

 

PROPORTION OF DIFFERENT NUTRIENT SUPPLEMENTS

Proportion of Different Nutrient Supplements………………………………xx