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
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
DEDICATION
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
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
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
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
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
LIST OF FIGURES
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
UV WDF WHO
Ultra Violet
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
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
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
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
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
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
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
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
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
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
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
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
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;
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
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
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-PatientsKEY:
: Represents sampling interval range:
U :
Represents sampled patientsFigure 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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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 Staphylococcusaureus 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 >
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
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
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
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