Honey has been used for centuries as a treatment for infected wounds. It has antimicrobial properties due to its high osmolarity and acidity, which helps inhibit bacterial growth. Recent research has rediscovered honey's medical applications, especially for wounds that fail to heal with conventional treatments. Different types of honey from various floral sources and geographical locations have varying antimicrobial potency and effectiveness in wound healing. The objective of the study is to assess the antibacterial action of forest honey against pathogens like E. coli, Proteus, Klebsiella, and Staphylococcus aureus by screening its antibacterial properties, isolating clinical samples, and determining zone of inhibition compared to standard antibiotics.

1. 1
INTRODUCTION
Honey is an ancient remedy for the treatment of infected wounds, which has
recently been ‗rediscovered‘ by the medical profession, particularly where conventional modern
therapeutic agents fail. The first written reference to honey is a Sumerian tablet writing, dating
back to 2100-2000 BC, mentions honey‘s use as a drug and an ointment. Aristotle(384-322 BC),
Since ancient furies honey is used as a remedy for curing ulcers, wounds and skin infections. It is
also effective in treating gastroenteritis in children. This honey not only used in Asia also used
worldwide. In the honey some types are more valuable than the others as it carries more
antimicrobial effect and cures wounds sooner. As usually honey which is raw has bacterial flora
composed of gram positive sporing Bacilli example Bacillus.
The source of microbes on honey can be by honey comb, honeybee, pollen, air,
nector, the most common species. Seen in behave environment are; Bacillus, Clostridium,
Corneybacterium, Pseudomonas which are capable of living in high sugar concentration
antioxidant seen in honey are known for there cytoprotective effects. As, there is increase in
antibiotics many microbes have become resistant to particular antibiotics leading to the infection
not get cured. So, natural remedies like honey can be an alternative against the antibiotics. Honey
is a thick sweet liquid made by bees from nectar of flowers. It contains water, glucose, fructose,
vitamins, proteins, minerals. There are two types of honey apiary and forest honey.
Honeys produced by a Apis mellifera in apeares are called apiary honey. They
are transparent and some foreign substance. This produced by rock bees Apis dorsata are
collected by crude method of squeezing the comb and are known as forest honey. They are turbid
owing to presence of beewax, polan and other materials. It is therefore necessary to filter honey
before doing the procedure. Water is critical as it can effect the storage of honey. The color and
consistency of honey is storage of honey varied depending upon nectar, flowers and bees
collecting honey has an acidic pH 3.2 – 4.5. which low and inhibits pathogens. Moreover it can
be used on skin grafts and infected skin donor graft successfully. The antibacterial activity of
honey was first recognized by van kevel in1882. Honey has the capacity, when applied on the
wound, honey uptakes the water from wound leaving it dry and making it heal faster. Honey has
been reported to have in inhibitory effect around 60 species of aerobes as well as anaerobes.

2. 2
It has been used as a treatment of infection of wounds more than hundred years ago even before
the discovery of bacteria as a causative agent. Honey can reduce the effect of aflatoxin B1 and
B2. It is recognized that honey from different floral sources and geographical locators vary
considerably not only in their antibacterial activity but also color and wound healing potential
free radicals are reactive molecules that arise from the products of oxidation reduction of oxygen
and hydrogen. The high oxidative reductive capability of these radicals can damage cell
membranes by culturing the oxidative states of certain compounds such as DNA. Two of the
most physiologically important free radicals are hydroxyl and superoxide radicals. Honey‘s
antimicrobial effect is contributed to high osmotic effect and acidic nature. Hydrogen peroxide
concentration and its chemical composition. Nectar is a sugar solution produced by the glands of
flower that has function in attraction of insects and birds.
Honey is the natural sweet substance obtained from the secretions of the living parts or
excretions of plants which the honey bees (Apis mellifera) collect and store (Moore et al. 2001).
Though honey is used widely in traditional medicine, its use in modern medicine is limited
(Geenwood 1993). Honey is used for the treatment of many infections, and also used effectively
as wound dressing including surgical wounds, burns and skin ulcers. Mainly because it speeds up
the growth of new tissues and help to heal the wound, reduces pain and odor quickly (Lusby et
al. 2002).
Honey is a complex substance made up of at least 181 different substances. The
presence of glucose and fructose in honey is due to action of bee enzyme invertase on sucrose
molecules thus producing glucose and fructose in ratio 1:2. Today honey is mainly known for its
sweetening capacity and as a desirable natural food product in ancient turns. It was regarded as
an important treatment for all problems. Honey and other bee products have been important to
mankind for many years, where it has been used by many others like a food or medical purpose
consequently bees gained a sacred place in some regions. It‘s difficult to be accurate about when
the relation between man and bee first started, but since man started to express himself through
cave paintings, bees are pictured as an act of honey.

3. 3
Honey is a super saturated sugar solution with high osmolarity. Hence has a broad
spectrum antimicrobial activity. However honey has a limited use in medicine due to lack of
scientific support. Although several invitro studies have demonstrated the antibacterial properties
of honey few have examined the action against fungi. The incidence of fungal infection is
increasing and the causative agents for Candida species.the production of honey produced by
honey bees is dependent on the flowers from which bees carry nectar to produce honey may
contribute in difference of antimicrobial activity. As a source of energy, the beneficial character
of honey are its high nutritional value of fast absorption consumption.
A common feature of all of the reports in the medical literature on the use of honey
as an antibacterial agent is that no consideration is given to the selection of type of honey for
therapeutic use. Aristotle, c 350 BC6, and Dioscorides, c AD 5046, rec-ommended that honey
collected in specific regions and seasons (and therefore pre-sumably from different floral
sources) be used for the treatment of different ail-ments. Such considerations have continued into
present-day folk medicine: the strawberry-tree (Arbutus unedo) honey of Sardinia is valued for
its therapeutic properties(Floris and Prota , 1989); in India lotus (Nelumbium sceciosum) honey is
said to be a panacea for eye diseases (Fotidar and Fotidar1945) 39. In modern clinical practice,
however, these views have gone unnoticed, as have the laboratory findings of large differences in
the antibacterial potency of honey from different floral sources

4. 4
AIM&
OBJECTIVES

5. 5
AIM AND OBJECTIVE
Aim
To assess the antibacterial action of forest honey against selected pathogens like
Escherichia coli, Proteus sp, Klebsiella sp and Staphylococcus aureus.
Objective
o To screen the antibacterial properties of forest honey
o Isolation of clinical samples like Escherichia coli, Proteus sp, Klebsiella sp and
Staphylococcus sp.
o To determine the zone of inhibition of microbes and compared with standard
antibiotics.

6. 6
REVIEW
OF
LITERATURE

7. 7
Review of literature
Honey is a supersaturated sugar solution with a high osmolarity; it will exert a high
osmotic pressure on bacteria because water molecules will be largely bound to sugar molecules,
making them unavailable for the growth of most microorganisms. Hence honey has a broad
spectrum antimicrobial activity. Honey contains trace amount of vitamins, flavonoids,
antioxidant components and unidentified plant derived elements (phytochemical components). It
also possesses trace amounts of other beehive products like propolis, royal jelly and wax of
which the first two are recognised as antimicrobial agents as well. Honey is also rich in organic
acids, with at least 30 different organic acids being recovered from this product, among the most
common are gluconic acid, acetic acid, citric acid, lactic acid, succinic acid and formic acid.
Understanding the mode of action of a new antimicrobial agent is necessary for it to be used
appropriately and safely. Although honey is not a new antimicrobial agent, its use in
conventional medicine is recent, with the introduction of the first manuka honey impregnated
wound dressing on to drug tariff. Further research into the mechanism of action of manuka honey
on relevant wound infecting pathogens is considered necessary. ( Desalegn Amenu 2013)
The biochemical components and antibacterial efficacy of pure honey produced by
honeybee (Apis mellifera) was investigated. Biochemical analysis revealed the presence of
reducing sugar, saponins, glycosides, alkaloids and flavonoids and absence of phenols and
tannins. Antibacterial activity of pure honey on some medically important bacteria including
Staphylococcus aureus, Salmonella typhi, Escherichia coli, Klebsiella pneumonia and
Pseudomonas aeruginosa was determined using the agar well diffusion method. vc The result
shows that pure honey exhibit strong antibacterial activity producing zones of inhibition against
the tested bacteria.Also, honey sample used in this study showed antibacterial activity than the
commercially available antibiotics. Our findings shows that honey,apart from their roles as food
and supplements, could be suitable for the treatment of various infection caused by bacteria
hence should be used as a preventive and curative measure to common diseases related to the test
organisms.(Elijah et al., 2015)

8. 8
The aim of the present research work to investigate antimicrobial activity of some honey
samples six winter honeys six summer honeys collected from different regions of Western Ghats.
The microbes used in this study are Staphylococcus aureus, Streptococcus pyogenes, Escherichia
coli, Pseudomonas aeruginosa, and Proteus mirabilis. Antibacterial activity of the honey was
assayed using the Disc diffusion method. Noticeable variations in the antibacterial activity of the
different honey samples were observed. Among the microbes Staphylococcus aureus is the most
sensitive against all honey samples shows the maximum inhibitor zone compare to summer
honeys. (Mahendran & kumarasamy 2015)
Honey has previously been shown to have wound healing and antimicrobial properties,
but this is dependent on the type of honey, geographical location and flower from which the final
product is derived. We tested the antimicrobial activity of a Chilean honey made by Apis
mellifera (honeybee) originating from the Ulmo tree (Eucryphia cordifolia), against selected
strains of bacteria. Ulmo 90 honey was compared with manuka UMF® 25+ (Comvita®) honey
and a laboratory synthesised (artificial) honey. An agar well diffusion assay and a 96 well
minimum inhibitory concentration (MIC) spectrophotometric-based assay were used to assess
antimicrobial activity against five strains of methicillin-resistant Staphylococcus aureus
(MRSA), Escherichia coli and Pseudomonas aeruginosa. Initial screening with the agar
diffusion assay demonstrated that Ulmo 90 honey had greater antibacterial activity against all
MRSA isolates tested than manuka honey and similar activity against E. coli and P. aeruginosa.
The MIC assay, showed that a lower MIC was observed with Ulmo 90 honey (3.1% - 6.3% v/v)
than with manuka honey (12.5% v/v) for all five MRSA isolates. For the E. coli and
Pseudomonas strains equivalent MICs were observed (12.5% v/v). The MIC for artificial honey
was 50% v/v. The minimum bactericidal concentration for all isolates tested for Ulmo 90 honey
was identical to the MIC. Unlike manuka honey, Ulmo 90 honey activity is largely due to
hydrogen peroxide production. Due to its high antimicrobial activity, Ulmo 90 may warrant
further investigation as a possible alternative therapy for wound healing. (Orla Sherlock et al.,
2010)

9. 9
Honey is well known for its antibacterial activity, which was first reported in 1892 (as
cited by Dustmann in 1919). Since an- cient times, honey has been used for treatment and
prevention of wound infections. With the advent of antibiotics, the clinical application of honey
was abandoned in modern Western medi- cine, though in many cultures it is still used. For all
antibiotic classes, including the major last resort drugs, resistance is increasing worldwide (1, 2)
and even more alarming, very few new antibiotics are being developed. The potent activity of
honey against antibiotic-resistant bacteria (3–5) resulted in renewed interest for its application.
Several honeys have been approved for clinical application. The incomplete knowledge of the
antibacterial compounds involved and the variability of anti- bacterial activity are however major
obstacles for applicability of honey in medicine. In recent years, the knowledge on the
antibacterial compounds in honey has expanded. In this review, we will give an overview of the
current knowledge on the anti- bacterial components in honey, and we will discuss the implica-
tions for standardization of the antibacterial activity of honey. (Paulus et al., 2012)
Bacterial colonisation of a wound is not regarded as detrimental to the wound healing
process. However colonisation may lead to chronic infection when the bacteria persistently
utilize host resources to a point where they out-compete the host‘s immune defense system
(Wolcott et al., 2010). Chronic wound infections are responsible for considerable patient
morbidity and an associated decrease in patient quality of life (Jørgensen et al., 2006). Chronic
wounds contribute significantly to escalating health care costs (Siddiqui et al., 2010).
Between 2005 and 2006 the cost to the UK National Health Service of caring for patients
with chronic wounds was estimated to be around £3.1billion (Posnett & franky 2008). In the
United States, chronic wounds affect 6.5 million patients annually and have an associated annual
treatment cost of $25 billion a year. These figures already represent a significant financial burden
but worldwide costs associated with chronic wounds are set to increase further due to an aging
population and a sharp rise in the incidence of diabetes and obesity (Sen et al., 2009).
Currently antibiotics are often used in the routine treatment of bacterial infections
however, the large number of long term chronic wounds referred to above demonstrate that,
antibiotics alone are not always an effective treatment method for the management of bacterial
infections. Many antibiotics have a narrow spectrum of action and therefore do not effectively

10. 10
treat multispecies wound infections. In addition the prevalence of bacteria with single and multi
antibiotic resistance mechanisms is increasing (Kumarasamy et al., 2010).
Few novel antibiotics are under development and it is now generally accepted that
bacteria will eventually develop resistance mechanisms to novel antibiotics, with a high volume
usage being a driving factor in the development of resistance characterisitcs. In order to improve
the treatment of chronic wounds and to address the ever increasing financial burdens associated
with these wounds alternative effective treatments are required. Two topical broad spectrum anti-
microbial agents are medical honey and silver. For many years it has been known that honey
demonstrates broad-spectrum antibacterial activity (Molan 2006).
Medical grade honey is recommended for use on open wounds because non sterilised
honeys can contain pathogenic organisms that have the potential to further infect vulnerable
patients (Cooper et al., 2009). Medical grade honey has been commercially available to wound
care professionals in the EU since 2002. (Westgate 2013)
The purpose of the present study was to investigate and compare the demonstrated variation in
antimicrobial activity of honey produced by introduced A mellifera and the stingless bee,
Tetragonisca angustula, commonly kept in hives in Costa Rica. There was no difference in
activity of honey produced by Apis mellifera and T. angustula against the 5 microbes tested.
Honey from different phytogeographic regions exhibited differential antimicrobial activity and
susceptibility of yeasts to honey of either species was greater than that of bacteria. (Jason et al.,
2004)
Honey is gaining acceptance by the medical profession for use as an antibacterial agent
for the treatment of ulcers and bed sores, and other surface infections resulting from burns and
wounds4,135. In many cases it is being used with success on infections not responding to
standard antibiotic and antiseptic therapy. Its effectiveness in rapidly clearing up infection and
promoting healing is not surprising in light of the large number of research findings on its
antibacterial activity. None of the reports in the medical literature, however, mention any
selection of the honey used for the treatment of infections. Although it is recognized that honey
has antibacterial activity, it is not generally realized that there is a very large variation in the
antibacterial potency of different honeys, and that the antibacterial properties can be easily lost

11. 11
by inappropriate handling and storage of honey. the research that has been done on these aspects:
giving regard to these findings should result in a more rational usage of honey in medicine and
allow its full potential as an antibacterial agent be achieved. (Peter Molan)
Honey is a natural sweetener, but it is not just a sweetener it‘s a nature‘s gift to mankind.
Natural honey has various ingredients in it, that contribute to its incredible properties. It‘s
antimicrobial properties have attracted researchers towards itself and now we can find many
research papers published on this topic. Honey is normally used in our daily life for treatment of
hearing loss, bad breath, fatigue, weight loss, pimples, influenza, ingestion, heart diseases,
toothache, hair loss, bladder infections, infertility etc. Honey is used as a mixture with many
natural products such as lemon, clover, milk, cinnamon and water for treatment of various
ailments and other health disorders. Beside this, honey is now used in various industries that
exploit nature‘s wonderful gift (honey). Commercially honey is used as moisturizer, hair
conditioner, laxative, aphrodisiac, rooting hormone, cleansers etc. Honey mixed with ground
almonds makes an excellent facial cleansing scrub. (Singh et al., 2012)
The increased incidence of bacterial resistance to antibiotics has generated renewed
interest in ―traditional‖ antimicrobials, such as honey. This paper reports on a study comparing
physico-chemical, antioxidant and antibacterial characteristics (that potentially contribute in part,
to the functional wound healing activity) of Cameroonian honeys with those of Manuka honey.
Agar well diffusion was used to generate zones of inhibition against Escherichia coli,
Pseudomonas aeruginosa and Staphylococcus aureus while broth dilutions were used to study
the minimum inhibitory concentrations (MICs). Non-peroxide activity was investigated by
catalase for hydrogen peroxide reduction. The Cameroonian honeys demonstrated functional
properties similar to Manuka honey, with strong correlations between the antioxidant activity
and total phenol content of each honey. They were also as effective as Manuka honey in
reducing bacteria load with an MIC of 10% w/v against all three bacteria and exhibited non-
peroxide antimicrobial activity. These Cameroon honeys have potential therapeutic activity and
may contain compounds with activity against Gram positive and Gram negative bacteria.
Antibacterial agents from such natural sources present a potential affordable treatment of wound
infections caused by antibiotic resistant bacteria, which are a leading cause of amputations and
deaths in many African countries. (Joshua Boateng & Keshu Nso Diunase 2015)

12. 12
Roma is an important quality factor in foods. The aroma of bee honey depends on volatile
fraction composition, which is influenced by nectar composition and floral origin. Honey of
unifloral origin usually commands higher commercial value, thus the floral determi- nation and
certification of unifloral honey plays an important role in quality control. This review concerns
investigations made on the volatile fraction of bee honey by gas chromatography/mass
spectrometry. Recent advances in extraction methods, results achieved, and comparisons of
alternative dependable methods for determining floral origin of bee honey are discussed. We
emphasize solid phase micro-extraction gas chromatography (SPME/GC) methodology and
present some of the results obtained to date, plus the advantages and drawbacks of SPME/GS in
comparison with other methods. 2006 Elsevier Ltd. All rights reserved. (Luis et al., 2007)
The authors studied the effect of storage period and heat on the physical and chemical
properties of honey and proceeded to study the antibacterial effect of honey on Escherichia coli
and Salmonella typhimurium. In samples of honey (Egyptian clover honey) that were heat-
treated and stored over a long period of time, water content decreased, hydroxymethyl furfural
(HMF) was produced and increased in concentration, and enzyme activity decreased. Colour,
measured in optical density, was markedly affected in honey samples stored over long periods of
time, as was the refractive index, but electrical conductivity remained unaffected by storage or
heating. Similarly, the storage period had no effect on pH value. To study the therapeutic effect
of honey on E.coli and S. typhimurium , 25 isolates of E. coli O157:H7 (18.5%) and 49 isolates
of S. typhimurium (36.2%) were isolated from 135 samples taken from children and calves (30
stool samples from children and 105 samples from calf organs and faecal swabs). Most E. coli
O157:H7 and S. typhimurium isolates were highly resistant to most antibiotic discs. In vitro , the
antibacterial effect of honey was more pronounced on E. coli O157:H7 than on S. typhimurium .
Water content, pH value, HMF and the presence of H2O2 all played an important role in the
potency of clover honey as an antibacterial agent. In vivo , mice were used as a model for
studying the parenteral usefulness of honey as an antibacterial agent against both pathogens. The
antibacterial activity of honey that had been stored over a long period of time decreased and high
concentrations of honey proved more effective as antibacterial agents. In this study there was
lower mortality among mice treated with honey but the parenteral application of honey and its
therapeutic properties require further investigation. (Badawy et al., 2004)

13. 13
Chronic wound infections and antibiotic resistance are driving interest in antimicrobial
treatments that have generally been considered complementary, including antimicrobially active
honey. Australia has unique native flora and produces honey with a wide range of different
physicochemical properties. In this study we surveyed 477 honey samples, derived from native
and exotic plants from various regions of Australia, for their antibacterial activity using an
established screening protocol. A level of activity considered potentially therapeutically useful
was found in 274 (57%) of the honey samples, with exceptional activity seen in samples derived
from marri (Corymbia calophylla), jarrah (Eucalyptus marginata) and jellybush (Leptospermum
polygalifolium). In most cases the antibacterial activity was attributable to hydrogen peroxide
produced by the bee-derived enzyme glucose oxidase. Non-hydrogen peroxide activity was
detected in 80 (16.8%) samples, and was most consistently seen in honey produced from
Leptospermum spp. Testing over time found the hydrogen peroxide- dependent activity in honey
decreased, in some cases by 100%, and this activity was more stable at 4uC than at 25uC. In
contrast, the non-hydrogen peroxide activity of Leptospermum honey samples increased, and this
was greatest in samples stored at 25uC. The stability of non-peroxide activity from other honeys
was more variable, suggesting this activity may have a different cause. We conclude that many
Australian honeys have clinical potential, and that further studies into the composition and
stability of their active constituents are warranted. (Julie Irish et al., 2011)
Phenolic compounds of dark and clear honeys from Trás-os-Montes of Portugal were
extracted with Amberlite XAD-2 and evaluated for their antioxidant and antimicrobial activities.
The antioxidant effect was studied using the in vitro test capacity of scavenge the 2,2-diphenyl-
1-picryhydrazyl (DPPH) free radical and of reducing power of iron (III)/ferricyanide complex.
The antimicrobial activity was screened using three Gram-positive bacteria (Bacillus subtilis,
Staphylococcus aureus, Staphylococcus lentus) and three Gram-negative bacteria (Pseudomonas
aeruginosa, Klebsiella pneumoniae and Escherichia coli). The results obtained from the partial
identification of honey phenolic compounds by high-performance liquid chromatography with a
diode array detector showed that p-hydroxibenzoic acid, cinnamic acid, naringe- nin,
pinocembrin and chrysin are the phenolic compounds present in most of the samples analyzed.
Anti- oxidant potential was dependent of honey extract concentration and the results showed that
dark honey phenolic compounds had higher activity than the obtained from clear honey. In the
biological assays, results showed that S. aureus were the most sensitive microrganisms and B.

14. 14
subtilis, S. lentus, K. pneumo- niae and E. coli were each moderately sensitive to the
antimicrobial activity of honey extracts. Neverthe- less, no antimicrobial activity was observed in
the test with P. aeruginosa. (Letícia Estevinho)
Antimicrobial activity of honey has been attributed to hydrogen peroxide, which is
produced by naturally occurring glucose oxidase, and phenolic compounds, although lethality of
and inhibition by these and other components against microorganisms vary greatly, depending on
the floral source of nectar. This study was undertaken to compare honeys from six floral sources
for their inhibitory activity against Escherichia coli O157:H7, Salmonella typhimurium, Shigella
sonnei, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. A disc assay
revealed that development of zones of inhibition of growth depends on the type and
concentration of honey, as well as the test pathogen. Growth of B. cereus was least affected. The
inhibition of growth of S. sonnei, L. monocytogenes, and S. aureus in 25% solutions of honeys
was reduced by treating solutions with catalase, indicating that hydrogen peroxide contributes to
antimicrobial activity. Darker colored honeys were generally more inhibitory than light colored
honeys. Darker honeys also contained higher antioxidant power. Since antimicrobial activity of
the darker colored test honeys was not eliminated by catalase treatment, non-peroxide
components such as antioxidants may contribute to controlling the growth of some foodborne
pathogens. The antibacterial properties of honeys containing hydrogen peroxide and
characterized by a range of antioxidant power need to be validated using model food systems.
(Peter et al., 2001)
The medicinal properties of honey have been known since ancient times. Ayurveda
(Indian medicine) describes honey as the nectar of life and recommends its use in various
ailments. There is renewed interest in honey treatment as evidenced by the number of reports
appearing in the scientific literature.' Honey has been useful in the treatment of surgical wounds,
burns, and decubitus ulcers, and the antibacterial and antifungal properties of honey have been
well documented.` In burns in particular, honey has been found to control wound infection and
accelerate wound healing." This study was undertaken in order to determine the minimum

15. 15
inhibitory concentration (MIC) of honey on bacteria isolated from burn wounds in our burn unit.
(Subrahmanyam et al., 2001)
Renewed interest in honey for various therapeutic purposes including treatment of
infected wounds has led to the search for new antibacterial honeys. In this study we have
assessed the antibacterial activity of three locally produced honeys and compared them to three
commercial therapeutic honeys (including Medihoney and manuka honey). Methods. An agar
dilution method was used to assess the activity of honeys against 13 bacteria and one yeast. The
honeys were tested at five concentrations ranging from 0.1 to 20%. Results. Twelve of the 13
bacteria were inhibited by all honeys used in this study with only Serratia marcescens and the
yeast Candida albicans not inhibited by the honeys. Little or no antibacterial activity was seen at
honey concentrations 1%, with minimal inhibition at 5%. No honey was able to produce
complete inhibition of bacterial growth. Although Medihoney and manuka had the overall best
activity, the locally produced honeys had equivalent inhibitory activity for some, but not all,
bacteria. Conclusions. Honeys other than those commercially available as antibacterial honeys
can have equivalent antibacterial activity. These newly identified antibacterial honeys may prove
to be a valuable source of future therapeutic honeys. (Patricia et al., 2005)
Honey has had a valued place in traditional medicine for centuries. Renewed interest in
honey for various therapeutic purposes, including treatment of infected wounds, has led to the
search for different types of honey with antibacterial activity. In this study, we have assessed the
antibacterial activity of different types of honey (manuka honey from Australia, heather honey
from the United Kingdom, and locally marketed Indian honey). The agar dilution method was
used to assess the antibacterial activity of honey against 152 isolates of Pseudomonas aeruginosa
by determining minimum inhibitory concentrations. The locally available (khadikraft) honey
produced the best activity against Pseudomonas aeruginosa and was found to be better than all
of the imported varieties of therapeutic honey. (Venkatachalam Mullai & Thangam menon
2007)
The nonperoxide antibacterial activity of honey and honey fractions was tested with
Staphylococcus aureus and Micrococcus luteus bacterial species. Antibacterial activity correlated
significantly with the honey acidity but did not correlate with honey pH. There were small

16. 16
differences between the antibacterial activities of different honey types: rhododendron,
eucalyptus and orange honeys had a relatively low activity, whereas dandelion, honeydew and
rape honeys had a relatively higher activity. These results suggest that a part of the antibacterial
activity might be of plant origin. However, the antibacterial activity of sugar-adulterated honeys
was the same as that of control honeydew honeys produced in the same apiary suggesting that
the major part of the antibacterial activity of honeydew honey is of bee origin. Ten different
honeys were fractionated into four fractions using column chromatography or vacuum
distillation: acidic; basic; nonvolatile, nonpolar; and volatile. The antibacterial activity of the
different fractions tested was: acids > bases = nonpolar, nonvolatiles > volatiles. This order was
the same using either Staph. aureus or Micrococcus luteus as test strains. An exception was
manuka honey from New Zealand where almost the entire activity was found in the acidic
fraction.(Stefan Bogdanov 1997)
Development of antibiotic-resistant strains of coagulase-negative staphylococci has
complic- ated the management of infections associated with the use of invasive medical
devices,and innovative treatment and prophylactic option s are needed.Honey is increasing ly
being used to treat infected wounds, but little is known about its effectiveness against coagulase-
negative staphylococci. The aim of this study was to determine the minimum active dilution of
two standardized, representative honeys for 18 clinical isolates of coagulase-negative
staphylococci. Anagarin corporation technique was used to determine the minimum active
dilution ,with dilution steps of 1% (v/v) honey [or steps of 5% (v/v) of a sugar syrup matching
the osmotic effect of honey]. The plates were inoculated with 10 mL spots of cultures of the
isolates. Results: The honeys were inhibitory at dilutions down to 3.6–0.7% (v/v) for the pasture
honey, 3.4–0.5% (v/v) for the manuka honey and 29.9–1.9% (v/v) for the sugar syrup. Typi cal
honey sare about eight times more potent against coagulase-negative staphylococci than if
bacterial inhibition were due to their osmolarity alone. Therefore, honey applied to skin at the
insertion points of medical devices may have a role in the treatment or prevention of infections
by coagulase-negative staphylococci. (French et al., 2005)
Honey, propolis, and royal jelly, products originating in the beehive, are attractive
ingredients for healthy foods. Honey has been used since ancient times as part of traditional
medicine. Several aspects of this use indicate that it also has functions such as antibacterial,

17. 17
antioxidant, antitumor, anti-inflamatory, antibrown- ing, and antiviral. Propolis is a resinous
substance produced by honeybees. This substance has been used in folk medicine since ancient
times, due to its many biological properties to possess, such as antitumor, antioxidant, an-
timicrobial, anti-inflammatory, and immunomodulatory effects, among others. Royal jelly has
been demonstrated to possess numerous functional properties such as antibacterial activity, anti-
inflammatory activity, vasodilative and hypotensive activities, disinfectant action, antioxidant
activity, antihypercholesterolemic activity, and antitu- moractivity.Biological activities of honey
,propolis ,androyal jellya remain lyattributed to the phenolic compounds such as flavonoids.
Flavonoids have been reported to exhibit a wide range of biological activities, including antibac-
terial, antiviral, anti-inflammatory, antiallergic, and vasodilatory actions. In addition, flavonoids
inhibit lipid per- oxidation, platelet aggregation, capillary permeability and fragility, and the
activity of enzyme systems including cyclo-oxygenase and lipoxygenase. (Viuda martos et al.,
2008)
More than two thousand bacterial strains isolated from six US domestic honeys and two
manuka honeys from New Zealand were screened for production of antimicrobial compounds. A
high incidence of antimicrobial inhibition determined by deferred inhibition assays was observed
with the bacterial isolates from all eight honey samples. In total, 2217 isolates out of 2398 strains
(92.5% of total isolates) exhibited antimicrobial activity against at least one of the tested
microorganisms. Antifungal activity by bacterial isolates originating from the eight honeys
ranged from 44.4% to 98.0%. Bacterial isolates from manuka honey (MH1) exhibited
antimicrobial activity against Bacillus subtilis ATCC 6633 and Bacillus cereus F4552, at 51.5%
and 53.3% of the isolates, respectively. However, less than 30% of the bacterial isolates from the
other manuka honey (MH2) and six domestic honey sources exhibited anti-Bacillus activity.
Listeria monocytogenes F2-586 1053 showed higher overall rates of sensitivity to between 11
and 66% of the bacterial isolates. The high rate of antimicrobial activity exhibited by the
bacterial strains isolated from different honey sources could provide potential sources of novel
antimicrobial compounds. (Hyungjae Lee et al., 2008)
Indeed, medicinal importance of honey has been documented in the world's oldest
medical literatures, and since the ancient times, it has been known to possess antimicrobial
property as well as wound-healing activity. The healing property of honey is due to the fact that

18. 18
it offers antibacterial activity, maintains a moist wound condition, and its high viscosity helps to
provide a protective barrier to prevent infection. Its immunomodulatory property is relevant to
wound repair too. The antimicrobial activity in most honeys is due to the enzymatic production
of hydrogen peroxide. However, another kind of honey, called non-peroxide honey (viz., manuka
honey), displays significant antibacterial effects even when the hydrogen peroxide activity is
blocked. Its mechanism may be related to the low pH level of honey and its high sugar content
(high osmolarity) that is enough to hinder the growth of microbes. The medical grade honeys
have potent in vitro bactericidal activity against antibiotic-resistant bacteria causing several life-
threatening infections to humans. But, there is a large variation in the antimicrobial activity of
some natural honeys, which is due to spatial and temporal variation in sources of nectar. Thus,
identification and characterization of the active principle(s) may provide valuable information on
the quality and possible therapeutic potential of honeys (against several health disorders of
humans), and hence we discussed the medicinal property of honeys with emphasis on their
antibacterial activities. (Manisha Deb Mandal & Shyamapada Mandal 2011)
Honey is an effective antiseptic wound dressing, mainly the result of the antibacterial
activity of hydrogen peroxide that is produced in honey by the enzyme glucose oxidase. Because
the rate of production of hydrogen peroxide is known to vary disproportionately when honey is
diluted, and dilution of honey dressings will vary according to the amount of wound exudate, it is
important to know more about the production of hydrogen peroxide at different concentrations of
honey. The rates of hydrogen peroxide production by honey with respect to honey dilution were
measured in eight different samples of honey from six different floral sources. Honey Research
Unit, Waikato University, Hamilton, New Zealand. Main The maximum levels of accumulated
hydrogen peroxide occurred in honey solutions diluted to concentrations between 30% and 50%
(v/v) with at least 50% of the maximum levels occurring at 15–67% (v/v). This is equivalent to a
10 cm 3 10 cm dressing containing 20 mL of honey becoming diluted with 10 to 113 mL of
wound exudate. Maximum levels of hydrogen peroxide reached in the diluted honeys were in the
range of 1–2 mmol/L. Significant antibacterial activity can be maintained easily when using
honey as a wound dressing, even on a heavily exuding wound. Concentrations of hydrogen
peroxide generated are very low in comparison to those typically applied to a wound, thus,
cytotoxic damage by hydrogen peroxide is very low. (Lynne et al., 2003)

19. 19
MATERIALS
AND
METHODS

20. 20
MATERIALS AND METHODS
The present study was broadly divided into three major steps
 Purification of honey
 Isolation and identification of human pathogens from various samples.
 Antimicrobial effect of honey against selected pathogens.
ISOLATION OF MICROORGANISMS:
Sputum, stool and urine, samples were collected aseptically and isolated and purified in the
Divine mother paramedical college laboratory, Pondicherry.
A loopful of sample was taken and streaked to the nutriend agar and incubated at 370
C for 24
hours. After incubation gram staining was done and the colonies were streaked to in differential
& selective media for isolation of bacteria.
MEDIA COMPOSITION
Nutrient Agar :
Peptone - 5g
Beef extract - 1.5g
Yeast extract - 1.5g
Sodium chloride - 5g
Agar - 15g
Distilled water - 1000ml
pH - 7.4

21. 21
IDENTIFICATION OF THE ISOLATES:
Isolated culture was preliminarily identified based on colony morphology, physiological and
biochemical characterization was performed by the following standard physiological and
biochemical procedures.
GRAM STAINIG:
The method Is named after its inventor, the Danish scientist hans Christian gram (1853 - 1938),
who developed the technique while working with carl Friedlander in the morgue of the city
hospital in berlin in 1894. Gram divised his technique not for the purpose of distinquishing one
type of bacterium from another but to make bacteria more visible in stained sections of lung
tissue (gram, HC 1884).
The isolated bacteria stains were made into a thin smear in clean glass slide. Air dried then heat
fixed. The primary stain crystal violet was flooded on the smear and washed with running tap
water. The mordant stain gram‘s iodine was flooded on the smear and left for one minute and
then washed with water. Decolorizing agent alcohol was added drop by drop until the dye gets
removed from the smear and then washed with tap water. The slide was air dried and observed
under microscope.
BIOCHEMICAL TESTS:
Indole test:
The bacterial culture was inoculated into tryptone or peptone broth and incubated at 370
C for 24-
78 hrs. After incubation 0.5 ml of kovac‘s reagent was added and gently agitated. Examined for
the presence of red color in the upper layer of liquid. Positive result was indicated the formation

22. 22
of red color (occurring within few seconds) and negative result was indicated by the formation of
yellow color.
Methyl red test:
The bacterial culture was inoculated into MR-VP broth and it was incubated for 24-48 hrs at
370
C. After incubation few drops of methyl red indicator was added into the culture medium.
Positive result was indicated by the formation of red color and negative result was indicated by
the formation of yellow color.
Voges proskauer test:
The isolated bacterial culture was inoculated into the MR-VP broth and incubated at 370
C for 48
hrs. After incubation, 0.3 ml of alpha naphthol reagent and 0.1 ml 40% KOH solution were
added. Positive result was indicated by the formation of strong pink color and negative result was
indicated by no color change.
Citrate utilization test:
Sodium citrate slant was inoculated with the pure culture and the tubes were incubated at 370
C
for 24 hrs. After incubation color change from green to blue color indicated the positive result.
Negative result was indicated by no color change and the medium remained green.
Urease test:
The urea agar was inoculated with the test organism and incubated at 35-370
C for 24-48 hrs.
After incubation purple/pink color indicated the positive and no color change indicated the
negative result.
Catalase test:

23. 23
One drop of hydrogen peroxide solution was placed on a slide, already loaded with culture.
Vigorous bubbling occurring within 10 seconds indicated positive result. No bubble formation
indicated negative result.
Oxidase test:
Aseptically transfer a large mass of pure culture to the oxidase disk. The disk was observed up to
3 minutes. If the area of inoculation turns dark to maroon to almost black, then the result was
positive. If the color does not occur within three minutes, the result was negative.
Esculin hydrolysis:
The esculin medium was inoculated with the test organism. Incubated at 35-370
C for up to 48
hrs in aerobic or in anaerobic condition. After incubation the plates were observed for a black
color development, indicating the positive result. No color change of medium was a negative
result.
Carbohydrate fermentation test:
Carbohydrate fermentation broth was prepared along with bromocresol purple indicator. To each
of the test tube durham‘s tube was added in an inverted position and sterilized at appropriate
temperature. After sterilization selected filter sterilized sugar was added into tube in an aseptic
condition.
After inoculation the tubes were incubated at 370
C for 24-48 hrs. After incubation the tubes were
observed for acid and gas production. Yellow color indicated the acid production. Bubbles in the
durham‘s tube indicated the gas production.
Coagulase test:
Coagulase test is used to differentiate Staphylococcus aureus (positive) from Coagulase Negative
Staphylococcus (CONS). Coagulase is an enzyme produced by S. aureus that converts (soluble)
fibrinogen in plasma to (insoluble) fibrin. Staphylococcus aureus produces two forms of
coagulase, bound and free.

24. 24
1. Slide coagulase test is done to detect bound coagulase or clumping factor.
2. Tube coagulase test is done to detect free coagulase.
GROWTH MEDIUM:
A growth medium or culture medium is a liquid or gel designed to support the growth of
microorganisms or cells, or small plants like the moss physcomitrella patens. There are different
type of media for growing different types of cells (Robert koch).
Selective media:
Selective media are used for the growth of only selected microorganism. Selective media such as
eosin methylene blue, macConkey agar, Hektoen enteric agar, xylose lysine deoxycholate agar
and salmonella shigella agar were inoculated with the culture for the identification of the
organisms .
Differential media:
Differential media or indicator media distinguish one microorganism type from another growing
on the same media. Differential media such as blood agar and macConkey agar (lactose
fermentation) were inoculated with the culture for the identification of the organism.
PURIFICATION OF FOREST HONEY:
Honey were collected from local markets in sterile screwed-cup container and kept in cool and
dry place in the laboratory for processing. Honey samples were first filtered with a sterile mesh
to remove the debris and were stored at 2–8 °C for further use.
ANTIMICROBIAL ACTIVITY:
Antibiotic evaluation test was performed on Mueller-Hinton agar using Kirby-Bauer method as
per clinical laboratory standards institute (Ho et al., 1998). Sterile disc were used to check the

25. 25
antimicrobial activity by diameter of the inhibition zone and was measured to determine bacterial
sensitivity as per clinical laboratory standards institute.
Test organisms
The clinical isolates such as Staphylococcus sp, E.coli, Proteus sp, Klebsiella sp were selected to
check the antibacterial study.the isolates were streaked and maintained in nutriend agar by
incubating at 370
C for 24 hours and stored in refrigerator for further use.
Determination of Antibacterial activity
Disc diffusion method was followed (Bauer et al., 1966) to determine the antibacterial
activity of honey. Petriplates containing 20 ml of Mueller-Hinton agar were seeded with 4 hours
old fresh culture of clinical isolates. By making use of template drawn extracts and (honey)
loaded discs were dispensed on the solidified Mueller Hinton agar with test organisms.
Ampicillin antibiotic disc (3µg/disc) obtained from M/s Hi-Media laboratories Ltd, Mumbai was
used as positive control for bacteria and empty discs were used as negative control. The plates
were incubated at 37°
C for 24 hours in an incubator .The test was performed in triplicates.The
zone of inhibition was measured by making use of Antibiotic zone scale (Hi - Media).
Various concentration of honey (20µl, 30 µl, 40 µl) were loaded on the disc with the respective
organisms.

26. 26
RESULT

27. 27
RESULT
Forest honey was collected and purified aseptically and stored in container for further
study. Plate- I & II shows and the purification of forest honey. Further the honey was checked
for its quality. Honey sample was streaked in Respective medium and there is no more microbial
growth. This shows the absence of contaminates hence the quality is good for further analysis
(plate III). Many articles reported that honey has a medicinal properties, which act against many
human pathogens.
Antimicrobial activity study was conducted to analyse the effect of honey against
pathogens. Clinical samples were collected and the study was carried out to identify the
pathogens. The organisms were finalized with the help of preliminary studies and biochemical
tests (Table 1) Staphylococcus sp, Proteus sp, Klebsiella sp and E.coli was confirmed. These
organisms were choosen to screen the antibacterial activity. Honey has been shown convincingly
to have a potent antibacterial activity, effective against a very broad spectrum of species. The
forest honey was choosen for our studies .it was purified by using sieve filter and heated at 37˚C
and stored for the analysis.
Staphylococcus sp shows high level of inhibition followed by Klebsiella sp, E.coli and
Proteus sp. Staphylococcus sp shows 25mm of zone in the highest concentration followed by
11 mm and 10mm in low concentrations. Followed by Staphylococcus sp, Klebsiella shows
14mm, 11mmand 10mm of zone of inhibition in various concentrations. E.coli recorded 12mm
in high concentration where as mild zone were observed in lower concentration. Comparing with
all the three microbes Proteus sp shows 2mm in lower concentration and 11mm in higher
concentration. For the entire organism 20, 30, 40 µl concentrations has been taken to find out the
antibacterial activity (Plate IV, Table 2, Fig 1 ).

28. 28
Our study reveals that forest honey shows the antibacterial effect against the human
pathogens. When comparing with the positive control honey shows excellent activity. In siddha
medicinal system honey act as a main source. Tribal peoples used this honey to cure many
disease and also it has wound healing properties. Hence honey is a best curative agent.

29. 29
DISCUSSION

30. 30
DISCUSSION
Nowadays we are facing challenges towards the diseases. Many allopathy medicines
have side effects and multidrug resistant pathogens are predominant. Ayurveda, Siddha, Unani,
homeopathy systems shows many curing process of disease without side effects. Honey is one
the agent which has been used as curative agents for wounds, Respiratory problems in Siddha.
So we choose Forest Honey for our studies.Lusby et al reported that honeys other than the
commercially available antibacterial honeys (e.g., manuka honey) can have equivalent
antibacterial activity against bacterial pathogens. Honey is effective when used as a substitute for
glucose in oral rehydration and its antibacterial activity shortened the duration of bacterial
diarrhoea. The organism we choosen shows sensitivity towards forest honey. We confirmed the
microbes with the help of various primary screening and biochemical test. These pathogens were
treated at various concentration of forest honey .Our study revealed that S. aureus, Proteus sp,
E. coli and Klebsiella sp, were more sensitive . This was supported by result of Getaneh et al.
from Ethiopia who conducted research on the in vitro assessment of the antimicrobial effect of
Ethiopian multi flora honey (Getaneh et al. 2013).The zone of activity was excellent in all the
choosen organisms. Staphylococcus sp and Klebsiella sp are highly sensitive when comparing
with E.coli and Proteus sp .
Our study reveals that honey has a excellent activity against some human pathogens.
This is preliminary analysis which shows the activity of forest honey against the pathogens.

31. 31
PLATE I -HONEY ON TREE

32. 32
PLATE II-HONEY PURIFICATION PROCESS

33. 33
PLATE III
QUALITY ANALYSIS
BLOOD AGAR

34. 34
PLATE IV
40µL
PROTEUS
40µL
30µL
20µL
N
P
E.COLI
P
30µL
20µL
N

35. 35
40µL 30µL
20µL
N
P
STAPHYLOCOCCUS
P
KLEBSHIELLA
40µL
30µL
20µL
N

36. 36
TABLE 1 - BIOCHEMICAL CHARACTERISATION OF
SELECTED PATHOGENS
S.NO TESTS Proteus Staphylococcus Klebsiella E.coli
1 Indole test + _ _ +
2 Methyl red test + + _ +
3 Voges proskauer test _ + + _
4 Citrate utilization test +/- + + _
5 Urease test + + _ _
6 Catalase test + + + +
7 Oxidase test _ _ _ _
8 Bile Esculin hydrolysis _ _ + _
9 Carbohydrate
fermentation test
+/- +/- +/- +/-
10 Coagulase test _ + _ _

37. 37
TABLE II - ANTIBACTERIAL ACTIVITY OF FOREST HONEY
S.NO ORGANISMS ZONE OF INHIBITION/mm/ µl
20 30 40 POSITIVE
CONTROL
NEGATIVE
CONTROL
1. Staphylococcus sp 10 11 25 M -
2.
Proteus sp 2 6 11 M -
3. Klebsiella sp 10 11 14 13 -
4. E.coli 2 6 12 10 -

38. 38
Figure 1 - ANTIBACTERIAL ANALYSIS
0
5
10
15
20
25
20µl
30µl
40µl

39. 39
SUMMARY
&
CONCLUSION

40. 40
Summary &conclusion
It has been shown that the potency of the antibacterial activity can vary very
markedly. The number of variable factors involved makes it impossible to predict with any
certainty that a particular honey will have a high antibacterial activity. Because of this, honeys
purveyed for therapeutic use should be assayed for their antibacterial activity as a form of quality
assurance. Consideration should also be given to the way that honey is processed if it is intended
for sale as an antibacterial product . Honey is often pasteurized, at a tem-perature of 70-75°C, to
destroy yeasts that can spoil a honey with a high water content, or to dissolve sugar crystals that
could initiate granulation in a liquid honey. In view of the short half-life of the antibacterial
activity at pasteurization temperatures, it is clear that pasteurization of honey is undesirable if the
honey is to be used as an antiseptic. It would also be advisable to keep any other warming of the
honey during processing to a minimum, and to store it at cool temperatures. Honey shows
excellent activity against the selected pathogens like Staphylococcus sp, Klebsiella sp,E.coli and
Proteus sp

41. 41
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