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  • Name: ANTIBACTERIAL ACTIVITY OF HONEY ON Staphylococcus aureusEscherichia coli and Streptococcus pyogen ISOLATED FROM WOUND
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Title Page i
Certification Page ii
Dedication iii
Acknowledgement iv
Table of Content v
List of tables viii
Abstract x
1.0 Introduction 1
1.1 Aims and objectives 3
2.0 Literature review 5

2.1 Wound infection 8
2.2 Definition of honey 9
2.3 Local test for honey 13
2.4 Classification of honey 14
2.5 Preservation of honey 16
2.6 Properties and active ingredient of honey 17
2.7 Mode of action of some antibacterial substance
present in honey 20
2.8 Clinical conditions for treatment with honey 22
2.9 Honey as an antimicrobial agent 24
2.10 Practical consideration for the clinical use of honey 27
2.11 Adverse reaction of honey 28
2.12 Research on honey 29

3.1 Source of Sample 32
3.2 Sources of honey 32
3.3 Identification of organisms 32
3.4 Gram Staining 33
3.5 Indole test 34
3.6 Catalase test 34
3.7 Coagulase test 35
3.8 Antibacterial sensitivity test 35
4.1 Result 37


5.1 Discussion 40
5.2 Conclusion 42
5.3 Recommendation 43
Appendix 1 51
Appendix 2 54


1.0 Introduction
Infections and other health related problems have been of great
concern to human beings and chemotherapy is the main approach in
the treatment of such conditions. Investigation into the microbial
flora of wound began in the late 19th century and since then;
improvements in techniques have facilitated the recovery,
identification and enumeration of a wide variety of microbial species.
Most wounds support relatively stable polymicrobial communities
(Bowkler, et.al; 2001) often without signs of clinical infection
(Hansson,et al; 1993).
However, potential pathogens may be present and the delicate
balance between colonized wound and an infected wound depends on
the interplay of complex host and microbial influences (Emmerson,
1998). The development of wound infection has deleterious effect on

patients by causing increased pain, discomfort, inconveniences and
can lead to life threatening conditions or even death.
Major challenges encountered with antibiotics in clinical use are
resistance to antibiotics which leads eventually to failure of the
treatment (Blair 2004). Infectious diseases are known to be treated
with herbal remedies throughout the history of mankind; even today,
natural substances continue to play a major role in primary health
care as therapeutic remedies in many developing countries (Jonathan,
et.al; 2007). Over the years, there have been reports of the production
of more potent antibiotics e.g. third and fourth generation of
cephalosporin by pharmaceutical companies which are not readily
available and expensive. Problems of various antibiotics include low
efficacy, side effect which has lead investigations into natural and
potent antibacterial seeming to be the right step to take. The invasion
of pathogenic organism is on the rise as a result, effects are been
made to develop antibacterial agent from natural sources for better

therapeutic effect (Gills, 1992). The therapies have drawn the interest
of both public and medicinal communities. Current research has been
focused on herbal and aromatherapy product. However, a number of
their product such as honey has shown therapeutic promise.
The presence in honey of various inhibins as described by (Doid and
Dzaio, 1937) has been reported by several investigators. Honey was
used to treat infected wound as long as 2000 years ago before
bacterial were discovered to be the cause of infection in c.50 AD,
Dioscorides described honey as been “good for all rotten and hollow
ulcers” (Gunther, 1959). More recently, honey has been reported to
have an inhibitory effect to around 60 species of bacterial including
aerobes and anaerobes, Gram positive and Gram negative (Molan,
1992). The current prevalence of the therapeutic use of ancient
remedies, include honey committee on science and technology.
1.1 Aims and objectives.
1. To determine antibacterial potential of honey.

2. To investigate the mechanism of antibacterial action of honey.
3. To determine the minimum inhibitory concentration of honey on
bacterial isolates from wounds of human beings.
4. To yield additional knowledge such as the possible dilution of
honey sample and activity of the honey sample in bacterial


2.0 Literature Review
The medicinal properties of honey have been reported and
documented by beekeepers and medical practitioners (Bankova, et.al;
2002). As a result of over use and abuse of antibiotics, there have
been increases in the number of diseases, which seem to evolve to
become more virulent with each generation. Investigations into
natural and potent antimicrobials seemed to be the right step to take.
The invasion of pathogenic organism is on the rise and as a result,
effects are being made to develop antimicrobial agents from natural
sources for better therapeutic effect (Gills, 1992). The therapies have
drawn the interest of both public and medical communities. Current
research has been focused on herbal and aromatherapy products.
Antimicrobial agents have been applied to wound for thousands of
years (Moellering, 1995) but many remedies have been discontinued
because the evidence to support their efficacy was anecdotal.

Continued use of systemic and topical antimicrobial agents has
provided the selective pressure that has lead to the emergence of
antibiotics-resistance strains which, in turn, has driven the continued
search for new agents. Unfortunately, the increased costs of searching
for such agents and the decreasing rate of their discovery
(Moellering, 1995) has made the situation increasing urgent and the
prevalence of antibiotics-resistant microbial species now justifies the
re-evaluation of former treatment (Anon, 1998). Honey has been used
as a medicine since ancient times in many cultures and is still used in
„folk medicine‟. The use of honey as a therapeutic substance has been
rediscovered by the medical profession in more recent times, and it is
gaining acceptance as an antibacterial agent for the treatment of
ulcers and bed sores, and other infections resulting from burns and
wound. In many of the cases in the cited reports, honey was used on
infections not responding to standard effective in rapidly clearing up
infection and promoting healing. Honey has also been found to be
effective in treating bacterial gastroenteritis in infants.

The medicinal use of honey in wound treatment is derived from
diverse ancient civilizations (Jones, 2001). The antibacterial
properties of honey were recognized more than a century ago and
have subsequently been extensively studied (Molan, 1992a, 1992b).
A wide range of microbial species has been shown to be inhibited by
honey but reported susceptibilities are not consistent. Failure to
identify the botanical sources of honeys used in many of those
studies, or to determine their antibacterial potency, makes
comparison of reported sensitivities unreliable. It is remarkable that
ancient physicians were selective in the honeys that they utilized in
their remedies (Jones, 2001), although the underlying principles
would have been obscure. Now it is possible to determine
quantitatively the antimicrobial activity of a honey (Allen, et. al;
1991) and also to discriminate between honeys whose mode of action
involves factors beyond their osmolarity in limiting bacterial growth
(Allen, et.al; 1991). In most honeys this depends on the enzymic
generation of hydrogen peroxide to varying degrees (Molan, 1992a)

but, in some honeys, there are additional phytochemical antibacterial
factors (Molan, 1992a). In recent studies, the susceptibility of wound
pathogens (Willix, et. al; 1992) and bacterial isolated from wound
(Cooper and Molan 1999; Cooper et. al; 1999) to honeys of known
floral source and defined antibacterial activity has been reported.
However, the inhibition f antibiotic-resistant bacteria by honey has
not been fully explored .using characterized honey, this study aims to
extend the range of wound pathogens whose susceptibility to honey
has been determined and to compare the susceptibility of antibiotic
sensitivity strains with those of antibiotics-resistant strains.
2.1 Wound infection
The moist environment of chronic wounds is an ideal growth medium
for bacteria (O‟Meara et. al; 2010) and infection is the prominent
cause of delayed healing. This has become an increasing problem
with the recent expansion of antibiotic-resistant bacteria (Leaper,
2006). Burns and chronic wound are particularly prone to infection

with 075% of dirty following burns involving infection (Thorn et. al;
2006). There is increasing interest in the use of topical antimicrobial
to wound cure. Compounds such as honey, iodine and silver have
been incorporated into dressings are simply the addition of an
antimicrobial to a pre-existing product (Thomas, 2003). Many in vitro
investigations into the efficacy of such product have been performed:
however there is a confusing mixture of evidence and differences in
research methodologies making interpretation and comparison of
results difficult. In addition, they are very few randomized controlled
trials comparing wound care product in clinical practice. A wound
may be defined as a breech in the epidermis or dermis due to trauma
or physiological change, activating the repair process (Benbow,
2005) wounds can be classified as either acute or chronic.
2.2 honey
Honey is the sweet substance made by bees using nectar from
flowers. Honey is made when the nectar and deposit from plant are

gathered, modified and stored in the honey comb by honey bees
(National Honey Board, 1996). The definition of honey stipulates a
pure product that does not allow for the addition of any other
substance such as water or other sweeteners. The flower from which
bees gather nectar largely determines the colour, flavor and aroma of
honey (Caron, 2004). Honey is also said to be highly variable like
must plant derived product and the chemical composition of honey
also depends on the flower from which it is made. Antibacterial effect
may therefore vary between different types of honey (Ovington,
1999). Honey is classified by the flower source and therefore is
divided according to their package and processing. Generally honey
is classified by the flora source of the nectar from which it was made.
Honey can be from specific types of flower nectar indeterminate
origin or can be blended after collection.
Honey originally used by the ancient Egyptians and Greeks, is a
viscous, saturated sugar solution now widely used in wound care

(Simon et. al; 2009). High osmolarity prevents the growth of bacteria
and encourages healing. This can be utilized for wound management
through the application of sugar paste or honey. In addition, honey is
believed to have specific antimicrobial properties, for example,
preventing the growth of staphylococcus aureus even when diluted
beyond the point at which osmolarity is no longer inhibitory (Moore
et. al; 2001). Studies have reported that it may modestly decrease
wound healing time, act as an anti-inflammatory, deodorize wounds,
and enhance cell proliferation and expansion in in-vitro (Du Tort,
2009). Studies in support of its use indicate that honey-based
treatment is preferential to silver or iodine, due to its comparative
lack of toxicity.
The medicinal properties of honey have been known since ancient
times. Indian medicines described honey as the nectar of life and
recommend it in various ailments. There is a renewed interest in
honey treatment as evidenced by the number of reputes appearing in

the scientific literature. Honey has useful on the treatment of surgical
wounds, burns; decubitus ulcer and the antibacterial and antifungal
properties of honey have been well documented. In burn in particular,
honey has been found to control wound infection and accelerate
wound healing. Honey bees transform nectar into honey by a process
of regurgitation and store it as a primary food source in wax
honeycombs inside the beehive. Beekeeping practices encourage over
production of honey so the excess can be taken from the colony.
Honey gets it sweetness from the monosaccharide fructose and
glucose and has approximately the same relative sweetness as that of
granulated sugar. It has attractive chemical properties for baking, and
a distinctive flavor that lead some people to prefer it over the sugar
and other sweetness. Most microorganisms do not grow in honey
because of its low water activities of 0.6. However, honey
sometimes contained dormant endospores of the bacterium
clostridium botulinum which can be dangerous to infants as the

endospores can transform into toxin-producing bacteria in the infant‟s
immature intestinal tract, leading to illness and even death. Honey is
also used in various medicinal traditions to treat ailments.
Honey is often eaten as an energy food (Caron, 2004) and yield about
64 centuries of energy which is high compared to other sweeteners
(National Honey Board, 1996) its simple sugar is absorbed into the
blood stream with digestion. The optimum storage temperature for
honey is below 520F (110C) or in the 70-300 range (21-270C) in an
air tight container (National Honey Board, 1996).
2.3 local test for real honeys
Honey can be adulterated by adding sugarcane syrup and other
ingredient which can be processed to give the colour and near texture
of honey. The aroma of the pure honey is not the same with that of
the adulterated one and the pure honey does not leave sediments
when diluted in water unless it was not properly filtered, although the
originality of honey can be tested using honey stained match stick

which brings light immediately when struck while the match stick
stained with adulterated honey does not bring light when struck.
2.4 Classification of honey
Crystallized honey: Is honey in which some of the glucose content
has spontaneously crystallized from solution as the monohydration.
Also called granulated honey” or candied honey” honey that has
crystallized can be returned to a liquid state by warming.
Pasteurized Honey: Is honey that has been heated in a pasteurized
process (161of(71.7oc) or higher). Pasteurization destroys yeast cells.
It also liquefies any microcrystals in the honey, which delays the
onset of visible crystallization. However, excessive heat exposure
also results in product deterioration as it increases the level of
hydroxymethylfurfur and reduces enzyme (e.g diastase) activity. Heat
also affects appearance.

Raw honey: Is honey as it exists in the beehive or as obtained by
extraction, settling or straining without adding heat. Raw honey
contains some pollen and may contain small particles of wax, local
raw honey is sought after by allergy suffers as the pollen impurities
are thought to lessen the sensitively to fever.
Ultrasonicated honey: Has been processed by ultrasonication, a
nonthermal processing alternative to honey when honey is exposed to
ultrasonication, most of the yeasts cells are destroyed. Those cells
that survive sonication generally lose their ability to grow, which
reduces the rate of honey fermentations substantially.
Honey is collected from wild bee colonies or from domesticated bee
hives. Wild bee nests are sometime located by following a honey
guild board. Collecting honey is typically achieved by using smoke
from a bee smoker to pacify the bees, this causes the bee to attempt to
save the resources of the hive from a possible forest of fire and make

them far less aggressive. The honey comb is removed from the hive
and the honey extracted after which it is filtered.
2.5 Preservation
Because of its unique composition and chemical properties, honey is
suitable for long –term storage and as easily assimilated even after
long preservation. Honey and objects immersed in honey, have been
preserved for decides and even centuries. The key to preservation is
limiting access to humidity. In its cured state. Honey has sufficiently
high sugar content to inhibit fermentation. If exposed to moist air, its
hydrophilic properties will pull moisture into the honey, eventually
diluting it to the point that fermentation can begin.
Honey should also be protected from oxidation and temperature
degradation. It should not be preserved in metal containers. However,
glass and plastic are now the favoured materials.


2.6 Properties and active ingredients of honey.
The good control of infection by honey is said to be attributed to the
high osmolarity while its hydrogen peroxide contents, lysozyme and
other unidentified substances from certain flora sources are
responsible for its responsible for its antibacterial properties (khali
et.al; 2001). The antibacterial activity of honey was also said to be
mainly due to enzymes, glucose oxidase in honey which
subrahamanyam said includes hydrogen-peroxide, flavoids and
phenolic acids plus many other unidentified properties
(subrahmanyam et. al; 2001). Also the chemical composition of
honey is said to comprise of seven tetracycline, fatty acids, lipids,
amylase, ascorbic acid, peroxidase and fructose all of which are
attributed to its antimicrobial activity together with high osmolarity,
low pH (3.6-3.7), content of phenol (inhibine), peroxidase glucose
and fructose in honey and the presence of tetracycline derivatives of
fatty acids (Al-jabril et. al; 2002).

A number of reasons have been suggested to include the shrinkage and
disruption of the bacterial cell wall due to the osmotic effect of the sugar
content, induction of an unfavorable environment with low water
activity thereby inhibiting bacterial growth and a low pH of 3.6 as well
as the fermentation of honey producing alcohol inside (subrahmanyam
et. al; 2001). However the relative importance of these factors is said to
depend on the sensitivity of the bacterial species and any additional
substance in the honey (Al-jibril et al 2002).

Table1. Composition of honey
Parameter average range standard deviation (SD)
Fructose (glucose) 1.23 0.76-1.86 0.126
Fructose (%) 38.38 30.91-44.26 1.77
Glucose (%) 30.31 22.89-40.75 3.04
Mineral (ash) % 0.169 0.020-1.028 0.15
Moisture (%) 17.2 13.4-22.9 1.46
Reducing sugar (%) 76.75 61.39-38.72 2.76
Sucrose 1.31 0.25-7.57 0.87
pH 3.91 3.42-6.10 –
Total activity 29.12 8.61-56.49 10.33
(Meg /100g)
True protein (mg/100g)168 57.5-67.5 70.9
Source: National Honey Board, 1996

2.7 Mode of action of some of the antibacterial substances in
Honey contains various substances/properties which are responsible
for the antibacterial properties observed with its use. Some of the
modes of action of these agents include the following.
High osmotic pressure
Honey due to its high osmotic pressure is said to draw water from
other sources such as tissue or bacterial cells. When it draws the
water from bacteria, it kills them (Ovington, 1999).
Low water activity
Honey is a super saturated sugar solution with low water activity
(Aw), which means that there is little available to grow if the water
activity is below 0.94-0.99 and the water activity of ripened honey
(0.56-0.62) does not support the growth of yeast. Diluted honey with
high water activity will not be effective against those species of

bacteria that grow most rapidly at water activity of 0.99 (Molan,
Glucose oxidase enzyme
Glucose oxidase is an enzyme contained in honey. This enzyme is
said to produce hydrogen peroxide, which kills bacteria when it
breaks down to form oxygen radicals (Ovington, 1999).
When honey is used topically, hydrogen peroxide is produced by
dilution of the honey with body fluids. As a result, hydrogen peroxide
is released slowly and acts as an antiseptic.

Low pH/ acidic environment
The natural acidity of honey will inhibit many pathogens. The
minimum pH values for some species that commonly infect wound
ranges from 4.0-4.5. Since honey characteristically has a pH range of

3.2-4.5, the acidity of honey is a significant factor in its antibacterial
activity (Molan, 2001).
2.8 Clinical conditions that respond to treatment with honey
Honey not only possesses significant antibacterial activity, it has also
been shown to actively promote healing (Blair, 2004). Some of the
clinical conditions that respond to treatment with honey include the
The use of honey as regards to wounds- Honey acts as highly vicious
barrier preventing bacteria penetration and colonization of wound
surface (Subrahmanyam et al; 2001).
The use of honey to treat severe acute post operative wound
infection- Topical application of crude undiluted honey have been
used in the treatment severe acute post operative wound infection due
to gram positive and gram negative bacteria following caesarean
sections and hysterectomies. This was said to lead to lead to faster

eradication of bacterial infection, reduced period of antibiotic use and
hospital stay, accelerated wound healing, prevent dehiscence and
used for restructuring and results in minimal scar formation (Al-wali
and Sallom, 1999).
Treatment with honey in chronic meningococcal skin lesions- chronic
infected meningococcal skin lesions have also been said to have been
successfully treated with honey (Dunford, et al; 2000).
Honey on gastritis/ diarrhoea- infantile gastroenteritis and diarrhea
are usually treated with hydrating glucose and electrolyte solutions,
when honey at 5%(v/v) concentration replace the glucose in
dehydration fluid, the duration of diarrhea is shortened in patience
with bacterial gastroenteritis compared to control group on standard
therapy (National Honey Board, 1999). Besides the antibacterial
activity of honey, the anti fungal activity has also been reported and
honey has been used in treatment of vagina yeast (Candida albican )

and tinea e.g, ringworm and athlete foot caused by dermatophytes (
National Honey Board, 1996)
Lastly, to assume the effectiveness of honey as an antimicrobial
agent, its exposure to heat and light should be limited.
2.9 Honey as an antibacterial agent
Work on the antibacterial activity of honey has been going on since
the 18th century and various researchers have shown that honey exerts
antibacterial activity against various organism including gram
negative and gram positive bacteria (subrahamanyam et al; 2001).
An in-vitro experiment to determine the antibacterial effect of Omani
and Africa honey against staphylococcus aureus, Escherichia coli
and Pseudomonas aeruginosa using standard antibacterial assay
showed that both honey samples were active against these organism.
Omani honey was reported to have anti-staph. aureus and E.coli

activity against pseudomonas which previously was reported not to
be susceptible to honey (Al-jabri et al; 2002).
An in-vitro study to confirm the potential antimycobacteria failed to
grow in plates containing honey with a conc. of 100% and 20% as a
result, Avilenna recommended honey in the treatment of tuberculosis
(Avilenna,1991).in one study to investigate the none peroxide
antibacterial activity of honey against staphylococcus and
micrococcus lateus using quantitative turbidimetric assay involving
serial dilution of the honey sample. The non peroxide antibacterial
activity in honey samples was found to correlate significantly with
aid content of the honey but not with its pH (Bogdanod, 1997).
Honey is produced from many different floral sources and its
antibacterial activity varies with origin and processing dioscorides
stated that pale yellow honey from Africa was the best. Aristotle
when discussing different honeys, referred to pale honey as being
“good as a salve for sore eye and wounds” substantial amount of

honey need to be applied to a wound to achieve adequate potency
although honey may be very viscous and even solid at room
temperature. Honey becomes very fluid at body temperature and even
more fluid when diluted with proportionally small volume of
exudates. It is therefore very important that sufficient honey should
be applied to a wound and kept in a place if good therapeutic effect is
to be obtained. For the optimal minimal inhibitory concentration
(MIC) of the antibacterial components of honey to be reached at the
deepest sites of infection there need to the high conc. possible on the
surface and a “reservoir” of sufficient quality that it is not
substantially depleted by diffusion into the wound tissues.
Honey produced as a food often is not well filtered and may contain
various particles in it. Also, although honey does not allow vegetative
bacteria to survive, it does contain viable spores, including clostridia.
This processing kills clostridia spores without loss of any of the
antibacterial activity.

In medicine, historically, honey has been used by humans to treat a
variety of ailments, from gastric disturbances to ulcers, wound and
burn through ingestion or topics application, but only recently have
the antiseptic and antibacterial properties of honey been chemically
explained. Different honeys have different properties which were
known since ancient times.
2.10 Practical consideration for the clinical use of honey
The amount of honey required on the wound relates to the amount of
fluid exuding from the wound diluting it. The frequency of dressing
change required will depend on how rapidly the honey is being
diluted by exudates. If there is no exudates, dressing needs to be
changed thrice weekly to maintain a “reservoir” of antibacterial
components as they diffuse into the wound tissue.
To achieve best results, the honey should be applied to an absorbent
dressing prior to application. If applied directly to the wound, the

honey tends to run off before the secondary dressing is applied to
hold it in place.
Honey will not soak readily into absorbent dressing, soaking is
facilitated by warming the honey to body temperature and or adding 1
part water to 20 parts honey to make the honey more fluid. Any
depression or cavity in the wound bed need to be filled with honey in
addition to using a honey impregnated dressing, this is to ensure that
the antibacterial component of the honey diffuse into the wound
tissue. Since infection may lie in the tissue underlying the wound
margins, honey dressings need to extend beyond the inflamed area
surrounding the wound (Postmes et al; 1997).
2.11 Adverse reaction of honey
Allergic reactions to honey are rare and have been attributed in some
cases to a reaction of some specific pollen in the honey (Molan,
1992). Honey processed for use in wound care is passed through fine

filters which removes most of the pollens. in more than 500 published
reports on the clinical usage of honey in open wounds, there have
been no adverse reactions noted other than a localized stinging
sensation described by some patients. This may be due to the acidity
of honey as it has not been reported when the acidity is neutralized
(Brady et al; 1997). A number of histological studies examining
wound tissues also support a safe use of honey.
2.12 Resent research on honey
Honey originally used by d ancient Egyptians and Greeks is a
viscous, saturated sugar solution now widely used in wound care
(Simon et al; 2009). High osmolarity prevents the growth of bacteria
and encourages healing. This can be utilized for wound management
through the application of sugar paste or honey. In addition, honey is
believed to have specific antimicrobial properties for example,
preventing of staphylococcus aureus even when diluted beyond the
point at which osmolarity is no longer inhibitory (Moore et al; 2001).

Studies have reported that it may modestly decrease wound healing
time, act as anti-inflammatory deodorize wounds, and enhance cell
proliferation and expansion in vitro (Du toit, 2009). Studies in the
support of the use honey indicate that honey-based treatment is
preferential to silver or iodine due to its comparative lack of toxicity.
Recently, further research was carried out at Waikato hospital
looking at the effect of honey on their collection of multiple
resistance ( MRSA) strains of staphylococcus aureus that cause ward
closures in hospital because they are resistant to most or all of the
commonly used antibiotics (Wilix, et al;2009). All of the strains were
found to have their growth halted completely by the honeys diluted to
5-10%. In the last few years, it has been recognized that dyspepsia
and stomach ulcers are frequently caused by infection of the stomach
by specie of bacteria (Helicobacter pylori). The possibility that the
healing infect of honey on the stomach may be through its action on
this bacterium which was suggested by Niaz-Al-somai (1999) at the

University of Waikato. In collection with microbiologist at the
Waikato hospital, he tested strains of H. pylori isolated from biopsy
samples of stomach ulcers, using the same honey that had been tested
on the wound-infected species of bacteria. It was found that the
honey with hydrogen peroxide did not prevent the growth of cultures
of H.pylori when added at concentration up to 50%, but the manuka
honey completely halted growth of the bacteria at a concentration of
50% (Wilix et al;2009). The source of the nectar used in the
production of the honey may have caused difference in the
antimicrobial activities of honey from different source.


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