ABSTRACT
This work investigates the antimicrobial activity of honey on Staphylococcus aureus and other bacterial pathogens associated with wound infection. The samples collected from patients with various kinds of wound at the Federal Medical Center (FMC), Umuahia, were inoculated at specific agar media that supported the growth of the pathogens associated with wound infection. Honey type sensitivity agar media prepared at decreasing concentration of 100%, 80%, 60%(control), 40%, 20%, 10% and 5%, containing the isolates as the inocula were incubated at 37oc for 24 hours and the Minimum Inhibitory Concentration (MIC) at the honey concentrations of interest were measured. The MIC was observed to decrease with decrease in concentration of honey. This implies that honey has some measurable level of antimicrobial activity on Staphylococcus aureus and other bacterial pathogens associated with wound.
TABLE OF CONTENTS
Title
page i
Certification
ii
Dedication iii
Acknowledgement iv
Table of
contents v
List of
Tables ix
List of
Figures x
Abstract. xi
CHAPTER ONE:
INTRODUCTION 1
1.1 Historical
Background 2
1.2 Problem
Statement. 4
1.3 Purpose
of the Study. 4
1.4 Specific
Objectives. 4
1.5 Scope
and Limitation of the Study. 5
1.6 Significance
and Relevance of the Study. 5
CHAPTER TWO: LITERATURE REVIEW.
2.1.1 Description
of wound. 6
2.1.2
Classification of wound. 7
2.1.3
Wound infection and stages. 11
2.1.4
Wound healing. 11
2.1.5
Wound
infection and prevention. 16
2.1.6
Description of Staphylococcus
aureus. 17
2.1.7
Classification of Staphylococcus
aureus. 17
2.1.8
Morphology of Staphylococcus aureus 18
2.1.9 Clinical
significance of Staphylococcus aureus. 18
2.2.1 Medical
laboratory diagnosis of Staphylococcus
aureus. 20
2.2.2 Natural
habitat/epidemiology of Staphylococcus
aureus. 23
2.2.3 Pathogenesis
of Staphylococcus aureus. 23
2.2.4
Antimicrobial resistance of S.
aureus. 28
2.2.5
Honey: the medicinal property and antibacterial activity. 30
2.2.6.
Medicinal property of honey. 31
2.2.7
Antibacterial activity of honey - Potential antibacterial activity. 32
2.2.8
Mechanism of antibacterial activity. 34
2.2.9 Factors affecting antibacterial nature of
honey. 35
CHAPTER THREE: MATERIALS AND METHODS.
3.1.1 Study area and time. 36
3.1.2 Study population. 36
3.1.3 Sample collection. 36
3.1.4
Sample collection method. 36
3.1.5
Isolation of the bacterial strain and growth condition. 37
3.1.6 Morphological characterization and
identification. 37
3.1.7 Biochemical characterization and
identification. 38
3.1.7.1 Catalase Test. 38
3.1.7.2 Coagulase Test. 38
3.1.7.3 Citrate Utilization Test. 38
3.1.7.4 Indole Production Test. 39
3.1.7.5 Oxidase Test. 39
3.1.7.6 Motility Test. 39
3.1.7.7 Methyl Red/Voges Proskauer Test. 39
3.1.7.8 Urease Test. 40
3.1.7.9 Hydrogen Sulphide Test. 40
3.1.8 Carbohydrate Utilization Test. 40
3.2.1 Percentage dilution of the antimicrobial
agent and preparation
of the colony suspension. 41
3.2.2
Culture and sensitivity. 42
CHAPTER FOUR: RESULT AND DISCUSSION.
RESULT.
4.1.1
Identification of staphylococcus
aureus and other bacterial
pathogens isolated from wound. 43
4.1.2 Antimicrobial
activity of honey on the pathogens. 44
4.1.3 Minimum
inhibitory concentration (MIC) of the
honey
on the organisms. 44
4.2.1
Identification of staphylococcus
aureus and other bacterial
pathogens isolated from wound. 45
4.2.2 Antimicrobial
activity of honey on the pathogens. 45
4.2.3 Minimum
inhibitory concentration (MIC)
of the honey on the organisms. 45
CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS.
5.1 Summary of achievement. 46
5.2 Problems encountered and solutions. 46
5.3 Conclusion. 46
5.4 Recommendation. 47
REFERENCES
APPENDIX
LIST OF TABLES
Table 1: Shows
the identification of the isolated test organisms. 43
Table 2: Shows
inhibition zones (in milimetre, mm) of the honey
on the organisms. 44
Table 3: Shows
the MICs of the honey on the test organisms. 44
LIST OF FIGURES
Fig
1: Laceration;
an open wound. 9
Fig 2: Burn;
an open wound. 9
Fig 3: Ulceration;
an open wound. 10
Fig 4: Superficial
wound (SSSS). 10
CHAPTER
ONE
INTRODUCTION
There
has been a quest for good health and the organisms that shift people from a
normal state of health to dereliction have been in great surge. Most of these
organisms are microscopic (microorganisms) or macroscopic (macroorganisms). The
ones that are microscopic can be living (bacteria and fungi) or non-living
(viruses, prions and viriods). Microorganisms are ubiquitous. This ubiquity has
led to colonization of our physical environment, internal and external
environment of our body. Most of this colonization is beneficial while others
are harmful. As many factors are always ready to predispose us to infection
caused by these microorganisms, there has always been a great demand of agents
or substances which can be used to kill or stop the harmful effect of these
microorganisms. Such agents or substances are referred to as antimicrobials.
Antimicrobials are chemo therapeutic agents that are effective in treating
microbial infections (Nester et al.,
2007). The antimicrobials were discovered from plant extracts, dyes and other
substances. Before the synthesis and discovery of these novel antimicrobials,
ancient physicians had a way of taking care of the ill and infirmed amongst
them. Antimicrobials have specificity of action (Prescott et al., 2013). Some are administered in vivo while others are
administered in vitro. The ones administered in vitro are usually applied to
the wound. A wound is a type of injury which happens relatively quickly in
which skin is torn, cut or punctured (an open wound), or where blunt force or
trauma causes a contusion (a closed wound). A wound can also be referred to as
a sharp injury which damages the epidermis of the skin. Open wounds are
predisposed to colonization by microorganisms. There are many microorganisms that
can colonize and infect the wound, notably amongst them is Staphylococcus aureus (Murray et
al., 2005). Staphylococcus aureus
is a Gram positive coccus (Ryan and Ray, 2004). The genus Staphylococcus is
composed of 33 species (Bergey and Holt, 1994). Most Staphylococci constitute
the normal flora of the skin and mucus membrane (Madigan, 2005). Some are
aerobic while others are anaerobic and can grow at high salt concentrations,
reaching up to 10% (Murray et al.,
2005). Catalase positive and resides harmlessly as a normal flora of the skin.
This bacterium gets into the abraded skin tissues and causes toxic shock
syndrome; wound infections with high fever, muscle aches, life threatening
shock and pyogenic discharges.
Antimicrobial agents can be grouped according to the
microorganisms they act primarily against. In instance, antibiotics are used
against bacteria and antifungal are used against fungi. Microbicidals kill
microbes while biostatic inhibits growth. The main classes of antimicrobial
agents are disinfectants (“non selective antimicrobials” such as bleach), which
kill a wide range of microbes on non-living surfaces to prevent the spread of
diseases, antiseptics (which are applied to living tissue and help reduce
infection during surgery) and antibiotics (which destroys microorganisms within
the body). Centuries ago, before the synthesis of these antimicrobials, the
ancient physicians administered honey in vivo and in vitro. When administered
in vitro, it was mostly applied to wounds. Wounds can be open or closed. The
open wound are classified into incised wound (caused by sharp edged object),
lacerated (irregular tear-like wounds caused by some blunt objects or trauma),
puncture (caused by an object with sharp-pointed mouth such as a nail entering
and coming out from the skin). If wounds are not properly cleaned or dressed,
microorganisms begin to colonize and proliferate in/on the wound causing sepsis
and putrefaction of the tissue which leads to purulent discharge.
Antimicrobials are used to stop the colonization,
proliferation of the microorganisms associated with wound.
1.1 Historical Background.
Early days, the most administered antimicrobials were
mostly natural. The important natural antimicrobial in question to this work is
honey. Honey is an ancient remedy for treatment of infected wounds and has been
‘rediscovered’ by the medical profession, particularly where conventional
(antibiotics and antiseptics) modern therapeutic agents fail. The first written
reference to honey, a Sumerian tablet
writing, dating 2100-2000 BC, mentions honey’s use as a drug and ointment.
Aristotle (384-322BC); when discussing different honey referred pale honey as
being “good as a salve for sore eyes and wounds”. Manuka honey has been
reported to exhibit antimicrobial activity against pathogenic bacteria such as Staphylococcus aureus and Helicobacter pylori, making this honey a
functional food for the treatment of wounds or stomach ulcers. In most ancient
cultures, honey has been used for both nutritional and medical purposes. The
belief that honey is a nutrient, a drug an ointment has been carried into our
days, and thus, an alternative medicine branch, called apitherapy has been
developed in recent years offering treatments based on honey and other bee
products against many diseases including bacterial infections. The notably amongst them is honey. Honey
produced by Apis mellifera, is a viscous, sweet substance made by bees and some
related insects in bee family (Wikipedia). Bees produce honey from sugary
secretions of plants (floral nectar) or from secretions of other insects (such
as honeydew), by regurgitation, enzymatic activity and water evaporation. Honey
gets its sweetness from the monosaccharide fructose and glucose, and has about
the same relative sweetness as granulated sugar. Honey is a popular folk
treatment for burns and other skin injuries. Preliminary evidence is that it
aids in healing of burns 4-5 day faster than other dressings, and moderate
evidence suggests that post-operative infections treated with honey heals
faster and has fewer adverse effect than with antiseptics or gauze. The healing
properties of honey can be ascribed to the fact that it offers antibacterial
activity, maintains a moist wound environment that healing, and has a high
viscosity which helps to provide a protective barrier to prevent infection.
Honey is composed of approximately 82.4% total carbohydrates (38.5% fructose,
31.0%glucose and 12.9% form of carbohydrates including maltose, sucrose, and
other sugars.) (Khan et al., 2007,
Vallianou et al., 2014). The natural
ingredients of honey show different activities against various microorganisms.
Its activity is likely to be dependent on the grazing grounds and the weather
conditions where the bees are raised, and on the natural structure of the
blossom nectar (Abd-El Aal et al.,
2007). Honey has increasing effect on levels of antioxidants, iron and rare
elements in blood (Theunissen et al.,
2001).
Abd-El Aal et al.
(2007) showed that honey had a more pronounced inhibitory effect (85.7%) on Gram
negative bacteria (Pseudomonas aeruginosa,
Enterobacter, Klebsiella) in comparism to commonly used antimicrobial agents.
Honey has great antimicrobial activity on Staphylococcus species which are
methicillin resistant or sensitive. Honey applied with other antimicrobial
agents achieves a synergistic effect in both Gram positive and Gram negative
bacteria (Alsomal et al., 1994).
Honey has more than 181 constituents (Bogdanov and Martin, 2002, Gheldon et al., 2002, Mandal, 2011, Vallianou et al., 2014). Components of honey under
preliminary research for their potential antibacterial properties include:
methylglyoxal, hydrogen peroxide and defensin-1.
The isolation of Staphylococcus
aureus from wound and its culture and application to determine the Minimum
Inhibitory Concentration (MIC) of honey on the bacterium, which can then be
used as an effective antimicrobial agent
used for regaining the normal state of health.
1.2 Problem Statement.
Wound infections are associated with increased
morbidity and mortality. The prevalence of wound infection in 2006, 2007, 2008,
2009 and 2010 was 71.4%, 76.2%, 74.5%, 61.5%, 67.0% respectively. The overall
prevalence of wound was 70.1% in all the years studied, according to African
Health Services. Sequel to this, the research conducted by this health
organization revealed that Staphylococcus
aureus was the prevalent pathogen in the patients used as the guinea pig.
Most of the patients I was opportune to meet during the sample collection
reported of chronic wound infection which most of the antimicrobial agents
administered in vitro were not able to heal. We suspected that the case was due
to the inability of those antimicrobials to penetrate the purulent wound so as
allow granulation tissue formation and epithelialization. Many people that had
minor skin surgeries on/in the skin, that later had wound infection came to be
a problem as most of these wounds weren’t susceptible to most synthetic
antimicrobials used on them. The death case of patient with wound infections
caused by; Clostridium perfringees,
Psuedomonas aeruginosa, or Staphylococcus
aureus, has statistically increased.
1.3 Purpose of the Study.
The sole aim of
this work is to determine the antimicrobial activity of honey on Staphylococcus aureus and other bacterial pathogens associated with wound
infection.
1.4 Specific Objectives.
·
To determine the antimicrobial
activity of honey on Staphylococcus aureus and other bacterial pathogens associated
with wound infection.
·
To compare the
antimicrobial activity of honey with other synthetic antimicrobial agents.
·
To determine the
antimicrobial activity of different dilutions of honey on antimicrobial
activity of honey on Staphylococcus aureus and other bacterial pathogens associated
with wound infection
1.5 Scope and Limitation of the Study.
There are natural substances that confer immunity to
the host against foreign substances from microorganisms. Some are plant
extracts while others are from organisms which can be microscopic or
macroscopic. But this work is limited to
the antimicrobial activity of honey on killing or inhibiting the growth of
pathogenic microorganisms in the wound, notably amongst them is antimicrobial
activity of honey on bacteria associated with wound infection.
1.6 Significance and Relevance of the Study.
The importance
of honey in treating infection cannot be belittled. Honey has been used
throughout the ages and has recently been reintroduced to modern medical
practice. Laboratory studies and clinical trials have shown that honey is an
effective broad-spectrum antibacterial agent. Honey antimicrobial action
explains the internal and external use of honey. Honey has been used to treat
adult and neonatal postoperative infection, burns, necrotizing fasciitis,
infected and non healing wounds and ulcers, boils, sepsis, venous ulcers, and
diabetic foot ulcers. These effects are ascribed to honey’s antimicrobial
action, which is due to its acidity, hydrogen peroxide content, osmotic effect,
nutritional and antioxidants content and stimulation of immunity. As the
importance of honey to health is up-surging, this work is carried out so as to
provide an in-depth knowledge to everyone but four-square to those that may
find it grim to get most synthetic antimicrobials, owing to their lack of knowledge
to those synthetic antimicrobials or their lack of money to get access to them.
Also, this work is carried to evaluate the importance of most natural product precisely
honey, to our health.
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