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JAMES MAGONDU GITHINJI B.Sc. (Hons)

REG: I56/CE/I0966/2006

A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF MASTER OF SCIENCE (MICROBIOLOGY),

KENY ATTA UNIVERSITY.

JULY 2012

Githinji, James Invitro antibacterial

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DECLARATION

This Thesis is my original work and has not been presented for a degree in any

other University or any other award.

Githinji James Magondu

Department of plant and microbial sciences ,

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-We confirm that the work reported in this Thesis was carried out by the candidate under our supervision.

Dr. John Maingi

Department of plant and microbial sciences Kenyatta University

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Dr. Francis Kariuki

Department of plant and microbial sciences Kenyatta University

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DEDICATION

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ACKNOWLEDGEMENT

It is difficult to overstate my gratitude to my MSc. Supervisors. Dr. John

Maingi who accepted me as an MSc student. He managed to teach me how to

work independently which is very important, but at any time, his useful advice

was available to me. Dr. John Maingi thank you so much. Dr. Francis Kariuki

was accessible and very resourceful in research layout and ecological science.

It is through his guidance that I was able to categorize the habitats that were

likely to provide the important floral source with medicinal value. His

suggestion that I incorporate phytochemical analysis in my research was very

challenging but equally instrumental. With their enthusiasm, inspiration, and

great efforts to explain things clearly and simply, they helped to make my

research work possible for me. Throughout my thesis-writing period, they

provided encouragement, sound advice, good teaching, good company, and lots

of good ideas. In my opinion, this ishow ideal supervisors should be. I treasure

their technical support. Iwould have been lost without them.

I thank Dr. Machocho and Ruth from the chemistry department for their

technical support in TLC procedures. Iwould also like to thank Mr Nzau the

chief technician for his immeasurable supports in my laboratory procedures

and Madam Ndundu for her directions. Mr Ndegwa the chief technician inthe

school of health sciences guided me through the TLC procedures as well,

thanks a lot. I cannot forget Mr Mathaka who supported me in my TLC

procedures all along. Itwas very challenging butwe conquered.

I was delighted to interact with Dr Matofari of Egerton University. His insight

to diagnostic microbiology was very instrumental. He sets an example of a

world-class researcher for his rigor and passion on research. Prof. P.C Molan

of Waikato University responded to my email promptly. I continued to use his

research literature for reference frequently in my research work. Iam grateful

to him.

I am indebted to Dr Ngugi former medical superintendent at Nyeri Provincial

General Hospital for facilitating the administrative consent that allowed my

specimen collection possible in the hospital. Iam equally indebted to Ann the

chief Nurse and her staff at the hospital. Miss Susan and Mr. Gitonga both of

ward 7, 8and 9 were particularly helpful and kind.

Iam grateful to my course mates who participated in part of my research work.

Iwill live with precious memories of the late Anthony Gikunju who reminded

me of some of the basics. Without them the research would not have been

possible. Their tolerance, good humor, and insight added much. I hope they felt

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TABLE OF CONTENTS

Title .

Declaration :... II

Dedication... III Acknowledgement... IV Table of contents... vi

List of Tables ix

List of Figures x

List of Acronyms... XI Abstract... .. xiii CHAPTER ONE

1.0 INTRODUCTION 1

1.1 Background 1

1.2 Problem statement 3

1.3 Justification 4

1.4 Research questions 4

1.5 Hypotheses 5

1.6 Research Objectives 5

1.6.1 General objectives 5

1.6.2 Specific objectives 5

CHAPTER TWO ••

2.0 LITERATURE REVIEW 7

2.1 Nature ofhoney 7

2.2 Antimicrobial properties of honey 7

2.3 Therapeutic potential of honey 9

2.4 Immune system activation 10

2.5 Antibiotic resistance of Staphylococcus aureus 10 CHAPTER THREE

3.0 MATERIALS AND METHODS 14

3.1 Study site and population 14

3.1.1 Experimental Design and Sample size... 14

3.1.2 Patient's profiles 16

3.2 Collection of cl inical specimen 16

3.3 Specimen collection and processing 17 3.4 Culture media preparation... 18

3.5 Honey sampling and preparation 19

3.5.1 Honey sampling 19

3.5.2 Preparation of honey 19

3.6 Bacterial culture and confirmatory tests.. .... .... .. .... .. 19

3.6.1 Inoculation and incubation 19

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3.6.2.1 Preparation of the smear and gram staining 21

3.6.2.2 Catalase test.. 21

3.6.2.3 The slide coagulase test.. 22 3.6.2.4 Quantification ofStaphylococcus aureus 22 3.7 Thin Layer Chromatography (TLC) experiment. 22

3.7.1 Extraction of phenolic

components from honey for detection 24

3.7.2 Detection ofbioactive compounds

present in honey samples 24

3.7.3 Contact bioautography 25

3.8 Bioassay methods 26

3.8.1 Inoculum preparation 27

3.8.2 Honey and sugar disks preparation 28 3.8.3 Preparation of Antibiotic stock solutions 28

3.8.4 Preparation of sugar syrup 29

3.8.5 MIC and MBC determination of the Antibiotics 29 3.9 Determination of (MIC) and (MBC) using macro dilution

broth tube method 30

3.10 Preparation of sucrose forStaphylococcus aureus storage 31 3.11 Storage ofpure cultures of

Staphylococcus aureus using sucrose 31

3.12 Statistical analysis of data 31

CHAPTER FOUR

4.0 RESULTS 32

4.1 Frequency and load of

Staphylococcus aureus inwounds 32

4.2 Phytochemicals present inhoney 32

4.3 Contact bioautography 35

4.4 In vitro antibacterial activity of honey at different honey

concentrations using disk diffusion method 36

4.5 MIC for honey samples 38

4.6 MBC for honey samples 39

4.6 In vitro activity of honey samples after inactivation of

hydrogen peroxide using disk diffusion method 39

4.7 MIC and MBC for control antibiotics 40

4.8 In vitro activity ofhoney samples after inactivation of

hydrogen peroxide 41

4.8.1 Zones of inhibition for hydrogen peroxide inactivated honey against Staphylococcus aureus

ATCC 25923 44

4.9 Inhibitory activity of sugar syrup using disk

diffusion method 44

4.9.1 Inhibitory activity of honey disks having sugar

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CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION 46

5.1 Discussion 46

5.1.1 Frequency and bacterial load of

Staphylococcus aureus in the clinical samples... 46 5.1.2 Antibacterial components present in honey ,

samples 50

5.1.3 Potent honey samples 61

5.1.4 Antibiotic resistance 62

5.2 Conclusion 64

5.3 Recommendations 65

References... 66

Appendix i: Comparison of Healthcare Associated and

Community Associated MRSA 74

Appendix ii: In vitro Susceptibility Test Quality Control Ranges

for Penicillin 10 Units 75

Appendix iii: Acceptable limits for quality control strains used to monitor accuracy of Disk diffusion testing using

Mueller Hinton (CLSI) 75

Appendix iv: Suggested Antibiotic Dilution Ranges for

MIC Testing 76

Appendix v: Anova table for Single Factor test using whole honey samples at varying concentrations

using disk diffusion assay 77

Appendix vi:In vitro antibacterial activity of hydrogen peroxide inactivated honey against Staphylococcus aureus 78 Appendix vii: Phytochemical analysis and reactions 79 Appendix viii Flavonoids fractions in honey samples

in replications 80

Appendix ix: Alkaloids fractions in honey samples

in replications 80

Appendix x: Saponins fractions in honey samples

in replications 81

Appendix xi: Terpenes fractions in honey samples

in replications 81

Appendix xii: Retention factor values for the

phytochemical fractions 82

Appendix xiii: Minimum Inhibitory Concentration for vancomycin

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LIST OF TABLES

Table 4.1 Staphylococcus aureus streak plate levels and CFU counts 32 Table 4.2 Phytochemical fractions ...•... 33 Table 4.3 Rrvalues for phytochemical fractions in their

elution order indicating molecular variation 34

Table 4.4 Mean zones of inhibition (mm) formed by tlavonoids and alkaloids analytes against

Staphylococcus aureus clinical isolates 35 Table 4.5 Mean zones of inhibition (mm) formed by Alkaloids,

flavonoids and control antibiotics against S.aureus isolates

and S.aureus ATCC 25923 36

Table 4.6 Variation in zones of inhibition with increase in honey

Concentration (%) using S. aureus isolates 37

Table 4.7 Variation in zones of inhibition (mm) with increase in honey concentration (%) using Staphylococcus aureus

ATCC 25923 38

Table 4.8 MIC using honey samples against Staphylococcus aureus 39 Table 4.9 MBC using of honey samples against Staphylococcus aureus .40

Table 4.10 The MIC ranges using control antibiotics .40 Table 4.11 The MBC ranges using control antibiotics 41 Table 4.12 Zones of inhibition (mm) using hydrogen peroxide inactivated

honey at different concentrations (%) against

Staphylococcus aureus .43

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LIST OF FIGURES

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ACRONYMS AND ABBREVIATIONS AIDS Acquired Immunodeficiency Syndrome

ANOV A Analysis of Variance

ATCC American Type Culture Collection c -DNA Complementary Deoxyribonucleic acid C. V Critical Value

CA-MRSA Community Associated Methicillin Resistant Staphylococcus aureus CA-MSSA Community Associated Methicillin Susceptible Staphylococcus aureus CDC Centre for Disease Control

CFU Colony Forming Unit

CLSI Clinical and Laboratory Standards Institute DCM Dichloromethane

DNA Deoxyribonucleic acid EPS Extra cellular polysaccharide

HA-MRSA Healthcare-acquired methicillin-resistant Staphylococcus aureus

ET Enterotoxin

HIV Human Immunodeficiency Virus HSD Honest Significant Test

IR Infrared

KEMRI Kenya Medical Research Institute LukF Leukocidin Fast eluting

LukS Leukocidin slow eluting

MBC Minimum Bactericidal Concentration MHA Mueller-Hinton Agar

MIC Minimum Inhibitory Concentration m-RNA Messenger Ribonucleic acid

MRSA Methicillin Resistant Staphylococcus aureus

MSCRAMM Microbial Surface Component Recognizing Adhesive Matrix Molecules MSE Mean Square Error

NAPRECA Natural Product Research Network for Eastern and Central Africa NCCLS National Committee for Clinical Laboratory Standards

NMR Nuclear magnetic resonance PBP Penicillin Binding Protein PCR Polymerase Chain Reaction PH Potential of Hydrogen PVL Panton Valentine Leukocidin

QC Quality Control

RIRDC Rural Industries Research and Development Corporation

Ribonucleic acid

Reactive Oxygen Species Staphylococcal Enterotoxin Thin Layer Chromatography Test Statistic

Toxic Shock Syndrome Toxin Unique Manuka Factor

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UV WDF WHO

Ultra Violet

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ABSTRACT

The present study evaluated the antibacterial potency of some selected honey

types in Kenya against Staphylococcus aureus. This bacterial species causes wound infections, impetigo, breast abscesses, boils, osteomyelitis and toxic

shock syndrome. It is also known to cause other more serious diseases like Staphylococcal pneumonia. It can produce exotoxins that cause food poisoning as well. The aim of this study was to determine the antibacterial efficacy of some selected honey samples in Kenya using in vitro bioassay. Ninety two wound dressing swabs were collected from in-patients at Nyeri Provincial General Hospital targeting Staphylococcus aureus. Bacterial cultures were

obtained. Six different types of multi floral honey were used for bioassay in this study. Three of these were refined commercial honeys while the other three were raw. Antibacterial susceptibilities were done using disk diffusion. Both Minimum Inhibition Concentrations (MICs) and Minimum bactericidal Concentrations (MBCs) were determined by serial dilution by macro dilution broth method. Contact bioautography was also done for the phytochemical chromatograms. The antibiotics used as the controls in the contact bioautography were Penicillin with a disc potency of 10 IU (455 ug) and Vancomycin 30llg. Staphylococcus aureusATCC 25923 strain was used as the

standard strain. Standard bacterial suspensions were prepared using the 0.5

McFarland's standard. Each type of honey was tested against the eighty one

bacterial isolates and the data used for analysis at 0.05 level of significance.

Alkaloids and flavonoids present in honey demonstrated significant (p <0.05) antibacterial effect against Staphylococcus aureus compared to the other phytochemicals according to the in vitro tests. Both the flavonoids and alkaloids had significantly (p < 0.05) larger zones of inhibition than penicillin but significantly (p <0.05) smaller than vancomycin disks. Hydrogen peroxide

inactivated honey samples revealed significantly smaller zones of inhibition (p < 0.05) compared to the hydrogen peroxide active honey samples. Honey

from Mt. Kenya forest and Kitui had the lowest MBC and MIC levels that were significantly (p <0.05) different from those of the other honey samples in the study. The MIC and MBC in vitro potency of the two honey samples did not

vary significantly (p >0.05).The control bacterial strain was more susceptible to both the honey samples and the control antibiotics than the clinical isolates.

The MIC of the honey samples ranged from' 6 - 8 % v/v on the test bacterial organisms, and MBC ranged betweenTs - 16 % v/v signifying its high potency. The results obtained in this study have confirmed that Staphylococcus aureus is a frequent clinical isolate. The invitro bioassay results revealed that honey samples obtained from Kitui and Mt. Kenya forests had higher potency

levels against the Staphylococcus aureus clinical isolates as well as the standard strain. At low dilution, commercial honeys retained a significant antibacterial potency because of the presence of hydrogen peroxide. Raw

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For centuries honey had a valued place in traditional medicine, being used in

the treatment of wounds and other diseases. The use of traditional medicine to

treat infection has been practiced since the origin of mankind, and in the past it

was the only method available (Jones, 2001). Currently, due to lack of

sufficient modern health care system particularly in rural areas, people prefer to

visit traditional healers or use herbal medicines. The integration of traditional

and modern medicine is gaining increased recognition globally (WHO, 2000).

Although very well known as a food, honey is not well recognised as being

medicinal, yet it is one of the oldest medicines known (Zumla and Lulat, 1989)

and has continued to be used as such throughout the ages (Jones, 2001).

However, honey has been rediscovered as being therapeutic in more recent

times by the medical profession (Zumla and Lulat, 1989). The antimicrobial

property of honey has been demonstrated and there is a hypothesis regarding

honey and its ability to bring healing in septic wounds (Kiistala et al., 1995).

Septic wounds could be treated successfully following a well outlined

procedure of treatment using honey as the key antimicrobial agent (Mavric et

al., 2008).

Staphylococcus aureus are the bacteria most frequently isolated from b~rns and

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indigenous microbiota (Ayliffe et al., 1998). This bacterial species has a

combination of various virulence factors (Kwakman etal., 2008).

Since the 1970s, Staphylococcus aureus strains resistant to the

penicillinase-stable penicillins that include cloxacillin, dicloxacillin, methicillin, nafcillin,

and oxacillin have emerged (Boyce, 2001). It has now become necessary to

search for an alternative and equally effective method of dealing with

increasing numbers of antibiotics resistant strains.

The use of honey as a therapeutic substance is gaining acceptance as an

antibacterial agent for the treatment of various clinical ailments caused by

among other bacterial pathogens, Multidrug Resistant (MDR) Staphylococcus

aureus (Elbagoury and Fayed, 1985). It is therefore a suitable agent in search

of treatment of antibiotic resistant strains of Staphylococcus aureus (K wakman

et al.,2008). Honey has been shown to be effective against both Gram positive

and Gram negative organisms, aerobic and anaerobic bacteria, as well as

inhibiting spore germination of Bacillus cereus. Since, the type of honey

produced by bees is dependent on the different vegetative flowers (Gedif and

Hahn, 2003), honeys from different ecological regions may therefore have

broad range of antimicrobial activities and their potency may vary.

This research investigated the antimicrobial activity of honey from different

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Staphylococcus aureus isolated from wounds on patients in Nyeri Provincial

General Hospital.

1.2 Problem statement

Indiscriminate use of antibiotics has led to increase in reported cases of infections by antibiotic resistant Staphylococcus aureus (Tonks et al., 2003).

The management of skin diseases and wounds is also becoming a priority due to the association of skin opportunistic infectious and HIV / AIDS. Estimates

show that 92% of HIV -infected individuals have cutaneous and mucosal complications (Geber et al., 2006).

The emergence of multidrug resistance by Staphylococcus aureus has been reported in Nyeri Provincial General Hospital in Kenya according to the

records at the hospital laboratories. Records in the hospital also indicate a rising trend in the number of patients suffering from staphylococcal wounds leading to cutaneous and mucosal complications. This has necessitated broad

spectrum antibiotic therapy. Prolonged broad spectrum antibiotic dosage may lead to serious side effects which include urine system disorders, nerve tissue impairment and pseudomembranous colitis (Kwakman et al., 2008). Broad

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1.3 Justification

In Kenya, access to modem health facilities and affordable and effective

treatment is still a challenge to many. Apitherapy would be a cost-effective

approach in the management of wounds compared to conventional therapies.

The threat of wound infections caused by antibiotic-resistant strains of

pathogens including Staphylococcus aureus is sufficient reason to consider

modifying our present reliance on antibiotics. Bacterial resistance to first

generation antibiotics like penicillin indicates that gradually through natural

selection antibiotic resistant strains emerge. Progressively, significant

antibiotic resistance would emerge for second and third generation antibiotics.

Alternative approaches are needed to address the emergence of wound

infections by antibiotic-resistant bacteria. There is therefore the need to

investigate the efficacy of various honey types from different ecological zones.

1.4 Research questions

I. IsStaphylococcus aureus a frequent pathogen in wound exudates?

2. Do the individual biochemical analytes present in honey samples from

different ecological zones in Kenya have antibacterial properties against

Staphylococcus aureus?

3. What are the in vitro sensitivity levels for selected Kenyan whole and

processed honey samples against Staphylococcus aureus isolates?

4. Are the honey samples from different ecological zones effective

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5. Do the antibacterial activities of different honey samples vary from that

of control antibiotics?

1.5 Hypotheses

1. Staphylococcus aureus is not a frequent clinical isolate In wound

exudates.

2. Honey samples obtained from different ecological zones in Kenya do

not have significant and varying in vitro antibacterial effects against

clinical strains of Staphylococcus aureus and standard Staphylococcus

aureus strain.

3. There is no significant difference in the in vitro antibacterial effect

between different honey samples and the control antibiotics namely

vancomycin and penicillin G.

1.6 Research objectives

1.6.1 General objective

The general objective of this study is to assay honey from different ecological

zones for in vitro antibacterial properties against Staphylococcus aureus.

1.6.2 Specific objectives

1. To determine the frequency and bacterial load of Staphylococcus

aureus in wound exudates of different patients in Nyeri Provincial

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2. To identify specific bioactive compounds in honey with antibacterial

effect against Staphylococcus aureus.

3. To assess the in vitro antibacterial activity of whole and processed

honey samples from different ecological zones against Staphylococcus

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CHAPTER TWO

LITERA TURE REVIEW

2.1 Nature of honey

Honey produced by bees is one of the ancient traditional medicines used for the

treatment and prevention of various illnesses (Mulu, 2004). Honey is formed from nectar collected by bees foraging a wide variety of plants. It is composed

of a saturated or supersaturated solution of sugars, consisting typically of 17% water, 38% fructose, 31 % glucose, 10% other sugars, and a wide range of

micronutrients (vitamins, amino acids and minerals), with a pH below 4

(Somerfield, 1991).

For the processing of nectar during its ripening into honey, the bees add some

enzymes (Willix et al., 1992). One of these is an invertase which breaks down the sucrose in the nectar into glucose and fructose, so that the sucrose content

of honey is typically only 1% of the total sugar content (Somerfield, 1991).

2.2 Antimicrobial properties of honey

The antimicrobial property of the honey may be attributed to phytochemicals contained in honey (Willix et al., 1992). Using contact bioautography, the

antimicrobial potency of individual phytochernicals can be assessed in using in vitro bioassay. Bioautography is a highly efficacious assay for the detection of antimicrobial compounds because it allows localization of bioactivity even in a

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antioxidants (Frankel et al., 1998), which would protect living tissues against

any free radical damage that could possibly result from the hydrogen peroxide

formed in honey. Flavonoids are the major antioxidants in honey (Lowy,

2000). Flavonoids have been singled out as one of the most significant

biochemical compounds present in most honey samples and they have both

antimicrobial and antioxidant activity (Halliwell and Cross, 1994). Flavonoids

consist of 6 major subgroups: chalcone, flavone, flavonol, flavanone,

anthocyan ins and isoflavonoids (Halliwell and Cross, 1994). The combinations

of different flavonoids often improve considerably their biological activities,

such as bactericidal and bacteriostatic action (Fukai et al., 1996; Fukai et al.,

2002; Zeng et al., 1992). Flavonoids are thought to trigger the production of

natural enzymes that fight disease causing organisms (Gedif and Hahn, 2003).

Glucose oxidase which forms gluconic acid and hydrogen peroxide from the

glucose is inactive in ripened honey but is reactivated when honey is diluted,

giving honey antibacterial activity (Molan, 1992). The low levels of hydrogen

peroxide produced by honey are sufficient to make honey quite potently

antibacterial (Molan, 1992) most bacteria cannot survive in the presence of

oxygen or hydrogen peroxide (Wahdan, 1998).

Hydrogen peroxide is a well-known antimicrobial agent, initially hailed for its

antibacterial and cleansing properties when it was first introduced into clinical

practice. The antimicrobial action of honey in infected wounds does not depend

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the tissues (Cooper et al., 1999). In more recent times, hydrogen peroxide has been considered undesirable for wound treatment because of inflammation and

damage to tissue (Halliwell and Cross, 1994). However, the hydrogen peroxide

concentration produced in honey activated by dilution is typically around 1 molll (Molan, 1992), about 1000 times less than in the 3% solution commonly

used as an antiseptic. The harmful effects of hydrogen peroxide are further reduced because honey inactivates the free iron which catalyses the formation of oxygen free radicals produced by hydrogen peroxide and its antioxidant

components help to mop up oxygen free radicals which damage membranes (Frankel et al., 1998).

It is speculated that sugars present in honey increase the osmotic effect of the media which consequently increases its antibacterial activity. Amylase present

in honey is partly responsible for antibacterial activity observed in honey and concluded that increase in concentration of sugar increases antibacterial ability

of the honey (Laid et al., 2008).

2.3 Therapeutic potential of honey

Besides its antimicrobial properties, honey can clear wound infection in a number of ways, including boosting the immune system, having anti-inflammatory and antioxidant activities and by stimulation of cell growth

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treatment with conventional antibiotics and antiseptics known to have allergic

effects to some individuals (Efem, 1988).

2.4 Immune system activation

In addition to its antibacterial activity, honey clear infection through

stimulating the activity of leukocytes (Tonks et al., 2001). Low concentrations

of honey stimulate proliferation of lymphocytes and activate phagocytes

(Abuharfeil et al., 1999), and stimulate monocytes to release cytokines which

are activators of the immune response to infection which influence the wound

healing process (Tonks et al.,2001).

2.5 Antibiotic resistance ofStaphylococcus aureus

The emergence of new strains of bacteria that resist the antimicrobial therapy

makes it essential to look for alternative treatments to control the

multidrug-resistant organisms (Kwakman et al., 2008). Vancomycin has been the

preferred antibiotic of choice the world over for the treatment of multi-drug

resistant strains. However, Aston et al. (2009) in their surveillance study

established that there are risk factors associated with treatment failure in

patients receiving vancomycin for hospital acquired methicillin resistant

Staphylococcus aureuspneumonia.

The emergence of antibiotic resistant Staphylococcus aureus poses a major

challenge to healthcare. Antibiotic resistant Staphylococcus aureus such as

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resistance (MRSA) is a major cause of hospital-acquired infection throughout the world and is now also prevalent in the community as well as nursing and

residential homes (Ayliffe et al., 1998). Of the Staphylococcus aureus isolates in the United Kingdom in 2005, 43.6% were found to be MRSA and a point prevalence survey showed that 16% of intensive care patients were either

colonized or infected with MRSA (Hails et al., 2003). In most parts of the world, mortality attributable to MRSA bacteraemia has been estimated to be

22% (Cosgrove, 2005). Increasing reports of resistance to antibiotics and antiseptics have sparked a wave of research to find alternative antimicrobial strategies (Deurenberg et al.,2007). This could form the basis of an alternative

approach for the eradication of such bacteria from superficial wounds.

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most important pathogens that cause postoperative infections and it accounts

for up to 40% of nosocomial Staphylococcus aureus infections in large hospitals and 25%-30% in smaller hospitals in the USA (Murray etal., 2003).

Methicillin-resistant Staphylococcus aureus emerged in London Hospital in

1982 (Duckworth et al., 1988). This strain was associated with significant

morbidity and mortality. The particular strain, termed epidemic methicillin-resistant Staphylococcus aureus, was recognized by its characteristic antibiogram, phage-type and plasmid profile. In this outbreak various means of control were attempted. Side room isolation did not curtail spread of the

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extended screening, mupirocin for treatment of carriage and the use of an

isolation ward. It reflects high survival rates for this bacteria species. Failure of

antibiotic therapy in the treatment of open wounds that result from acute

complicated traumatic orthopedic injuries is being reported as a clinical

problem all over the world (Kwakman et al., 2008). However, some

antimicrobials have been tested and found to have antimicrobial effect against

Staphylococcus aureus (Anguzu and Olila, 2007). Several hypotheses may

explain the emergence of high virulence and pathogenecity levels similar to the

ones observed in MRSA (Mohammad and Mohammad, 2007). Research by

Dowd et al. (1983) found that a large number of Staphylococcus aureusstrains

resistant to gentamicin, methicillin and other antibiotics, isolated in several

Dublin hospitals during a 4-year period, had uniform plasmid profiles and were

also susceptible to honey.

The resistance observed in Staphylococcus aureus could be attributed to

irrational use of antibiotics for conditions that may not clinically indicate their

use, over-the-counter sale of antibiotics in pharmacies without prescription by

authorized practitioners, some new drug formulations which may be of poor

quality and dumping of banned products into the market where the public may

get access to them (Ayliffe et al., 1998). In view of the resistance observed,

infections caused by MRSA can be expensive in terms of costs of treatment;

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Cases of antibiotic resistance are frequent and constantly rising. Mechanisms

of microbial inhibition of cellular target sites have not yet been fully

investigated but multiple, non specific sites are predicted. Both antibiotic

susceptible and antibiotic resistant strains of Staphylococcus aureus and other

bacteria have detectable susceptibility to honey suggesting that honey may be

having an alternative antibacterial potency (Cooper et al., 2002). Antibiotic resistance among microbes urgently necessitate the development of novel

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CHAPTER THREE

MA TERIALS AND METHODS

3.1 Study site and population

The study was carried out at Nyeri Provincial General Hospital after the

consent was granted by the hospital administration. The population comprised

of in-patients, before starting the antibiotic therapy treatment process in the

hospital. These patients were suffering from septic superficial wounds

following serious skin infections, burns or general injuries. The patients were

of both sexes at different ages.

3.1.1 Experimental design and Sample size

In-patients in three hospital wards were enrolled for the study. The wards were

identified as wards 7, 8 and 9. Each ward has a bed capacity of thirty. Due to

the scarcity of health facilities, some beds accommodated two patients. Within

the study period of four months, a total of one ninety patients were hospitalized

at different times with most of them sharing beds. The study population was

defined by considering only those in-patients that had not started the in-patient

antibiotic treatment in the hospital. Logically it excluded the post operation

patients whose bacterial strains may have been weakened by the antibiotics.

/ /

The population size was therefore one twenty thus giving a population sample

of ninety two. The specimens were collected using systematic random

sampling. The sample size was determined using a simplified formula for

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Thus;

n = N/ {I +N (e) 2} Where n = Sample size = 92;

N = Population size = 120; e =Level of precision (0.05). T'he interval of

specimen collection was determined using the formula K= N/n = 1.30. Since

the K value is a non integral value, the number line concept was incorporated to

determine the patients to be sampled.

Thus;

o

to 1.30 1.30 to 2.60 Intervals

~ ~

o

1 2 3 4 5 6 92-Patients

KEY:

: Represents sampling interval range:

U :

Represents sampled patients

Figure 3.1: Systematic random sampling using the number line

In the above number line concept illustration, 1.30 represents the first station

along the number line using the K= 1.30 interval value and 0 being the lowest integral value. Since by rounding off to the nearest whole number the answer is

I, then patient number I was the first patient to be sampled. The second patient

to be sampled was patient number 3 and the systematic random sampling

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sampled are shown using vertical arrows, while the intervals are shown using

horizontal arrows in the illustration above.

3.1.2 Patients' profiles

A total of ninety two in-patients presenting superficial wounds were enrolled in

the study between September 20 I 0 and January 20 II. These were initially

out-patients that had shown clinical signs of septic recalcitrant wounds due to

Staphylococcus aureus infection. Out of ninety two patients studied, 59% were

males and 41%were females. Below the age of 18 years were 12 males and 6

females. Above the age of 18 were 42 males and 32 females. The ages of study

groups ranged from 5 - 77 years. The premedication stay in hospital ranged

from I - 4 days. The causes of these wounds were physical injuries,

neuropathy due to diabetes mellitus, skin infections, burns and cutaneus and

sub cutaneus secondary infections.

3.2 Collection of clinical specimen

Swabs were sampled from wound exudates to serve as the inocula. This was

done by ward nurses during wound dressing sessions at Nyeri Provincial

General Hospital involving in - patients in wards 7, 8 and 9. The pus swabs

were obtained using sterile swabs from wound dressings before the wounds

were cleaned using an antiseptic solution. These swabs were collected one at a

time to avoid cross contamination between wound dressings obtained from

different patients. The wound dressings were later disposed in accordance to

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name, age and sex of the patient under study. This made it possible to avoid

duplication of specimen collection. In order to protect the privacy, of the

patients, the names and other personal details of the patients were not revealed

in this study. However, the specimen collection and processing was done

successfully and routine monitoring effected using labels SPI, SP2 , SP3,

SP4 SP92 to represent specimen collected in sequential order.

3.3 Specimen collection and processing

Amies transport media (Cheesbrough, 2006) was dispensed into disposable

sterile screw-cap tubes for specimen collection and transport. The transport

media ensured that bacterial pathogens likely to be present in the swabs

remained viable. Ninety two swabs obtained from wound dressing were

collected systematically over a period of 17 weeks for isolation of

Staphylococcus aureus from in-patients at Nyeri Provincial General Hospital.

Swabs supposedly containing bacterial contaminants were obtained from

different wound dressings immediately after the patients were undressed. These

swabs were inoculated into Amies transport medium in a screw-cap tube and

labeled accordingly.

In order to avoid cross contamination, each patient's wound dressings were

used only once and then disposed immediately. The specimens were handled

using standard procedures to avoid the spread of infection. In order to achieve

this, specimen bottles with well fitting caps were used. The outer surface of

(31)

methylated spirit before and after specimen collection in order to prevent contamination and spread of these pathogenic bacteria. Disposable gloves were

,

also used in all the stages of specimen collection and processing.

The process of culturing and identification of Staphylococcus aureus bacteria

was done in the department of Plant and Microbial Sciences (PMS) laboratories

at Kenyatta University within 24 hrs of specimen collection. Specimens were processed according to CLS[ guidelines (Cheesbrough, 2006) that involved the following key stages;

(i) Specimen inoculation on nutrient agar followed by incubation at 37 DC for

24 hrs.

(ii) Sub culturing on Mannitol salt agar to obtain a pure culture of

Staphylococcus sp. This also facilitated identification of the Mannitol

fermenting bacterial species.

(iii) Carrying out catalase and coagulase tests as the main biochemical tests to confirm the identity ofStaphylococcus aureus.

(iv) Carrying out microscopy to confirm the gram stain reactions and cell

morphology of Staphylococcus aureus.

3.4 Culture Media Preparation

Preparation of nutrient agar (oxoid), mueller- hinton agar (oxoid), mueller

(32)

3.5Honey sampling and preparation

3.5.1Honey sampling

Three raw honey samples were collected from traditional log hives in each of

the three different habitats. These are Lake Ol-bolossat basin representing

samples from aquatic ecosystem, Mount Kenya forest representing terrestrial

highlands and Kitui region representing terrestrial lowlands. Honey was

sampled from hives by simple random sampling.

3.5.2 Preparation of honey

Six different types of honey were used in this research. Three of these were

raw honey while the other three were refined. The unrefined honey designated

M, Land K were collected using sterile syringes and then sieved and stored in

colored bottles to avoid photo-chemically induced reactions. No further

processing was done to the refined honey designated C, Sand P. All the honey

samples were stored at room temperature under sterile conditions. The honey

samples were then diluted to form the various dilutions for disk diffusion. The

hydrogen peroxide inactivated honey samples were prepared by addition of

catalase using the method described by Molan (1997).

3.6Bacterial culture and confirmatory tests. 3.6.1 Inoculation and incubation

Inoculation using the wound exudates swabs from the hospital was done within

48 hrs of specimen collection. This was achieved by streaking specimen swabs

(33)

culturing procedure was carried out inside a laminar flow cabinet. Every

culture plate was labeled accordingly for the purpose of reference and

identification. The specimen swabs were also preserved in their respective

specimen bottles for temporary storage.

The streaked plates were incubated at 37°C for a period of 24 hrs. This

allowed the growth of the targeted bacteria (isolates). Other untargeted bacteria

were able to grow. However, the distinguishing colony morphology of

Staphylococcus aureus bacteria, which gives large golden colonies, enabled the

first step of identification. The suspected colonies were sub cultured in

mannitol salt agar to obtain pure isolates which were subjected to the

confirmatory tests.

3.6.2 Confirmatory Tests

Preliminary identification of bacteria was based on colony characteristics of

Staphylococcus aureus. These colonies were first identified for each specimen

at the end of the incubation period. The large golden smooth colonies were

isolated using sterile wire loops and streaked on Mannitol salt agar which is the

recommended selective media for the Staphylococcus sp and Micrococcus sp.

It is also differential since out of these bacterial species only Staphylococcus

aureus and S. saprophyticus are able to grow and ferment mannitol after

incubation. The phenol red dye in the media turns yellow due to acid formation

by mannitol fermenting bacteria. Biochemical tests and other microscopy

(34)

identification of the isolates. The following confirmatory tests and procedures

were used to confirm the identity of Staphylococcus aureus.

3.6.2.1 Preparation of the smear and gram staining

The bacterial smear was prepared by picking one colony in a pure plate culture

grown on the Mannitol salt agar. A sterilized wire loop was used to prepare a

smear on a clean microscope slide having a drop of physiological saline. The

smears were air dried and then fixed using heat by passing it mildly over a

burning flame thrice. The slides were then gram stained. The gram stain

procedure facilitates the identification of bacterial type by observing its cell

morphology. Staphylococcus aureus appears as gram positive cocci In grape

like clusters.

3.6.2.2 Catalase test

This test was carried out as a confirmatory test for Staphylococcus aureus after

successfully isolating the bacteria from mannitol salt agar and having

demonstrated its ability to ferment mannitol. Staphylococcus aureus reveals

gram positive cocci in grape like clusters on gram staining. A colony is picked

from a pure culture using an inoculating wood stick. The colony was smeared

on a clean slide. Drops of hydrogen peroxide were added on the bacterial

smear. The bacterial colonies of Staphylococcus aureus were able to degrade

hydrogen peroxide into oxygen and water due to their ability to produce

(35)

of the tested Staphylococcus aureus clinical isolate tested negative for catalse

test to warrant an alternative to catalase test.

3.6.2.3 The slide coagulase test

All the bacterial isolates that were able to give positive results for all of the

above confirmatory tests were subjected to coagulase tests using the rabbit

plasma. Using a sterile inoculating wood stick, pure bacterial colony isolate

was smeared on the slide. Rabbit plasma was added drop wise and mixed.

Observation was made for any signs of plasma coagulation within a period of

10 minutes.

3.6.2.4 Quantification of samples containing Staphylococcus aur.eus

The swab method and quantification was used according to the procedure of

Bowler et al. (200 I). After streaking and incubating the plates, the cultures

were observed for colony presence that ranged from the first quadrant to the

fourth (1+ to 4+). The probability of occurrence of Staphylococcus aureus was

determined by comparing the number of specimen that tested positive for this

bacteria species and total number of specimen collected. This was calculated as

follows;

Total number of specimen found with S. aureus =Probability of occurrence Total number of specimen collected

3.7 Thin layer chromatography (tIc) experiment

Thin layer chromatography was conducted to facilitate separation of individual

(36)

the stationary phase with calcium sulphate being the binding material.

Alluminium was used as the carrier TLC sheet. Suitable solvent system was

prepared after a series of trial experiments using solvent systems of different

polarity in order to establish the most suitable constitution. Commercial TLC

plates (Silica gel G 60, Merck), 20 x 20 ern, were cut at the chosen width with

a glass cutter. Micropipettes were used to measure the solvents. All-glass tank

with ground glass cover was used for the procedure. The solvents used were

Oichloromethane (OCM) and Methanol at a ratio of 1: I (50% t050%).

The procedure that was followed involved pre-run trials in improvised tanksto

select most suitable solvent system starting with 2 parts ethyl acetate to 3 parts

hexane. The other solvents used in the pre-run trials were chloroform and

Diethylether. Spotting using dissolved honey samples in the solvents was done

at 1.5 em from the bottom of the plate and separated from each other by I cm.

Plates were then placed immediately in the elution tank with the height of

solvent at 5 mm. The sample was introduced onto the TLC plate using micro

capillary tubes and then the solvent was allowed to ascend. A 10 ern migration

was allowed for the separation of analytes. The chromatogram was then

removed from the tank to allow the solvent evaporation in the fume hood. The

TLC runs were performed under laboratory conditions. For visualization of the

analytes, p-Anisaldehyde, Antimony (III) chloride and Oragendorff reagent

(37)

3.7.1 Extraction of phenolic components from honey for detection

Consisting of mainly sugars, honey as such cannot be subjected to TLC for

analysis of phenolic components, directly. The latter constituents present in

minor amounts can only be detected after concentration and without sugars

interfering. Therefore, phenolic constituents were extracted from honey prior to

TLC analysis, as follows: Honey (25 g) was dissolved in 75 ml of water, and

the solution was extracted with ethyl acetate. The combined ethyl acetate

extracts were dried over anhydrous magnesium sulfate. After filtration, and

evaporation of the solvent, the residue was taken up in 10 ml of ethanol (96%)

and subjected to TLC analysis (www.freepatentonline).

3.7.2 Detection of bioactive compounds present in the honey samples

Honey samples were spotted and separated on aluminium-backed plates using

the method described by Rahalison et al. (1991). Analytes were separated in

bands on intact TLC plates. Thin-layer chromatography (TLC) was performed

on precoated 20 x 20 cm TLC plates coated with 0.25 mm layers of silica gel

60 F254 (Merck).

Constituents were extracted from honey before TLC analysis as follows: 5 ml

honey was dissolved in 75 ml of extraction solvents namely OCM and

Methanol After application of the honey extract samples, spoting was done

using capillary tubes for all the six different types of honey. The TLC

chromatography development runs were done using the most suitable solvent

(38)

Two chromatography solvents were used: DCM and Methanol at a final ratio

of 50:50. Molecules identity was done by the method adopted by Fukai et al.

(1996) whereby analytes were identified by visualization. Some analytes were

visible using fluorescence UV light. The number of analytes in a

phytochemical group e.g. alkaloids, was determined by calculating the Rr

value. This helped to determine the level of biochemical diversity. The R;value

was worked out using the following formula:

Rr. =Distance travelled by component Distance travelled by solvent

3.7.3 Contact bioautography

Isolated compounds were evaluated by bioautography technique.

Derivatization reactions modify or destroy the structure of the compounds

detected in TLC procedures (www.erowid.org). Contact bioautography

therefore reduces the probability of denaturing the analytes further since no

more chemical related procedures are involved. This technique allowed the

localization of antimicrobial activity directly on a chromatographic plate where

the organism is applied. In contact bioautography antimicrobials diffuse from a

TLC plate or moist paper disk to an inoculated agar plate. The chromatogram

derived disk was placed face down onto the inoculated agar layer with

Staphylococcus aureus and left for two hours to enable diffusion. The disk was

(39)

3.8 Bioassay methods

In this study, antimicrobial susceptibility tests of isolated Staphylococcus,

aureus were done by disc diffusion method using Kirby-Bauer techniques

(Bauer et al., 1966) and as per recommendation of CLSI. Minimum Inhibition

Concentration and Minimum Bactericidal Concentration were also determined.

Identification of isolates was done using standard microbiological methods as

described by Cheesbrough (1984) and Cowan (1993). All the containers and

glass equipment used in the processing of the individual honey samples were

correctly labeled to avoid contamination or mixing of these honey samples.

This ensured that the results that were obtained were a true reflection of

bioassays and tests of distinct honey types. Staphylococcus aureus ATCC

25923 was used as the standard strain (Appendix ii,).

The dilution of the honey for the in-vitrotest was done to confirm the presence

of antibacterial components in the different honey samples and evaluate their

potency at varying concentrations. The Disk diffusion assay was carried at

20%, 40%, 60% and 80% concentration for each honey sample using Mueller

Hinton agar. Overnight fresh cultures of Staphylococcus aureus bacteria were

prepared and inocula of 0.5 MacFarland (1 x 108 CFU/ml) microorganisms

were prepared and inoculated onto a Mueller-Hinton agar (MHA) plate.

Inoculation was done within 15 mins of preparation time followed by

incubation at 37°C for 24 hours. Agar plates containing 15 ml of culture

medium were used and this volume was kept constant in all the four levels of

(40)

The amount of inhibition was recorded by measuring the diameter of the zone

of inhibition, in millimeters (mm); this was measured using a zone reader. The

values representing the zones of inhibition and their corresponding standard

deviations were recorded.

3.8.1 Inoculum preparation

The plates were prepared containing 20 ml of sterile Mueller-Hinton agar for

the purpose of employing the disk diffusion using Kirby-Bauer technique. The

inoculum was prepared by picking parts of the pure colony culture of

Staphylococcus aureus with a sterile wire loop. This was suspended in sterile

peptone water (broth) and incubated up to two hours to allow organisms to

reach their log-phase in growth. The density of suspension to be inoculated was

determined by comparison with opacity standard on McFarland 0.5 Barium

Sulphate solution (Elmer et al., 1997). A sterile swab was dipped into the

suspension of the isolate in peptone water, squeezed free from excess fluid

against the side of bottle and then spread over the agar plate.

Staphylococcus aureus bacteria were isolated in pure cultures by sub culturing

using mannitol salt agar from nutrient agar. The Staphylococcus aureus isolates

were able to grow and ferment mannitol salt agar; the recommended selective

medium. For the biochemical tests confirmation, each isolate tested positive for

both catalase and coagulase tests. In the microscopy procedures, the

Staphylococcus aureus isolates consistently produced gram positive cocci in

(41)

reference strain for identification and quality control purposes. The obtained

pure isolates were then used for bioassay procedures.

3.8.2 Honey and sugar disks preparation

Different concentrations of each honey type constituting, 20% v/v, 40% v/v,

60%v/v and 80% v/v were made in sterile distilled water. This was achieved by

dissolving the respective volumes: 0.2 ml, 0.4 ml, 0.6 ml, and 0.8 ml of each

honey into corresponding volumes of sterile distilled water to give a Iml

preparation. Filter paper disks of 6 mm diameter were prepared by the method

of Cheesbrough (2006). The disks were impregnated with different

concentrations of each type of honey by allowing them to settle in honey for

about 30 mins and then allowing them to drip excess honey for exactly 30 mins

to ensure uniformity before placing them on the agar plates for the disk

diffusion test. The same procedure was followed to impregnate disks using

sugar syrup at similar concentrations.

3.8.3 Preparation of Antibiotic stock solutions

Commercially prepared powders of Vancomycin with a potency of 900 ug/mg

and Penicillin with potency of 1667 ug/mg were accurately weighed to give 8

mg and 15.36 mg respectively and to make up volumes of 50 ml and 900 ml

for vancomycin and penicillin respectively. Stock solutions were prepared

using the formula given by Bauer et al.(1966) in order to obtain the standard

antibiotic dilution MIC ranges (Appendix iv). Sterile glass wares were used in

(42)

3.8.4 Preparation of sugar syrup

To obtain 80% v/v of sugar syrup concentration, whose antibacterial potency

would be compared with that of 80% v/v honey concentrations (the highest in

disk diffusion assay), analytical sucrose and glucose were used at equal

proportions and diluted using distilled water.

3.8.5 MIC and MBC determination of the antibiotics

One milliliter of sterile nutrient broth was pipetted into each of the sterile

test-tubes using a micropipette. One milliliter of the antibiotic was introduced into

the first test tube and mixed thoroughly. One milliliter of the mixture was then

pipetted and transferred into the second test-tube thus reducing the

concentration downwards serially until the nth test tube after which the excess

1ml was discarded. Broth cultures having SOIlI of the test bacteria were

inoculated into each test tube - including nthtube. One tube was left with just

the broth and everything was incubated at 37°C for 24 hrs and the plates were

read. The work was done in triplicates and under aseptic conditions. The MIC

was recorded as the lowest concentration of antibiotic concentration that did

not give any visible bacterial growth in the broth. The lowest concentration of

the antibiotic that did not permit any visible growth in the tubes even after sub

(43)

3.9 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) using macro -dilution Broth tube method

In this research the procedures were performed on the confirmed clinical isolates of Staphylococcus aureus. These bacterial suspensions were standardized using 0.5 McFarland constant in order to obtain the most suitable

turbidity levels. Other CLSI guidelines and recommendations were observed. Appropriate volumes of honey, were added to sterile tubes containing Mueller

Hinton broth using micropipettes at the following final concentrations of 32%v/v, 16%v/v, 8%v/v, 4%v/v, 2%v/v ,1%v/v ,0.5%v/v and 0.25v/v. This procedure was carried out for all the six different honey types after all of them

revealed an in vitro antimicrobial ability.

The antibiotics concentrations were also prepared. Using a volumetric pipette, 50111of the test bacteria broth cultures was added into each of the tubes. The

tubes containing the bacterial cultures were incubated at 37°C for 24 h, after which they were observed to determine the lowest concentration of the test

honey that did not permit any visible growth in the tubes and even after sub culturing on nutrient agar. This was recorded as the MBC. It was compared with that of the control. Two controls were employed; one was a row of positive control tubes containing Staphylococcus aureus ATCC 25923 strain

inoculated in the growth medium. The other was a negative control that consisted of a row of tubes containing different concentrations of honey that were not inoculated. The MlC was recorded as the lowest concentration of

(44)

The MIC and MBC for Staphylococcus aureus ATCC 25923 strain against the

six different types of honey was also determined.

3.10 Preparation of sucrose for Staphylococcus aureus storage

The confirmed isolates of Staphylococcus aureus were stored In sucrose

solution for future reference. The following procedure was followed in

accordance to a formulation given by the department of dairy technology at

Egerton University. Analytical sucrose was weighed and dissolved in 100 ml

distilled water. A volume of 0.5 ml was dispensed in each vial and autoc\aved

at 121°C for 15 mins. After cooling, the solution was stored in vials at 4 °C

until the time of use.

3.11 Storage of pu re cultu res ofStaphylococcus aureus using sucrose

Pure colonies were harvested using sterile cotton swab from nutrient agar and

suspended in 0.25 molar sucrose solutions in vials with caps. These were stored

at-23°C in deep freezer.

3.12 Statistical analysis of data

Statistically, significant means were separated using Tukeys (a = 0.05). For

comparison with control antibiotics and the standard Staphylococcus aureus

strain, T - test statistics and Anova (a = 0.05) were worked out and the

(45)

CHAPTER FOUR

RESULTS

4.1 Frequency and load ofStaphylococcus aureus in wounds

Staphylococcus aureus bacteria were isolated in 88.04% of sampled specimens.

In 2.17 ± 0.3% of culture plates, Staphylococcus aureus were present in low

bacterial load represented by quadrant streak level of I+ while 3.26 ± 0.3% and

10.86 ± 0.4 % of the culture plates had moderate bacterial load of 2+ and 3+

quadrant streak level respectively. A high bacterial load of 4+ quadrant streak

level was observed in 71.75 ± 0.5% of the clinical specimens that tested

positive forStaphylococcus aureus (Table 4.1).

Table 4.1:Staphylococcus aureus streak plate levels and CFU counts (n

=

81)

Percentage of number of plates with Quadrant CFU/g tissue biopsy

Staphylococcus aureus isolates level correlation

2.17 ± 0.3 % 1+ 102to 103

3.26 ± 0.3 % 2+ 102to 103

10.86 ± 0.4 % 3+ 102to 103

71.75±0.5% 4+ 105tol07

1+ - Presence of bacteria in the first quadrant; 2+ - Presence of bacteria in the first two quadrants; 3+ - Presence of bacteria in the first three quadrants; 4+ -Presence of bacteria in the all the four quadrants.

4.2 Phytochemicals present in honey

Alkaloids, Flavonoids, Saponins, Sugars and Terpenes were detected in all

honey samples (Table 4.2). Mount Kenya and Kitui honey had the highest

number of Alkaloids, 4 and 5 and Flavonoids, 5 and 6 analytes/fractions

respectively which were significantly (p < 0.05) higher than the rest of the

(46)

all honey samples had I fraction of each. In all the six honey samples, there

was no formation of distinct sugar fractions (Table 4.2).

Table 4.2: Phytochemical fractions (n

=

3)

Honey samples Number of fractions in honey samples

alkaloids flavonoids saponins terpenes sugars

Mount Kenya 4 5 1 1 No distinct fractions

Lake 01- No distinct fractions

bolossat

Kitui 5 6 No distinct fractions

Canan No distinct fractions

Sues No distinct fractions

(47)

Table 4.3: Rr values for phytochemical fractions in their elution order indicating molecular variation (n =3)

Honey samples Rr values

Phytochem icals

Mount flavonoids 0.40 0.55 0.66 0.72 0.77

-Kenya alkaloids 0.20 0.35 0.55 0.61

terpenes 0.25

-saponins 0.45

-Lake 01- flavonoids 0.30

-bolossat alkaloids 0.10

-terpenes 0.22

-saponins 0.46

Kitui flavonoids 0.40 0.55 0.61 0.66 0.77 0.80

alkaloids 0.20 0.38 0.5\ 0.61 0.69

-terpenes 0.28

-saponms 0.45

Canan flavonoids 0.30

-alkaloids 0.10

-terpenes 0.33

-saponins 0.45

-Sues flavonoids 0.30

-alkaloids 0.10

-terpenes 0.24

-saponms 0.45

-Peptang flavonoids 0.30

-alkaloids 0.\0

-terpenes 0.23

-saponins 0.45

-Based on retention factors (Rr)which is an indication of variation in molecular

(48)

the same type of flavonoid and alkaloid analytes while Mt. Kenya and Kitui

honey samples had a common alkaloid fraction type as well as 4 same

flavonoids fractions types (Table 4.3).

4.3 Contact bioautography

Flavonoids and alkaloids from all the six honey samples used in the study

against the 81 clinical isolates of Staphylococcus aureus demonstrated

significant (p < 0.05) antibacterial activity by revealing visible zones of

inhibition (Table 4.4). The mean zones of inhibition of both the flavonoids and

alkaloids were not significantly (p >0.05) different.

Table 4.4: Mean zones of inhibition (mm) formed by flavonoids and

alkaloids analytes against Staphylococcus aureus clinical isolates (n

=

81)

Honey samples Zones of Zones of

inhibition for inhibition for

alkaloids flavonoids

Mount Kenya 8.7 ± 0.2 a 9.1 ± 0.2 a

Lake 01- bolossat 8.6±0.3 a 9.0 ±0.3 a

Kitui 8.6 ± 0.3 a 9.1 ± 0.1 a

Canan 8.7 ±0.4 a 9.0 ±0.3 a

Sues 8.7 ± 0.2 a 9.0 ± 0.3 a

Peptang 8.7±0.2 a 9.0 ±0.2 a

Means within a column and within the same row followed by the same letter are not significantly different (p ::::0.05)

The mean zones of inhibition formed by the control antibiotics, alkaloids and

flavonoids against Staphylococcus aureus ATCC 25923 and 81 clinical isolates

(49)

Table 4.5: Mean zones of inhibition (mm) formed by alkaloids, flavonoids and the control antibiotics (penicillin 10 IV (455 Jig),vancomycin (30Jig) against the Staphylococcus aureus isolates (n

=

81) and Staphylococcus

aureus ATCC 25923(n

=

3) ,

Staphylococcus Zones of inhibition

aureus strains alkaloids tlavonoids penicillin vancomycin Staphylococcus 9.0 ± 0.3 b 9.1±0.2b 6.0 ± 0.2 a 12.0± 0.03 c

aureus ATee 25923

Staphylococcus 8.6 ± 0.3 b 9.0 ± 0.2 b 6.0 ± 0.2 a 12.0± 0.01 c aureus isolates

Means within a column and within the same row followed by the same letter are not significantly different (p 2: 0.05)

Both the tlavonoids and alkaloids had significantly (p <0.05) larger zones of inhibition than penicillin but significantly (p <0.05) smaller than vancomycin disks (Table 4.5).

Staphylococcus aureus ATee 25923 strain (Table 4.5) and the Staphylococcus

aureus clinical isolates did not have significantly (p > 0.05) different zones of inhibition from tlavonoids, alkaloids and the control antibiotics namely

penicillin and vancomycin.

4.4 In vitro antibacterial activity of honey at different honey concentrations using disk diffusion method

At 20%, 40%, 60% and 80% honey concentrations, the zones of inhibition for all the honey samples under investigation were not significantly different (p >

(50)

Canan, Lake Ol-bolosatt basin, Sues and Peptang commercial honeys in their

respective order (Table 4.6).

Table 4.6: Variation in zones of inhibition (mm) with increase in honey

concentration (%v/v) usingStaphylococcus aureus isolates (n

=

81).

Zone of inhibition at different concentrations

Honey samples 20% 40% 60% 80%

Mount Kenya 9.06 ± 0.1 a 9.60 ± 0.1 a 10.42 ± 0.1 a IIJ8 ± OJ b Lake Ol-bolosatt 9.0 I± 0.1 a 9.50 ± 0.1 a 10.23 ± 0.2 a 11.21± 0.2 b Kitui 9.04 ± 0.2 a 9.80 ± OJ a 10.48 ± 0.1 a 11.51± 0.3 b

Canan 9.02±0.2a 9.70±0.2a 10.44±0.2a 11.58±0.2b

Sues 8.84 ± 0.1 a 9.34 ± 0.1 a 9.98 ± 0.3 a 10.94 ± 0.3 b

Peptang 8.78 ± 0.1 a 9.30 ± 0.2 a 9.96 ± 0.2 a 10.82 ± 0.2 b

Ranges within a column and within the same row followed by the same letter

are not significantly different (p ::::0.05)

At 40% honey concentration level, the honey sample with the largest zones of

inhibition was that from Kitui followed by Canan honey sample then Mt.

Kenya honey sample. Honey sample from Lake Ol-bolosatt was next followed

by other honey samples namely Sues and Peptang respectively (Table 4.6).

At 60% honey concentration level, the most effective honey samples were the

Kitui honey sample then Canan honey sample followed by honey sample from

Mt. Kenya forest. Honey sample obtained from Lake Ol-bolosatt was next

followed by Sues honey sample and Peptang in that order (Table 4.6).

At 80% honey concentration level, Canan commercial honey sample had the

largest zones of inhibition followed by the Kitui honey sample. These were

(51)

and Peptang commercial honey samples had the smallest zones of inhibition in

their respective order (Table 4.6).

For clinical isolates and the standard strain (Table 4.7), the only significant

(p < 0.05) difference in zones of inhibition was between 80% concentration

level and the 20%,40% and 60% honey concentrations.

Table 4.7: Variation in zones of inhibition (mm) with increase in honey concentration (%v/v) using Staphylococcus aureus ATCC 25923 (n = 3).

Honey Zone of inhibition at different concentrations

samples 20% 40% 60% 80%

Mount Kenya 9.66 ± 0.1 a 9.70 ± 0.1 a 10.56 ± 0.1 a 11.40 ± 0.3 b

L.OI-bolosatt 9.30 ± 0.1 a 9.60 ± 0.2 a 10.30 ± 0.2 a 11.20 ± 0.3 b

Kitui 9.40 ± 0.2 a 9.80 ± 0.3 a 10.60 ± 0.1 a 11.55 ± 0.3 b

Canan 9.03 ± 0.2 a 9.40 ± 0.2 a 10.62 ± 0.2 a 11.60 ± 0.2 b

Sues 8.80 ± 0.1 a 9.38 ± 0.1 a 9.94 ± 0.3 a 10.78 ± 0.2 b

Peptang 8.80 ± 0.1 a 9.23 ± 0.2 a 9.90 ± 0.2 a 10.72 ± 0.4 b

Ranges within a column and within the same row followed by the same letter are not significantly different (p ~ 0.05)

In summary, the in vitro susceptibility test showed that the growth of all

isolates of Staphylococcus aureus were inhibited to varying degree by the

honey samples under investigation. There was notable increase in the in vitro

antibacterial activity for all six honey samples with increase in honey

concentration.

4.5 Minimum Inhibition Concentration (MIC) for the honey samples

The results of MIC showed that honey from Kitui and Mt. Kenya had lower

MIC while the lake Ol-bolossat as well as the commercial honey samples had

(52)

were not significantly (p > 0.05) different but were significantly (p < 0.05)

lower than those of Lake Ol-bolosatt, Peptang and Canan honey samples

(Table 4.8). There was no significant variation (p > 0.05) in MIC levels

between Canan Sues and Peptang honey samples.

Table 4.8: MIC using honey samples against Staphylococcus aureus isolates (concentration in % v/v, n

=

81)

Honey samples MIC

Mount Kenya Lake Ol-bolosatt Kitui

Sues Peptang Canan

6.0± 0.2 a 8.0 ±0.1 b 6.0 ±0.3 a 7.5± 0.5 b 7.5±0.3 b 8.0 ±0.4 b

Ranges within a column followed by the same letter (s) are not significantly different (p 2: 0.05)

4.6 Minimum Bactericidal Concentration (MBC) for the honey samples

Honey from Mt. Kenya forest and Kitui had the lowest MBC levels that were

significantly (p <0.05) different from those of the other honey samples in the

study. The Lake Ol-bolosatt Canan Sues and Peptang had the highest MBC

levels that were not significantly (p > 0.05) different. The level of MBC

between Mt. Kenya forest and Kitui honey samples were not significantly

(p > 0.05) different and similarly between Lake 01- bolosatt and the

(53)

Table 4.9: MBC using honey samples against Staphylococcus aureus isolates (concentration in %vlv, n

=

81)

Honey samples MBC

Mount Kenya Lake 01bolosatt Kitui

Sues Peptang Canan

12.0±0.3 c 16.0±0.3 d 12.0±0.2 c 16.0 ±0.2 d 16.0 ±0.2 d 16.0 ±0.5 d

Ranges within a column followed by the same letter (s) are not significantly different (p ::::0.05)

4.7 MIC and MBC for control antibiotics

The MIC and MBC results are represented in table 4.10 and table 4.11

respectively. Both the MIC and MBC for Penicillin were significantly (p < 0.05) higher than those of Vancomycin in both Staphylococcus aureus ATCC

25923 and the Staphylococcus aureus isolates.

Table 4.10: The MIC ranges using control antibiotics

(Staphylococcus aureus isolates n=81,Staphylococcus aureus ATCC 25923 n =3)

Staphylococcus MIC(~g/ml)

aureus

Penicillin Vancomycin

Staphylococcus 16.0 ±OJ c 0.5 ±0.01 a

aureus isolates

Staphylococcus 8.0±0.1 b 0.5 ±0.01 a

aureus ATCC

25923

Figure

Figure 3.1: Systematic
Figure 3.1: Systematic random sampling using the number line
Table 4.2: Phytochemical
Table 4.5: Mean zones of inhibition (mm) formed by alkaloids, flavonoidsand the control antibiotics(penicillin 10 IV (455 Jig), vancomycin (30Jig)againstthe Staphylococcusaureus isolates (n = 81) and Staphylococcusaureus ATCC 25923 (n = 3),
+5

References

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