ANTIBACTERIAL ACTIVITY OF HONEY ON STAPHYLOCOCCUS AUREUS, ESCHERICHIA COLI AND STREPTOCOCCUS PYOGENES ISOLATED FROM WOUNDS

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TABLE OF CONTENT
Title Page i
Certification ii
Declaration iii
Acknowledgements iv
Table of Content v
List of Tables ix
Abstract x
CHAPTER ONE 1
INTRODUCTION 1
1.1 Background to the Study 1
1.2 Statement of Problem 4
1.3 Justification of Study 4
1.4 Objectives of the study 6
CHAPTER TWO 7
LITERATURE REVIEW 7
2.1 Overview of Honey 7
2.2 Historical Review of Honey 8
2.3 The Contents of Honey 9
2.4 Therapeutic Uses of Honey (Mangala et al., 1994). 10
2.5 How Does Honey Work To Treat Infection (Bergman et al., 2001). 12
2.6 Precautions to Be Taken Before Using Honey 13
2.7 Wound 13
2.7.1 Skin: Overview 13
2.7.2Skin Related Infection 16
2.7.3Wound Infection and Inflammation 17
2.8Staphylococcus aureus 19
2.9Escherichia coli 22
2.10Streptococcus pyogenes 23
CHAPTER THREE 25
MATERIALS AND METHODS 25
3.1 Samples 25
3.2 Materials used 25
3.3 Collection of Samples 25
3.4 Source of Honey 26
3.5 Sample Processing 26
3.6 Identification of the Pathogen 26
3.6.1 Gram’s Staining 26
3.6.2 Biochemical Test 27
3.6.2.1 Indole Production Test 27
3.6.2.2 Methyl Red Test 28
3.6.2.3 Voges Proskauer Test 29
3.6.2.4 Citrate Utilization Test 29
3.6.2.5 Catalase Test 29
3.6.2.6 Oxidase Test 30
3.7 Antibacterial Sensitivity Testing 30
3.8 Antibacterial Activity of Honey 31
3.7.2 Agar Well Diffusion Method 31
3.8Preparation of serial dilution of honey 32
3.9Sterilization of honey 32
3.10Preparation of serial dilutions of reference antibiotics: 33
3.10.1Azithromycin: 33
3.10.2Ciprofloxacin 33
3.11Preparation of Standard Bacterial Suspension 33
3.12Modified Diffusion Technique 34
CHAPTER FOUR 35
RESULTS AND DISCUSSION 35
4.1Results 35
4.2Discussion 42
CHAPTER FIVE 44
SUMMARY, CONCLUSION AND RECOMMENDATION 44
5.1Summary 44
5.2Conclusion 44
5.2Recommendations 45


LIST OF TABLES
Table 4.1: Morphological and Biochemical identification of the isolates from wounds 36
Table 4.2: the inhibition zone diameter in (mm) and the minimum bactericidal concentration of Azthromycin (reference antibiotic) against standard bacteria tested 38
Table 4.3: the inhibition zone diameter in (mm) and the Minimum Bactericidal Concentration (MBC) of Ciprofloxacin (reference antibiotic) against Standard Bacteria tested 39
Table 4.4: the inhibition zone diameter in (mm) and the minimum bactericidal concentration (MBC) of honey against standard bacteria tested: 40
Table 4.5: the mean inhibition zone diameter in (mm) and the minimum bactericidal concentration (MBC) of honey against clinical isolates tested: 41

ABSTRACT
Twenty six (26) wound swab samples were collected from Uyo teaching hospital to determined antimicrobial activity of honey on three (3) selected pathogenic bacteria which were Staphylococcus aureus, Escherichia coli and Streptococcus pyogenes and it was brought to the laboratory for microbial analysis. The antimicrobial sensitivity test was carried out using Agar well diffusion method. The inhibitory efficiency of the honey sample on the growth of the tested organisms increase with increase in concentration from 20 – 100%. Staphylococcus aureus had 3.38 to 22.1mm diameter of inhibition and was sensitive to honey sample while Streptococcus pyogenes (1.26 – 13.6mm) was the least sensitive. The Zone diameter of inhibition of the organism at different concentrations of honey were found to be statistically significant. The inhibition efficiency of the honey samples on the growth of the tested organisms was found to be dependent on the concentration and the type of honey used as well as the nature of the tested organisms. The result also showed that most of the honey sample at 20 – 30% (w/v) and completely inhibited the growth of all of the tested bacteria. It was clear that the different types of honey were less effective against Escherichia coli than the other bacteria.

CHAPTER ONE

INTRODUCTION

1.1 Background to the Study

Honey is a sweet food. Honey bees are prepared by using nectar from flowers of different plants. Honey bees belong to genus Apis. Different varieties of honey are produced by different species of honey bees and these are collected by bee keepers. It is consumed by humans. Some people prefer honey over sugar and other sweeteners (Tonelli et al., 1990). Honey is sugar rich compound having the following constituents as glucose 38.19%, fructose 21.28%, maltose and other sugars 8.81%, enzymes and pigments 2.21%, ash 1.0% and water 17.20% (Finlay et al. 2003). I-loney has low water content due to which most of the microorganisms do not grow in honey. In 1892, Van Ketel first recognized the antibacterial property of honey (Dustmann, 1979). The antibacterial activity is only due to osmotic effect of its high sugar content but when it is used to cure wound or to control infection, it causes reduction in osmolarity. A well-known antimicrobial agent hydrogen peroxide is present, in small concentration, in honey (Puritt and Reiter, 1985). Antibacterial activity of honey is dependent on amount of hydrogen peroxide (Molan and Allen, 1996). Honey is acidic and its pH lies between 3.2 and 4.5 which inhibit the growth of a number of pathogens but some wound infecting species are able to grow at this pH range, such as Escherichia coli, Salmonella sp. and Pseudomonas sp. Microorganisms that are found in honey are very low in numbers. Botulinum endospores are naturally present in honey. Honey should not be given to the children under one year of age because their digestive system is not well developed and they are not able to destroy the spores (Snowdon and Cliver, 1996). Bacillus circulans is another species that is present in honey and produces lipases in honey (Elwan er al., 1983; Nasir et al., 2010). Wounds are common injuries that are produced due to different reasons e.g., accidents and road traffic (Armstrong and Athanasius, 1998). A wound is type of physical injury in which the skin is tom, cut or punctured and injured and which cause damage in the dermis of the skin (Bakker, 1998). In present study, antibacterial effect of honey on wound of diabetic person was studied.

Infections and other health related problems have been of great concern to human beings and chemotherapy is the main approach in the treatment of such conditions. Investigation into the microbial flora of wound began in the late 19th century and since then; improvements in techniques have facilitated the recovery, identification and enumeration of a wide variety of microbial species. Most wounds support relatively stable polymicrobial communities (Bowler et al., 2001) often without signs of clinical infection. However, potential pathogens may be present and the delicate balance between colonized wound and an infected wound depends on the interplay of complex host and microbial influences. The development of wound infection has deleterious effect on patients by causing increased pain, discomfort, inconveniences and can lead to life threatening conditions or even death. Major challenges encountered with antibiotics in clinical use are resistance to antibiotics which leads eventually to failure of the treatment (Blair 2004). Infectious diseases are known to be treated with herbal remedies throughout the history of mankind; even today, natural substances continue to play a major role in primary health care as therapeutic remedies in many developing countries. Over the years, there have been reports of the production of more potent antibiotics e.g. third and fourth generation of cephalosporin by pharmaceutical companies which are not readily available and expensive. Problems of various antibiotics include low efficacy, side effect which has lead investigations into natural and potent antibacterial seeming to be the right step to take. The invasion of pathogenic organism is on the rise as a result, effects are been made to develop antibacterial agent from natural sources for better therapeutic effect. The therapies have drawn the interest of both public and medicinal communities. Current research has been focused on herbal and aromatherapy product. However, a number of their product such as honey has shown therapeutic promise. The presence in honey of various inhibins has been reported by several investigators. Honey was used to treat infected wound as long as 2000 years ago before bacterial were discovered to be the cause of infection in c.50 AD, Dioscorides described honey has been “good for all rotten and hollow ulcers”. More recently, honey has been reported to have an inhibitory effect to around 60 species of bacterial including aerobes and anaerobes, Gram positive and Gram negative (Molan, 2006). The current prevalence of the therapeutic use of ancient remedies, include honey committee on science and technology.

1.2 Statement of Problem
The moist environment of wounds is an ideal growth medium for bacteria thereby causing delay in healing. Some of these bacteria may not be pathogenic while some could be pathogenic. The pathogenic bacteria could cause diseases such as sepsis which could likely result to severe infection. Antimicrobial agents (such as newly synthesized antimicrobial drugs) are essentially important in reducing the global burden of infectious diseases. However, the rapid increase in multiple drug resistant microorganisms associated with conventional agents has necessitated the search for new antimicrobial agents that are safer and less expensive. Therefore, a re-evaluation of the therapeutic use of ancient remedies, such as honey is clearly needed.

1.3 Justification of Study
Honey is one of the oldest traditional medicines considered as traditional remedy for microbial infections. It is also recognized as an efficacious topical antimicrobial agent in the treatment of burns and wounds (Brudzynski, 2006). This leads to the search for different types of honey with antibacterial activity (Mullai and Menon, 2007). The healing effect of honey could be due to various physical and chemical properties (Snow and Manley-Harris, 2004). The floral source of honey plays an important role on its biological properties (Molan, 2002). Honey is being used in a few hospitals, especially in the clinical treatment of ulcers, bedsores, burns, injuries and surgical wounds. The antibacterial properties of honey may be particularly useful against bacteria which have developed resistance to many antibiotics, e.g. Staphylococcus aureus, which is a major cause of wound sepsis in hospitals (Armstrong and Otis, 1995). Honey is thus an ideal topical wound dressing agent in surgical infections, burns and wound infections (Betts and Molan, 2002).The use of honey as a medicine has continued into the present-day medicine. It has been shown that natural unheated honey has some broad-spectrum antibacterial activity when tested against pathogenic bacteria, oral bacteria as well as food spoilage bacteria (Bassom et al., 1994, Mundo et al., 2004 and Lusby and Coombes 2005). The antibacterial potency of honey has been attributed to its strong osmotic effect, naturally low PH (Kwakman and Zaat, 2012), the ability to produced hydrogen peroxide which plays a key role in the antimicrobial activity of honey (Kacaniova et al., 2011 and Wahdam,1998)and phytochemical factors. Numerous reports and clinical studies have demonstrated the antimicrobial activity of honey against a broad range of microorganisms, including multi-antibiotic resistant strains, hence the need for this research.

ANTIBACTERIAL ACTIVITY OF HONEY ON STAPHYLOCOCCUS AUREUS, ESCHERICHIA COLI AND STREPTOCOCCUS PYOGENES ISOLATED FROM WOUNDS