TABLE OF CONTENTS
Title page
Certification page
Acknowledgement
Dedication
Abstract
Table of Content
List of Tables
List of Figures
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction
1.1.1 Statement of problem
1.1.2 Aim
1.1.3 Objectives of the study
1.2 Literature Review
1.2.1 General characteristics of Lactobacillus
1.2.2 Importance of Lactobacillus
1.2.3 Lactobacillus as normal micro flora of the vagina
1.2.4 Defence mechanisms of Lactobacillus
1.2.4.1 Organic acid production
1.2.4.2 Bacterial interference
1.2.4.3 Hydrogen peroxide production
1.2.5 Bacteriocin
1.2.5.1 Mode of action of bacteriocin
1.2.5.2 Production of bacteriocin
1.2.5.3 Purification of bacteriocin
1.2.6 Disadvantages of antibiotics
1.2.7 Group B Streptococci (GBS)
1.2.7.1 Diseases and complications of GBS
1.2.7.2 Routes of infection
1.2.7.3 Pathogenesis of GBS
1.2.7.4 Stages of the disease
1.2.7.5 Risk factors for colonization by GBS
1.2.7.6 Prevalence of GBS
1.2.8 Other vagina microbiota
1.2.8.1 Candida
1.2.8.2 Escherichia coli
1.2.8.3 Staphylococcus aureus
1.2.8.4 Gardnerella vaginalis
1.2.9 Circumstances that allow opportunistic infections
1.2.10 Effects of Lactobacillus bacteriocins of microorganism
CHAPTER TWO: RESEARCH METHODOLOGY
2.1 Specimen collection
2.2 Isolation of Lactobacillus
2.3 Isolation and identification of Group B Streptococcus
2.4 Bacterial identification
2.4.1 Gram staining
2.4.2 Catalase reaction
2.4.3 Cultural characteristics
2.4.4 Sugar fermentation test for Lactobacillus
2.4.5 Hydrolysis of arginine for Lactobacillus identification
2.4.6 CAMP test for GBS
2.5 Production of crude bacteriocin-like inhibitory substance (BLIS)
2.6 Production of partially purified BLIS
2.6.1 Determination of percentage ammonium sulphate saturation
2.6.2 Protein determination
2.6.2.1 Procedure for protein determination
2.6.2.2 Preparation of the component reagents for protein determination
2.6.2.3 Preparation of 2mg/ml Bovine Serum Albumin (BSA) Standard Protein
2.6.3 Ammonium sulphate precipitation of bacteriocin
2.6.4 Dialysis
2.7 Preparation of the Test Organism
2.8 Determination of inhibitory spectrum of bacteriocin
2.9 Effect of hydrogen ion concentration on BLIS
2.10 Effect of temperature on BLIS
2.11 Combined effect of two BLISes
CHAPTER THREE: RESULTS
3.1 Isolation and Morphological identification of isolates
3.2 Cultural and Biochemical Identification of Isolates
3.3 The Inhibitory activities produced by each bacteriocin-like inhibitory substance (BLIS) of Lactobacillus species on GBS
3.4 The inhibitory activities produced by crude BLIS of Lactobacillus species on GBS at different temperatures
3.5 The inhibitory activities produced by crude BLIS of Lactobacillus species on GBS at different pH levels
3.6 The inhibitory activities produced by partially purified BLIS of Lactobacillus species on GBS at different pH levels
3.7 The combined effects of BLIS produced by Lactobacilli on Group B Streptococci (GBS)
CHAPTER FOUR: DISCUSSION AND CONCLUSION
4.1 Discussion
4.2 Conclusion
References
Appendix
ABSTRACT
The vaginal normal flora of a healthy woman is dominated by species of Lactobacillus. The balance between the vaginal flora is maintained by the protective antagonizing compounds produced by Lactobacillus. Group B Streptococci (GBS) is an opportunistic organism usually carried asymptomatically by pregnant women but cause severe neonatal infections. Antibiotics chemotherapy poses a lot of threat especially to pregnant women. Thus, this study was aimed at evaluating the antimicrobial activity of bacteriocin-like inhibitory substance produced by vaginal Lactobacillus of pregnant women on GBS. Identification of isolates was by microscopy, cultural characteristics and biochemical tests. Lactobacillus isolates were tested for their ability to inhibit GBS using agar diffusion method. Partial purification was done using ammonium sulphate precipitation and dialysis. The result revealed that of the 24 Lactobacilli isolates obtained, 7 strains were bacteriocin producers. Bacteriocin-like inhibitory substances (BLIS) from the seven strains inhibited the growth of GBS. Partially purified extracts of Bacteriocin-like substances showed more inhibitory activity than the crude extract. The mean diameter of crude BLIS (16.9 mm) produced by isolate R5 was the highest, while isolate L2 had the lowest activity at 12.1mm. The partially purified BLIS from isolate R5 had the highest activity at 18.5 mm while L2 had the lowest activity at 14.2 mm. BLIS showed inhibition of the test organism at pH 5.5, 6.0, 7.0, 7.5 and 8.0 and at temperatures of 30°C - 100°C for 10 mins except for BLIS from isolate L4 t hat lost its activity at 100°C. The zones of inhibition were strongest at 30°C for all the is olates. Generally, their activities decreased with an increase in temperature although there was no significant reduction. At 30°C, inhibitory activity of isolate L6 was 15.0 mm and 13.4 mm; R6 was 17.4 mm and 16.7 mm; R5 was 17.4 mm and 15.4 mm; L3 was 14.0 mm and 12.8 mm; Rl was 14.3 mm and 12.0 mm; L3 was 16.0 mm and 14.8 mm; L4 was 16.1 mm and 15.3 mm for the partially and crude BLIS respectively. Crude BLIS produced by isolate L6, R6, L2, L3 and L4 showed strongest activity at pH 5.5. Isolate R5 and Rl showed strongest activity at pH 6.0. At pH 5.5, the partially purified BLIS produced by isolate L6, R6, L2 and L3 showed the strongest inhibition; isolate Rl and L3 was strongest at pH 7.0. The combined effect of BLIS produced by isolate R5 and R6 was 19.06 mm and 21.07 mm for crude and partially purified BLIS respectively; were stronger than the individual effect. The combined effect was strongest at pH 6.0 with 21.20 mm. At temperature 30°C - 100°C; the inhibitory effects were relatively stable. Hence the study showed that BLIS produced from the isolates have potential of being used as probiotics and antibiotics for the prevention of Group B Streptococcicolonization in pregnant women and subsequent reduction of neonatal infections.
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction
Bacteriocins are defined as ribosomally synthesized antimicrobial peptides which are generally active against microorganisms closely related to the producer strain (Turovskiy et al., 2009). The term bacteriocin-like substance is applied to antagonistic substances that are not completely defined or do not fit the typical criteria of bacteriocins. They have been reported to inhibit a wide range of both Gram-positive and Gram-negative bacteria as well as fungi (Ocana et al., 1999). They function mainly by creating pores across bacterial cell membrane.
Lactobacillus species are non-pathogenic Gram positive rods that are predominantly isolated from the vagina of healthy premenopausal women (McClelland et al., 2009). In this environment, they exert a protective effect against pathogenic microorganisms by using different mechanisms such as production of antimicrobial agents, which include organic acids, hydrogen peroxide, and bacteriocins (Ocana et al., 1999).The protective role of Lactobacilli in the vagina becomes evident when their concentrations drop, which may be as a result of the use of antibiotics or in an immunocompromised host, in which case the hydrogen ion concentration (pH) of their environment tends to increase. This, in turn, favors colonization by intestinal bacteria and the overgrowth of microorganisms indigenous to the vagina but usually present in low numbers. These include the etiological agents of bacterial vaginosis, yeast vaginosis, trichomoniasis, and the potential pathogen Streptococcus agalactiae (Rebeca et al., 2008).
Streptococcus agalactiae also known as Group B Streptococci inhabiting bothurinogenital and pharynegeal sites in healthy individuals have been associated with acute
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