ANTIBACTERIAL ACTIVITY OF BILE ON BACTERIA ISOLATED FROM FAECES, URINE AND BLOOD.

ABSTRACT

The antibacterial activity of  bile on bacteria isolated from  faeces, urine and blood  was determined in this study precisely Escherichia coli and Salmonella spp   . Bile from three animal origin(cow ,goat and chicken) was extracted using alcohol and water and their antibacterial activity tested using the agar well diffusion method  at different concentrations . The crude extracts exerted the highest inhibition zone diameter within the range of 20mm-49mm and 38mm-48mm against Salmonella spp and Escherichia coli respectively, followed by the alcoholic extract with inhibition zone diameter in the range of  6mm-35mm and 7mm-33mm against Salmonella spp and Escherichia coli respectively. While the aqueous   extract exhibited the least activity   in the range of 5mm-25mm and 7mm- 25mm against Salmonella spp and  Escherichia coli respectively.   The MIC of the bile extracts which was determined using the tube macrodilution method and the MBC showed  that  bile have  both bactericidal and bacteriostatic activity on the test organisms. The findings of this work has shown that bile contains some substances that can exert inhibitory effect on bacteria hence can be employed  in the treatment of diseases caused  by these groups of bacteria.

TABLE OF CONTENT

Title page i

Declaration ii

Certification iii

Dedication iv

Acknowledgment v

Table of content vi

List of tables vii

Abstract viii

CHAPTER ONE

 INTRODUCTION 1

1.1       Background of the Study               1

1.2       Aim of the Study 4

1.3       Specific Objectives 4

1.4       Significance 4

CHAPTER TWO

LITERATURE REVIEW 5

2.1      Cholanology 5

2.2      Chemical Structure of Bile And Evolution 5

2.3      Bile and microorganisms: Previous Studies 6

2.4      Overview of Enterobacteriaceae Cell Envelope 8

2.5      Mechanism of Action of Bile on Cells 10

CHAPTER THREE

MATERIALS AND METHODS 14

3.1        Materials 14

3.1.1     Equipments 14

3.1.2     Media, Reagents and Solvents Used 15

3.2        Methods 16

3.2.1     Bile Sample Collection 16

3.2.2     Bile Extraction 16

3.2.3     Preparation of Extract Concentration 16

3.2.3.1  Preparation of Aqueous Bile Solution 17

3.2.3.2  Preparation Alcoholic Bile Solution 17

3.2.3.3  Preparattion of the Crude Extract 17

3.2.4     Media Preparation 18

3.2.5     Isolation of the Test Organisms 18

3.2.6     Identification 18

3.2.6.1  Grams Reaction 18

3.2.6.2  Citrate Utilization Test 19

3.2.6.3  Indole Test 19

3.2.6.4  Oxidase Test 20

3.2.6.5  Methyl Red-Voges Proskauer Test. 20

3.3   Bile Activity Assay 21

3.3.1      Preparation of Standard Innoculums of Test Organisms 21

3.3.2      Antibacterial Susceptibilty Testing 21

3.3.3      Well Diffusion Method Assay 21

3.3.4      Determination Of Minimum Inhibitory Concentration (MIC) Using the         

             Macrodilution Method 22

3.3.5      Determination of Minimum Bactericidal Concentration (MBC) 22

CHAPTER FOUR

RESULTS 23

4.1       Morphological And Biochemical Properties of the Isolates 23

4.2       Antibacterial Activity of Bile on the Test Organisms Using

 Well Diffusion Method Assay 25

4.3       Antibacterial Activity of Bile on the Test Organism Using

 Tube Macrodilution Assay 29

CHAPTER FIVE

DISCUSSION 37

5.1      Conclusion 40

5.2      Recommendations 40

          REFERENCE        

          APPENDIX

                                        LIST OF TABLES

 1:     Morphological and biochemical properties of the 25

         isolates    

 2:     Antibacterial activity of aqueous bile solution

         from cow, goat and chicken against the isolates 27

3:      Antibacterial activity of alcoholic exracts of bile

         from cow, goat and chicken against the isolates 28

 4:    Antibacterial activity of crude extracts of bile

        from cow,goat and chicken against the isolates 29

 5a :   Mnimum inhibitory concentration (MIC) and minimum   

         bactericidal concentration (MBC) of the cow

         bile against salmonella spp   32

 5b:      Minimum inhibitory concentration (MIC) and minimum

            bactericidal concentration (MBC) of the goat

            bile against salmonella spp 33

 5c :     Minimum inhibitory concentration (MIC)  and minimum

            bactericidal concentration (MBC) of the chicken

            bile against salmonella spp 34

 6a:      Minimum inhibitory concentration (MIC) and minimum

            bactericidal concentration (MBC) of the cow

            bile extracts against escherichia coli 35

 6b:      Minimum inhibitory concentration (MIC) and minimum

            bactericidal concentration (MBC) of the goat

            bile against escherichia coli 36

 6c:      Minimum inhibitory concentration (MIC) and minimum

            bactericidal concentration (MBC) of the chicken

            bile against  escherichia coli 37

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

The digestive system typically combats potentially pathogenic microbe through the production of several bactericidal agents along the tract. Some of these bactericidal agents are gastric secretions, hydrochloric acid and bile (Megan et al .,2009). In vitro studies have demonstrated that bile salts have cytotoxic and bacteriostatic properties (Begley et al.,2005a, Sung et al.,1993). Although the antibacerial mode of action of bile acids has not been fully elucidated, it is clear that bile does play an important role in limiting the number of microorganisms in the intestine. a significant increase in the numbers of microorganisms in the small intestine is found in individuals that have a reduced capacity to secrete bile into the duodenum (Hofman et al.,2006).  The detergent action of conjugated bile acids; the principal active ingredients in bile, on microbial cell membrane which is the basis for bile solubility test is thought to be the basis for the cytotoxic and bacteriostatic action of bile (Begley et al.,2005). The ability of bile to solubilize polar lipids has generally been thought to perturb the cell envelop surrounding bacterial cells. Indeed, in vitro bile acids are known to have a negative impact on microbial growth and survival. However, in vitro studies show that unconjugated bile acids have a more potent inhibitory effect than conjugated bile acids (Sung et al 1993, Begley et al.,2005), a finding that suggests that conjugated bile salts may not be the most antimicrobial component of  bile in vivo, however, conjugated bile acids might also influence the gut microbiota in an indirect way. The farnesoid X receptor (FXR), a nuclear receptor is activated by conjugated bile acids. This activation triggers the production of antimicrobial factors including nitric oxide and interleukin 18(Inagaki et al.,2006). A model emerges suggesting that high concentration of conjugated bile acids helps to limit the microbiota cell numbers in the duodenum and proximal ileum while in the distal ileum, an FXR-mediated response to conjugated bile acids help to  restrict the population size. (Hofman et al.,2006).

 Apparent proof for this model/hypothesis came when it was shown that feeding of bile or conjugated bile acids in conditions of bile acid deficiency in the intestine abolished bacterial overgrowth and reduced bacterial translocation to intestinal lymph nodes ( Lorenzo-Zuniga et al.,2003). The antimicrobial effect of administered conjugated bile acids may be co-mediated by fatty acids partly present as soaps that are associated with the conjugated bile acids in mixed micelles in the proximal small intestine. Long chain fatty acids have been known to have potent antimicrobial effects (Zheng et al.,2005)

 Bile, a yellow/green aqueous solution of organic and inorganic compounds whose major constituent include bile acids, bile alcohols, phosphatidylcholine and a pigment bilverdin is an end product of cholesterol metabolism in vertebrates (Hofmn et al.,2008). The synthesis occurs in liver cells which synthesize primary bile acids(cholic acid and chenodeoxycholic acids) via cytochrome P450 mediated oxidation of cholesterol in a multistep process. The secondary bile acids are synthesized as a result of partial dehydroxylation of the primary ones. The end-products are:  the amphipathic multifunctional bile alcohols and bile acids. After their synthesis in the hepatocyte, they are converted to strong acids by conjugation at the terminal carbon of the side chain. Bile alcohols are conjugated by esterification with sulfates whereas bile acids are conjugated by N-acylamidation  with taurine or a taurine derivative or less commonly with glycine. Such conjugation results in the formation of molecules that are impermeant to the epithelium of the biliary tract and small intestine content. Bile alcohol sulfates and conjugated bile acids are collectively termed bile salts. Bile salts are reabsorbed from the distal intestine after secretion into the proximal intestine and returned to the liver which removes them and resecretes  them into bile. The end result is the accumulation of  a bile salts that circulates between the intestine and the liver, this molecular influx is termed enterohepatic circulation. Other substances such as immunoglobulin A, endogenous  substances such as lipovitamins, water- soluble vitamins, steroids, essential trace metals as well as exogenous substances undergoes this enterohepatic cycling. (Hofman et al., 2008).

Bile salt biosynthesis results from at least two complex biochemical pathways. The  synthesis is under negative feedback control modulated at least in part by the nuclear hormone receptor farnesoid X receptor (FXR) and the peptide fibroblast growth factor 18(Inagaki et al .,2005). The major  function of bile in vivo is to act as a biological detergent which emulsifies and solubilizes fats this also confers potent antimicrobial properties on bile and gives it an important role in the body’s physiochemical defense system. Bile is also excretory in function eliminating substances that cannot be efficiently excreted in urine because they are insoluble or protein bound for example, cholesterol which is derived from excess synthesis or the pigment bilirubin the end- product of heme metabolism which is carried in blood attached to albumin proteins (Hofmann,1999,Ridlon et al.,2006)

Nonetheless, irrespective of the mechanisms, these experiments provided strong evidence for a second physiological function of conjugated bile acids in the proximal small intestine to prevent bacterial overgrowth by their antimicrobial activity (Hofmann and Eckmann,2006)

1.2 AIM OF THE  STUDY 

To ascertain the antibacterial effect of bile extract against two isolates of Enterobacteriaceae

1.3 SPECIFIC OBJECTIVES

 The following specific objectives were achieved:

i. To isolate the Enterobacteriaceae group of organisms from urine,faeces and blood samples.

ii. To determine the antimicrobial potency of crude bile on the selected microbial group

iii. To determine the antimicrobial potency of alcoholic and aqueous bile extracts on the selected group of microbes.

iv. To determine the minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the crude bile extract as well as the aqueous and alcoholic extracts.

1.4    SIGNIFICANCE 

To the knowledge of the researcher, this study will widen the study of cholanology in combating infections caused by pathogenic microbes. It might be the first to deal with bile in combination with organic and inorganic solvents against the Enterobacteriaceae in my institution.

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