B.SC Project A review on Honeys antibacterial propertiess

Honeys
A Review on
Antibacterial properties
Said F. Fakhri
Harun Kucukyildiz
Shirin Ishaque
Supervisor: Sascha Liberti
Molecular biology
Roskilde University
2013-05-27
1
Abstract
Methicillin resistant Staphylococcus aureus (MRSA) can cause a wide range of
infectious diseases in health care facilities and community. There are only few
antibiotics that are useful against MRSA and that they may develop resistance to
these antibiotics is just matter of time. These issues emphasize the need to
develop alternative and effective methods to treat MRSA.
Honey has been used in many cultures through history to treat wound and in the
last decades honey has been rediscovered. There is a solid literature addressing
honeys antibacterial activity against a wide range of bacteria including many
MRSA strains.
This report sheds light on the extent of the problem with MRSA and discusses the
mode of action of honey and its potential to treat wounds infected with MRSA.
Honeys antibacterial activity is attributed to a multifactors, which includes
osmolarity, hydrogen peroxide, defensins, prostaglandins, acids and phenolics
and other non-peroxide compounds. Several active compounds in honey may act
in synergy with each other and thus enhance the property of honeys
antibacterial activity. Hereby honey has a good potential to be an effective
topical antibacterial agent for wounds infected with MRSA.
2
Abstrakt
Methicillin resistente Staphylococcus aureus (MRSA) kan forårsage en bred vifte
af infektionssygdomme i sundhedsinstitutioner og i samfundet. Der er få
antibiotika, der er brugbare mod MRSA, og det er sandsynligt at MRSA vil udvikle
resistens over for disse antibiotika før eller siden. Disse bekymringer
understreger behovet for at udvikle alternative og effektive metoder til at
behandle MRSA.
Honning er blevet brugt i mange kulturer gennem historien, til sårbehandling, og
i de sidste årtier er honningen blevet genopdaget. Der er adskillig litteratur, der
omhandler honnings antibakterielle aktivitet mod lang række bakterier, blandt
andre mange MRSA-stammer.
Denne rapport belyser omfanget af problemet med MRSA og diskuterer
virkningsmekanismen af honningen og dets potentiale til behandling sår
inficeret med MRSA.
Honnings antibakterielle aktivitet skyldes en multifactor, som inkluderer
osmolaritet, hydrogen peroxide, defensiner, prostaglandiner, syrer og phenoler
og andre ikke-peroxide forbindelser. Adskillige aktive komponenter i honning
kan virke i synergi med hinanden og dermed styrke honnings antibakterielle
egenskab. Herved har honning et godt potentiale til at være en effektiv topisk
antibakterielt middel til sår inficeret med MRSA.
3
Contents
Honeys................................................................................................................................................................... 1
Abstract................................................................................................................................................................. 2
Abstrakt ................................................................................................................................................................ 3
Introduction ........................................................................................................................................................ 5
Dermatology ....................................................................................................................................................... 6
Epidermis ........................................................................................................................................................ 6
The Stratums of epidermis .................................................................................................................. 6
Dermis .............................................................................................................................................................. 7
Wounds ................................................................................................................................................................. 8
Wound healing .............................................................................................................................................. 8
Hemostasis................................................................................................................................................. 9
Inflammation ......................................................................................................................................... 10
Proliferation ........................................................................................................................................... 10
Remodeling............................................................................................................................................. 11
Bacteria in wounds ....................................................................................................................................... 13
S. aureus ........................................................................................................................................................ 13
S. aureus infection and development ................................................................................................ 14
Development of resistance ................................................................................................................... 16
The Composition of Honey ........................................................................................................................ 21
Properties of honey.................................................................................................................................. 23
Osmolarity............................................................................................................................................... 24
Hydrogen Peroxide.............................................................................................................................. 24
Acids, phenolics and other compounds ...................................................................................... 24
Honey stimulates the immune system ........................................................................................ 25
Prostaglandins....................................................................................................................................... 26
Discussion ......................................................................................................................................................... 27
Conclusion ........................................................................................................................................................ 33
Perspective ....................................................................................................................................................... 34
References ........................................................................................................................................................ 36
4
Introduction
Staphylococcus aureus is a major cause of wound infection and the prevalence of
multidrug-resistant strains demands innovative interventions. This prevalence
of Staphylococcus aureus resistant strains with resistance determinants to
multiple antibiotics, both in healthcare settings and in the community,
represents a serious health threat. As consequence of increasing antimicrobial
resistance patient mortality and morbidity has increased [Lowy, 2003], which is
compounded by the lack of alternative antimicrobials in development. To
contribute to the combat of methicillin resistant Staphylococcus aureus (MRSA),
new perspectives are required to develop alternative and potent therapies.
There has been a renaissance in the use of honey for medical purposes in recent
times. Honey has been used as a medicine throughout the human history [Zumla
and Lulat, 1989]. It has recently been re-discovered into modern medicine. In
particular, there is a strong and supportive literature on the use of honey in
dressings used to treat infected wounds. In many studies honey was used when
antibiotic treatments had failed to clear the infection [Zumla and Lulat, 1989].
The aim of this report is to investigate and discuss the antibacterial activity of
honey in respect to Staphylococcus aureus on open wounds.
5
Dermatology
The skin is the largest of all the body organs accounting for about 16% of total
body weight [McLafferty et al., 2012]. The skin is arranged with different and
important functions. The thickness of the skin varies according to function and
area of the body from 0.5 to 4mm. In general the skin is 1-2mm thick [McLafferty
et al., 2012]. The composition of the skin is made of two distinct regions. The
superficial region, the epidermis, is thick epithelial tissue. Beneath epidermis is
dermis, a fibrous connective tissue [McLafferty et al., 2012].
Epidermis
The epidermis layer contains four different types of cells; keratinocytes,
melanocytes, Merkel cells and Langerhans cells. Epidermis gets metabolite
provision and removal of the metabolite waste products, by the blood vessels of
the underlying dermis layer [McLafferty et al., 2012].
Epidermis consists of five layers, which represent the different stages of cell
maturation and movement; stratum basale, stratum spinosum, stratum
granulosom, stratum lucidum and stratum corneum [McLafferty et al., 2012].
Figure 1: An overview of the skins composition, with the four layers. [McLafferty et al., 2012]
The Stratums of epidermis
The keratinocytes in the deepest stratum divides rapidly by mitosis and two
daughter cells forms and separates. One of them remains in the layer, while the
6
other moves up through the layers to the surface where it becomes keratinized,
which protects the skin from damages. As the cell migrates further away from
stratum basale it receives less nutrition from the blood supply of the dermis, and
as a result it dies. The process from mitosis to the migration of the daughter cell
up to the epidermal surface, takes approximately 28 days [McLafferty et al.,
2012].
When the migrating daughter cell from the stratum basale moves into stratum
spinosum, it lose the ability to divide, and as moving through the layer the cell
breaks down and reform to desmosomes. Desmosomes are intracellular bridges
that are daughter cells joined together. The arrangement of the cells contributes
to the tensile strength and flexibility of the skin [McLafferty et al., 2012].
In stratum spinosum, Langerhans cells which are a class of dendritic cells are
found, which are produced in the red bone marrow and following migrate into
the layer, where they act as immune response against foreign organisms which
may have invaded the epidermis [Sparper et al., 2010]. The immune response
occurs by the cells attract the microbes and starts phagocytosing them followed
by presenting their antigens to the T-lymphocytes. Hereby the T-lymphocytes
become activated and destroy the foreign cells.
As the cells migrate through the layers upwards, the keratinocytes become more
horizontally flattened and longer and makes up the stratum granulosum. The
cells die, and hereby lose their nucleus and become keratinized and become a
part of the protein keratin, keratohyalin [McLafferty et al., 2012].
The most external part of the epidermis is stratum corneum. This layer is of
keratin, which protects the skin from temperature, microbial or chemical
damage [McLafferty et al., 2012]. Moreover the lipid from the stratum
granulosum adheres to the cells and prevents the cells from drying out. As the
cells migrate outwards they lose their sticky-ness and parts in clumps
[McLafferty et al., 2012].
Dermis
This is the second major layer of the skin and as mentioned above it lies
underneath the epidermis. The dermis has lymphatics, nerve endings, hair
follicles and glands [McLafferty et al., 2012].
7
Dermis consists of two main layers; the reticular layer and the papillary layer.
The reticular layer is composed of strong connective tissue which contains
collagen and elastin fibres. The papillary layer is made up of nerves and
capillaries, which have the function to nourish the epidermis [Ventre et al.,
2009].
Wounds
The skin functions as a protecting agent for the internal tissues, and is exposed to
outer environmental germs and dangers, which can cause damage to the tissue. A
skin wound can be of two types; acute- or chronic wounds [Stadelmann et al.,
1998].
Acute wounds are those caused by exogenous exposure such as burns, cuts and
bites. These types of wounds are expected to heal after some time depending on
the type, depth and area.
Chronic wounds are associated with diabetes, vascular diseases, and immobility
resulting from strokes and traumatic paralysis, which all are by endogenous
mechanisms [Bowler et al., 2001]. Chronic wounds recover uncertainly and
slowly. Moreover chronic wounds are low in oxygen due to poor blood flow
[Bowler et al., 2001].
As a result of tissue damage, the body will respond by activating the survival
mechanism, which is an increase in blood pressure. The increase in blood
pressure depends on interaction between glucagon and adrenalin and reduction
in insulin level. This will raise the glycogenolysis in the liver [Stadelmann et al.,
1998].
Wound healing
The process of the wound healing has four stages; Hemostasis, inflammation,
proliferation and remodeling [Tredget et al., 1997]
8
Figure 2 The healing phases of the wound [Tredget et al., 1997]
Hemostasis
The hemostasis will rapidly occur when the tissue get damaged. It will response
by having vasoconstriction in approximately 5-10 minutes followed by
vasodilatation after 20 minutes of the damage [Stadelmann et al., 1998]. The
response is from the polyunsaturated fatty acid, which produces prostaglandins
and leucotrienes that has the vasodilatary- and the anti-inflammatory effect. This
makes the fatty acids responsible for the cell membrane structure and function
as a whole [Russell et al., 2001]. To prevent oxidative degradation of lipids in the
cell membranes caused by free radicals, vitamin E is necessary to act as an antioxidant and assist the healing [Russell et al., 2001].
Moreover, the hemostasis occurs with an increase of the capillary permeability
for the blood supply and any foreign objects to be pulled out [Stadelmann et al.,
1998]. The increase in the permeability leads to an increase in the plasma
proteins, albumin, from the serum, which also becomes available to enter the
interstitial area, which makes the wound healing easier [Stadelmann et al., 1998;
Russell et al., 2001].
Hereafter platelets initiate blood clotting, which has been mediated by the
prostaglandins. This aggregation of the plateletes helps to obtain hemostasis by
creating a fibrin clot [Tredget et al., 1997]. Moreover the platelets initiates their
release and activation of a cascade of immune system signal cells which then
triggers an immune response by recruiting anaphylatoxins which are a part of
the immune response and host defense [Tredget et al., 1997].
9
Inflammation
The immune response
Along with the capillary permeability there is an incoming flow of different
populations of cells. The polymorphnuclear leukocytes are most commonly the
first population in the wound, followed by the mononuclear leukocytes
[Stadelmann et al., 1998], where they mature into wound macrophages and
afterwards into leukocytes. The leucocytes and the macrophages use glucose for
the aerobe glycolysis, which provides energy for the production of the collagen
synthesis and fibroblast stimulating factors [Russell et al., 2001]. In the incoming
flow of the population of cells, vitamin A is included. Vitamin A helps fight wound
infections by replacing the glucocorticoids catabolic effect on the healing of
wounds [Russell et al., 2001]. The fibronectin, which is a glycoprotein, is
produced after 24-48 hours after the tissue damage. Fibronectin binds to the
collagen, fibrin and proteoglycans by which it creates a platform from where the
fibroblasts can migrate into the wound. The platform is for the formation of the
granulation tissue [Stadelmann et al., 1998].
Proliferation
The proliferation occurs 2-3 days after the wound formation, and starts
dominance within the first week [Stadelmann et al., 1998]. The first 2-3 days are
limited to only cellular replication and migration whereas collagen synthesis will
occur hereafter [Stadelmann et al., 1998].
Granulation tissue
This tissue is made up by the biosynthesis and degradation of the extracellular
matrix, which is made during the hemostasis –wound clotting [Tredget et al.,
1997]. The degradation of the matrix happens through collagenase,
proteoglycanase and other proteases, which are released by mast cells,
macrophages, endothelial cells and fibroblasts [Tredget et al., 1997].
The
fibroblasts are the dominant cells in wound healing, and give the primary
stimulus to the granulation tissue formation. The granulation tissue starts from
being rich in cells and ends up by being acellular matrix of collagen due to
apoptosis.
10
Collagen synthesis
Vitamin C is essential for the synthesis since the activity of the leucocytes and the
macrophages increase their activity level by uptaking vitamin C, at the tissue
damage, which leads to energy from the glycolysis [Russell et al., 2001].
Furthermore the collagen synthesis is stimulated by vitamin A, leading to tensile
strength and fast healing [Russell et al., 2001; Stechmiller, 2010].
The glycosaminglycans which are produced by the fibroblasts includes
hyaluronic acid, chondroitin-4-sulphate, dermatan sulphate and heparin
sulphate. These four glycosaminglycans makes up a gel-like substance called
“The ground substance”. This substance collects and moves the fibroblastproduced collagen fibers. The collagen is synthesized and secreted during the
first two-three days of the proliferation phase. The levels of collagen raises in
approximately 3 weeks until homeostasis are reached. The homeostasis reached
by the controlling collagenase, when the collagen degradation is equal to the
collagen synthesis [Stadelmann et al., 1998].
During the proliferation a process called angiogenesis occurs, which forms new
blood vessels from the pre-existing ones for the healing process’ metabolic needs
[Stadelmann et al., 1998].
The collagen synthesis starts as a monomer in an intracellular process, which
afterwards is secreted to the exogenous environment –the wound, where the
monomers polymerizes into collagen fibers. The appearance of the collagen
fibers is after one week, when it reaches the maximum rate [Stadelmann et al.,
1998].
Remodeling
Maturation
The remodeling phase starts after three weeks of the wound occurrence and can
continue up to two years [Stadelmann et al., 1998].
This phase depends on the factors of the host, including the age, duration of
inflammation, type and size of the wound and no exogenous environmental
contamination [Bowler et al., 2001].
11
Re-epithilization
This phase is a sign of the wound in good condition, as it represents the activity
of the epithelial cells [Stadelmann et al., 1998]. The epithelial cells increase when
mitosis maximizes, and here by the wound will be covered from one edge to
another edge [Stadelmann et al., 1998].
For understanding the mechanisms of honey in the healing process of wounds in
the skin, we need to study the bacterial activity relevant to wounds.
12
Bacteria in wounds
Several types of bacteria can attack skin wounds, which can infect these wounds.
The most common bacteria that contaminate skin wounds are Staphylococcus
aureus (S. aureus) and Streptococcus pyogenes.
In our study for the effect of honey for healing process on open wounds, we will
focus on S. aureus.
S. aureus
The increase of infections caused by multi-resistant bacteria, the so called
methicillin resistant S. aureus (MRSA), is a common problem in hospitals because
of their ability to get resistant against new developed drugs. This increase makes
it important to protect vulnerable patients more than ever and several
researches in this field strive to find cures or methods for prevention.
S. aureus exist almost all over the skin and in the nose. S. aureus can infect the
epithelial cells by colonizing the skin and soft tissue membranes in nose [Garzoni
et al., 2009].
Nearly 20 % of people worldwide are so called persistent carriers of S. aureus,
and 60% are intermittent carriers who have been carriers of some strains in part
of their life. The rest of the population is thought to never have any S. aureus in
their body [Kluytmans et al., 1997].
The persistent and intermittent carriers are more exposed to become infected by
MRSA than others and once infected, the infection can spread to all of the body
organs through the blood and hereby cause bone tissue damage and
cardiovascular diseases [Garzoni et al., 2009].
S. aureus interacts with cell surface proteins and make a firm interaction to
protect itself from the host’s body defense system which can eliminate it.
Adherence of the S. aureus to the cell surface requires physicochemical,
hydrophobicity and the affinities of bacterial cell surface moieties that recognize
the surface membrane receptor proteins of the cell [Kluytmans et al., 1997].
Adherence of S. aureus in the vestibulum nasi depends on the permeability of the
surface proteins of epithelial cells. The vestibulum nasi consists of soft tissue and
hair and there is no big barrier from the body’s defense system in this area
against S. aureus to infect the cells. Thus preventing or minimalizing the
13
spreading of S.aureus it is necessary to treat the area before the migration of the
bacteria to rest of the body. Once it has infected the rest of the body, it will cause
other diseases and will be complicated to treat.
S. aureus infection and development
S. aureus is one of the most frequent organisms to cause pathogenic infections in
human and animals. It is necessary to understand the mechanism of infection in
order to develop improved treatments.
Investigation of penicillin in the beginning of the 1940’s was the biggest
milestone for researchers to treat bacterial infections. In the following twenty
years the first penicillin resistant bacteria was discovered and isolated from
hospitals. S. aureus resistance to penicillin is sustained by an enzyme called βlactamase that is encoded by blaZ gene. This enzyme is produced when S. aureus
is attacked by β-lactam antibiotics [Lowy, 2003].
The resistance led to development of semi-synthetic derivatives of penicillin.
Methicillin has been used to treat infections as many other derivatives of
penicillin. However, recent years of investigations show that almost 60% of
isolates from hospitals are now MRSA. Another antibiotic; Vancomycin, is being
used to treat infections caused by MRSA but still in some places the bacteria has
been able to develop resistance [Honeyman et al., 2002].
Transmission of MRSA is simple and it is commonly spread in nursing homes and
hospitals. In most cases the infection spreads from hand to hand among
healthcare workers, other staff and patients. Intravenous drug users, burnwound patients and patients that have surgical wounds are highly exposed to
MRSA [Schmitz et al., 1997].
S. aureus infects cells with several coordinated actions of virulence to survive
since the cells are contaminating the food and environment for that purpose
[Honeyman et al., 2002].
MRSA contaminates penicillin-binding protein (PBP) or in some cases called
PBP2A or PBP` which have enzyme like lactamase that inhibit β-lactam
antibiotics to bind. This ability makes that antibiotics like penicillin cannot bind
efficient to their target on the S. aureus bacterial cell-wall. [Schmitz et al., 1997;
Hacbart et al., 1989].
14
Like all other bacteria the first step of infection to the host cell is related to the
ability of the binding to the host cell surface. S. aureus overcomes this barrier
with its cell surface proteins. Fibronectin is part of the cell surface matrix of S.
aureus as in epithelia and endothelia cells [Rohilla, 2010]. When bacteria are
attached to the cell they start to grow until they are ready to infect the cell and
protect themselves against the host’s body defense system. S. aureus manages
this with the protein A(ll) and capsules which contains polysaccharides (cf.
figure 3) [Honeyman et al., 2002].
Figure 3 The black circles are the capsules that surrounding S. aureus. These capsules contaminate
polysaccharides and protect S. aureus from environments such like host defence system and phagocytes. S.
aureus attached the host body with protein A and endocytose that may release into matrix of host-cell.
Leucocidins and hemolysins are the exoproteins toxins of the S. aureus and if needed they lyse the cells like
erythrocytes platelets which are part of host defence system [honeyman et al., 2001].
Vancomycin is a very efficient drug used now a days against MRSA. It is a
glycopeptide antibiotic. Glycopeptides are large molecules that inhibits bacterial
cell-wall synthesis leading to destruction of S. aureus. Studies show that
vancomysin is only efficient when interacted with other drugs if there are
vancomysin resistant strains. However, it must be used in a long term process to
have an effect against MRSA Vancomycin resistant strains. [Schmitz et al., 1997;
Honeyman et al., 2002;Tenover et al., 2004].
15
On other hand Vancomycin has a high level toxicity.As an alternative to
Vancomycin, e.g. teicoplanin is used for patients who cannot tolerate high
toxicity of Vancomycin [Schmitz et al., 1997].
The increase in number of patients infected with S. aureus and particularly MRSA
has led the attention to find a solution to prevent the bacteria spread.
Additionally, production of new drugs is required to treat infected patients with
the bacteria. Several studies have focused on factors responsible for spreading
the bacteria [Projan and Youngman, 2002; Projan, 2002].
Development of resistance
As mentioned earlier, years after penicillin was developed, new strains of
bacteria became resistant to the drugs, which caused difficulty in treating
diseases. It has become a big problem both for hospitals and other health-care or
general public-care centers to discover new drugs that may be efficient against
bacteria. This problem will continue the following years and therefore the
development of new drugs against multi-resistant bacteria is now more
necessary than ever.
Development of new drugs demands understanding of the mechanisms of the
bacteria and how they become resistant to antibiotics. The extent of the problem
will be clear and one can look forward for the solution.
S. aureus become resistant either by insertion of DNA from plasmids or
transposons in chromosomes or by having mutation in the chromosomes.
MRSA contaminate strains from plasmids that make it resistant or more stable
against drugs [Lowy, 2003]
Bacteria like every other living organism need nutrients to live and grow. The
anti-bacterial drugs are therefore very efficient if they can stop or destroy some
of those mechanisms.
As mentioned above MRSA are resistant to most of the antibiotics we have now.
Only few drugs that are efficient against MRSA is a matter of time that it also will
develop resistance to those drugs. Moreover, resistant bacteria are also very
16
expensive to treat and very dangerous for people with weakened immunity or
for older people and children.
The antibacterial drugs interfere with some of the targets to inhibit the cell
development of S. aureus. Some of these targets are intracellular and some are on
cell surface[Lowy, 2003]. (cf. figure 4)
Figure 4 shows the different antibiotics and their target side on S. aureus. The most efficient drugs like
Vancomycin target the bacterial cell-wall synthesis [Rohilla, 2010]
The drugs inhibit the cell-walls synthesis (β-lactam, vancomysin and teicoplanin
interfere
with
bacterial
cell
wall
synthesis),
nucleic
acid
synthesis
(fluoroquinolones, sulfonamids and trimethoprim(TMP)), protein synthesis
(macrolides, aminoglycosides and tetracyclines use differences between
bacterial and eukaryotic ribosomes) or metabolic pathways [Tenover et al.,
2004].
In order to develop resistance to all of the above mentioned drugs the bacteria
has to change some of their components and make it difficult for the drugs to
reach their target site and perform its function.
Vancomycin is one of the very efficient drugs against MRSA which is in use to
date.. It forms a complex with peptydoglycan side chain of D-alanyl-D-alanine
terminus. This is different from β-lactam antibiotics contaminating fem
membered ring. But there are reports which indicate resistant strains of MRSA
against it. Tenover et al., 2004 shows how MRSA become resistant to
Vancomycin.
17
As mention above Vancomycins side chain peptidoglycan called D-alanyl-Dalanine terminal and D-alanine is produced from L-alanine. Vancomycin resistant
MRSA changes binding site for this dipeptide to D-alanine-D-lactate and with this
way lover the binding-ability of Vancomycin with 1000 fold. (cf. figure 5 )
Figure 5 Shows how the bacteria develops resistance against Vancomycin. With changes in the DAla-D-Ala residue to D-Ala-D-Lac residue, the bacteria inhibits the possibilty of Vancomycin to bind
to it and hereby it allows continuation of the cell-wall synthesis [Lowy, 2003].
18
Figure 6 Shows two different cell types, Vancomycin susceptible and Vancomycin resistant cells. The
Vancomycin intermediate resistant S.aureus (VISA) strains bind to Vancomycin with D-Ala-D-Ala
residues. This mechanism prevents Vancomycin to reach its target and inhibits the cell division.
[Lowy, 2003].
On the other hand MRSA develop a new binding site for methicillin called
penicillin-binding protein (PBP2a) that contains mecA determinants. MecA has
high levels of gene vanA that lowers the binding affinity of the most drugs
containing β-lactam [Severin et al., 2005]. β-lactam contains a four membered
ring, which undergoes conformation change with trans-peptidases [Rohilla et al.,
2010].
Both mechanisms work together in newly found strains of Vancomycin resistant
S. aureus (VRSA).
Bacterial cell walls are covered with peptidoglycan which is very important for
their survival. If the peptidoglycan becomes damaged by drugs the bacteria will
lose the cell wall and die [Rohilla et al., 2010].
19
Figure 7 a) shows the mechanism of action by β-lactamase when S. aureus is attacked by penicillin.
BlaI (DNA-binding protein) binds to the operator region that induces repressing of RNA
transcription of blaZ and blaR1. Penicillin binds to BlaR1 and stimulates to autocatalytic activation.
Than BlaR1 cuts BlaI and this allows transcription of blaZ and blaR1. blaZ encodes β-lactamase and
hydrolyze β-lactam ring from penicillin. B) Shows the mechanism of MRSA when it is exposed by βlactam antibiotics. When the antibiotic is induced begins mecR1 synthesis and mecI become
inactivated. This leads to synthesis of PBP2a [Lowy, 2003].
20
The Composition of Honey
Honey is a complex mixture that contains many different substances, both from
organic and inorganic origin. The primary ingredients of honey are sugar and
water. The water content of honey is in average 17.2% but varies depending on
types. The low water activity of honey inhibits bacterial growth as well [Song
and Salcido, 2011].
The content of honey is particularly high in carbohydrate materials, with 95 to
99 percent of the solids being sugars. The identity of these sugars has been well
studied. Glucose and fructose are the major sugars in honey in average 38.2 and
31.3%, respectively. However many other sugars have been discovered (table 1)
[Doner et al., 1977; Stefan et al., 2008].
Ten Disaccharides: Ten Trisaccharides:
Sucrose
Melezitose
Maltose
3-a-isomaltosylglucose
Isomaltose
Maltotriose
Maltulose
l-kestose
Nigerose
Panose
Turanose
Isomaltotriose
Kojibiose
Erlose
Laminaribiose
Theanerose
a, B-trehalose
Centose
Gentiobiose
Isopanose
Table 1 Honeys primary sugars are dextrose and levulose. However 22 other sugars have
been found as well. Ten disaccharides, ten trisaccharides and two more complex sugars,
isomaltoteraose and isomaltopentaose have been identified. Most of these sugars are
present in very small quantities [Doner, 1977; Bogdanov et al., 2008].
In addition, honey contains amino acids, minerals, vitamins, acids, enzymes, and
nitrogen [de Rooster et al., 2008]. The amount of nitrogen in honey is quite low,
it ranges between 0.04-0.1 percent. Numerous amino acids are vital to life and
must be taken through food. However, the amount of free amino acids in honey is
quite small and a recent study has revealed that various honeys contain eleven to
twenty-one free amino acids [Carratú et al., 2011]. (table 2).
21
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
Alanine
Asparagine
Aspartic acid
Cysteine*
Glutamic acid
Glutamine*
Glycine*
Proline*
Selenocysteine*
Serine*
Tyrosine*
Arginine*
Histidine*
Table 2 Amino acids are the “building blocks” of the proteins. With chromatography it has
been revealed that various honeys contain 11 to 21 free amino acids. These are some most
essential to life. Isoleucine are the most common and with proline predominating.
It has been known for many years that honey contains small and variable
amounts of protein approximately 0.2 percent of bee and plant origin [Babacan
and Rand, 2005].
Enzymes are large biological molecules, and are one of the most essential
elements present in every cell in our body.
The main enzymes in raw honey are; invertase, diastase and glucose oxidase.
There are a number of other important enzymes that are reported to be present
in honey, including catalase and an acid phosphatase.
Honey is rich in minerals as well. Some scientists studied the mineral content of
honey and they reported the following minerals (table 3) [Bogdanov et al., 2007].
22
Mineral content in honey:
Potassium
Chlorine
Sulfur
Calcium
Sodium
Phosphorus
Magnesium
Silica
Iron
Manganese
Copper
Table 3 Honey contains varying amounts of mineral substances. But the amount of these
elements depends on environmental, geographical and botanical factors. [Bogdanov et al.,
2007]
Properties of honey
Honey has a complex composition and numerous interesting properties, which
depend on the type of honey [Sherlock et al., 2010; Tan et al., 2009]. The
antibacterial activity of honey is increasingly valued with an increase in the
development of antibiotic-resistant bacteria. It has been reported that honey
exhibits broad-spectrum antimicrobial activity that ranges to around 80 species
of bacteria including aerobes and anaerobes, gram-positives and gram-negatives
[Molan, 1992; Blair and Carter, 2005]. Recently, several publications describe
both in vitro and in vivo experiments suggesting inhibition and eradication of
wound pathogens by honey, with both antibiotic-resistant, including MRSA, and
antibiotic-sensitive strains exhibiting susceptibility to honey [Willix et al., 1992;
Cooper et al., 2002; Karayil et al., 1998; Cooper et al., 2002]. The antibacterial
activity of honey may be due to multiple mechanisms working either singularly
or synergistically. High sugar content, low acidity, low water content, hydrogen
peroxide production, phytochemicals, or other unidentified components support
the activity of honey on bacterial growth [Molan 1992; Mavric et al., 2008]. All of
these factors have toxic effects on bacteria that may affect their metabolism and
structure.
23
Osmolarity
The antibacterial activity of honey has been assumed to be a result of the osmotic
effect from high sugar content. Honey has a high osmolarity effect, which inhibits
bacterial growth. The sugar molecules tie up the water molecules and thereby
dehydrate the bacteria [Chirife et al., 1982; Chirife et al., 1983; Bose 1982]. The
osmolarity of honey is valuable in treatment of infected wounds to control
infection and to enhance wound healing [Bagdonov 1984; Knutson et al., 1981].
Furthermore, it has been shown that infected wounds with Staphylococcus
aureus quickly become sterile after treatment with honey [Efem 1988; Cavanagh
et al., 1970; Armon 1980; Cooper et al., 1999].
Hydrogen Peroxide
Hydrogen peroxide in honey is produced enzymatically by glucose oxidase which
catalyzes conversion of glucose in the presence of oxygen and water.
[Brydzynski, 2006]. Hydrogen peroxide is generally assumed to be the main
compound responsible for the antibacterial activity of honey [White et al., 1963;
Weston, 2000; Brudzynski, 2006]. Hydrogen peroxide has been shown to inhibit
a wide range of microorganisms, including some health-care-associated
pathogens e.g., S. aureus [Brydzynki et al., 2011].
The mechanism of hydrogen peroxide is by producing destructive hydroxyl free
radicals that can attack membrane lipids, DNA, and other vital cell components
[Brydzynski et al., 2011].
Another study made by Brydzynski et al., 2011 showed that the hydrogen
peroxide in honey was involved in oxidative damage causing bacterial growth
inhibition and DNA degradation. However, catalase that is produced by aerobic
organisms and anaerobes can stop the hydrogen peroxide activity. Catalase
protects cells from metabolically produced hydrogen peroxide by degrading it to
water and oxygen [White et al., 1963; Bang et al., 2003; Turner 1983; Block,
1977].
Acids, phenolics and other compounds
The acidity of honey plays a role in preventing the growth of many bacteria. The
pH of honey is usually between 3.2 and 4.4. This low pH of honey is due to the
presence of several different organic acids, which are formed in honey from
24
glucose into the hydrogen peroxide and gluconic acids by glucose oxidase
[Vandenbroucke et al., 2010].
Additionally, in pure unprocessed honey, there are a range of other, largely
uncharacterized compounds that may be missed in processing and fractionation.
However, some compounds have been identified including flavonoids, phenolic
acids, and methylglyoxal that exhibit a wide range of biological effects. Their
presence in honey explains its antibacterial activity. However, the precise
mechanisms are still unknown [Almahdi and Kamaruddin, 2003; Weston et al.,
2000; Russel et al., 1990].
Honey stimulates the immune system
Previous clinical data made by Tonks et al., indicate that honey stimulate
inflammatory cytokine production from human monocytes [Tonks et al., 2003].
The researchers from this group used the monocytic cell line, MonoMac-6
(MM6), as a model. Their study showed that honey significantly increased the
TNA-alpha, IL-1beta and IL-6 release from MM6 cells, compared with untreated
and artificial honey-treated cells. The results suggest that the effect of honey on
wound healing might partially be associated with the stimulation of
inflammatory cytokines from monocytic cells. These cell types are known to play
a critical role in healing and tissue repair [Tonks et al., 2003].
In addition, recent research shows that honey stimulates proliferation and
activation of peripheral blood B-lymphocytes and T-lymphocytes in cell culture
at a concentration as low as 0.1%. Further, phagocytes are activated by honey at
a concentration as low as 0.1% [Abuharfiel et al., 1999]. These results might shed
light on the activating the immune response to the infection.
Another current report made by Vandenbroucke et al. discovered bee defensins
in honey [Vandenbroucke et al., 2010]. Defensins are peptides that are abundant
and widely distributed in human and animal tissues. Defensins protect mucosal
epithelia and skin against microbial infections and are produced in large
amounts by neutrophils. Defensins function by interacting with the microbial
membrane, once embedded, they cause the formation of membrane wormholes
or pores which allow efflux of essential ions and nutrients from the cell [Ganz,
2003].
25
Prostaglandins
Prostaglandins are mediators of inflammation and pain. They are generally
considered as immunosuppressive, and reduce immunity by decreasing many Band T-lymphocyte functions [Phipps et al., 1991]. A study made by Al-Waili,
2005, revealed that honey can lower plasma prostaglandin concentrations in
healthy individuals. The inhibitory effect of prostaglandin was increased with
time. The mechanism of action might be due to either cyclooxygenase 1 (COX-1)
or cyclooxygenase-2 (COX-2), or both. However, they found that artificial honey
increased prostaglandin concentration [Al-Waili, 2005]. Hence, raw honey might
contain some active compounds that are capable of inhibiting prostaglandin
synthesis. Further, this ability of honey to lower prostaglandin concentration
could shed light on several biological and therapeutic effects, mostly those
related to inflammation, pain, immunity, and wound healing [Al-Waili, 2005].
26
Discussion
Within the past 20 years there has been an increasing focus on the global
problem of antibiotic resistant bacteria. Unfortunately, an increasing number of
pharmaceutical industries have reduced or completely eliminated the
development of new antibiotics. This is resulted by multiple factors, however,
financial considerations are the main reasons [Projan, 2002]. This has resulted in
clinicians and doctors around the world are losing the battle and hospitals will
soon run out of effective drugs. This is a concern because with fewer effective
antimicrobial agents to manage wound infections, results in an increased
morbidity, treatment costs and mortalities [Filius and Gyssens, 2002]. The
difficult therapeutic problem of MRSA is just one example of diminishing efficacy
of antimicrobial agents for treatment of bacterial infections. These issues
emphasize the need for the development of new antibiotics [Projan and
Youngman, 2002; Projan, 2002], but whenever a new inhibitory drug is
introduced, the resistance to these drugs will arise. This trend is especially
alarming and critical for Staphylococcus aureus because of the severity and
diversity of disease caused by this uniquely versatile pathogen. To combat the
resistant bacteria strains to multiple antibiotics, new perspectives are required
to develop alternative, effective non-antibiotic drug treatments.
Honey has recently become the focus of attention for treating certain diseases as
skin inflammation and burn wounds as well as stimulating overall health and
well being. Honey has well characterized properties: antibacterial, antimicrobial,
anti-inflammatory and wound healing [Lusby et al., 2005; Mundo et al., 2004;
Simon et al., 2009; Molan, 2006; Medhi et al., 2008; Cooper et al., 2010; Robson et
al., 2009]. Honey has been subjected to laboratory and clinical investigations
during the past few decades [Emsen, 2007; Zumla and Lulat, 1989; Allen et al.,
1991]. To date there are numerous studies reporting both in vitro and in vivo on
the therapeutics properties of honey, which have confirmed its activity against a
wide range of bacteria including pathogens such as MRSA [Taormina et al., 2001;
Willix et al., 1992].
The antibacterial activity of honey is attributed largely to high sugar content, low
acidity, low water content, hydrogen peroxide production, presence of some
27
phytochemicals, or other non-peroxide compounds [Molan, 1992; Mavric et al.,
2008]. Overall, honey has a restraining influence on the growth of most bacteria,
including some MRSA strains. Clinical observations on wounds showed many
beneficial qualities of honey. Topical use of honey has showed the rapid
clearance of infections [Braniki, 1981; Efem, 1993; Efem, 1988; Phuapradit and
Saropala, 1992; Armon, 1980], heal deeply infected surgical wounds [Cavangh et
al., 1970; Armon, 1980; McInerney, 1990; Bergman et al., 1983; Vardi et al.,1998;
Al-Waili and Saloom, 1999; Cooper et al., 2001], and to prevent progress of
necrotizing fasciitis [Efem, 1993; Hejase et al., 1996]. It has healed wounds,
which has not been succeed and not responding to conventional therapy with
antibiotics and antiseptics [Efem, 1988; Vardi et al., 1998; Cooper et al., 2001;
Wood et al., 1997], including wounds infected with MRSA [Al-Waili and Saloom,
1999; Natarajan et al., 2001; Dunford et al., 2000]. Also, honey has been reported
to rapid suppression of inflammation [Subrahmanyam, 1988], to reduce edema,
and exudate. In addition, using honey has been reported to minimization of
scarring, and to stimulate the growth of tissue granulation and epithelium [Efem,
1993; Efem 1988; Hejase et al., 1996; Subrahmanyam, 1996; Dumronglert, 1983;
Dunford
et
al.,
2000;
Subrahmanyam,
1991;
Subrahmanyam,
1988;
Subrahmanyam, 1994]. All these healing properties are due to the several
physical and chemical factors, which makes honey unique as a wound dressing.
A study made by Cooper et al., 2010 tested whether honey has the potential to
select for honey resistance. They isolated four cultures from wound inclusive
Staphylococcus aureus and MRSA and exposed to sub-lethal concentrations of
manuka honey in continuous and stepwise training experiments to determine
whether the susceptibility to honey diminished. Their study showed that
continuous exposure to sub-lethal concentrations of manuka honey for up to 28
days failed to select for honey-resistant mutants in both S. aureus and MRSA.
Staphylococcus aureus were successively cultured in stepwise increasing
concentration of honey, and were not recovered above their starting minimum
inhibitory concentration (MIC) values. However, reduced susceptibility to
manuka honey was observed throughout the long-term training periode in
MRSA. The MIC of honey increased in MRSA by factor of 1.6, respectively.
However, the susceptibility was increased again during cultivation in honey-free
28
nutrient broth and during storage at -80 degrees. It is unknown why the
susceptibility increased during the storage at -80 degrees. This study has
confirmed and extended those from Blair et al., 2009. Their findings show that
increasing topical use of honey in wounds, will the possibility of selecting for
honey-resistant wound pathogens arise [Cooper et al., 2010; Blair et al., 2009].
However, using a medical-grade honey killed eight species of problematic wound
pathogens, including MRSA by 4.0-14.8% honey [Blair et al., 2009]. Thus, this
concentration can be maintained in the wound environment. However, this will
not give rise to clinical failure, because the concentration of entitle honey e.g.
medical-grade manuka honey, that are contained in contemporary licensed
wound care products usually exceed 80% (w/v) and many are 95% (w/v)
[Cooper et al., 2010]. In addition, topical management of honey to wounds will
always result in dilution, depending on the extent of exudation. Thus, we suggest
regularly dressing changes to keep levels of honey high, especially in highly
exuding. Furthermore, honey is more difficult for bacteria to develop resistance,
compared to antibiotics [Blair et al., 2009]. Hence, it is suggested to use a whole
product of honey and because the active components which is purified and used
alone, is more likely to promote the development of honey-resistant bacteria.
The whole product of honey acts in a unique and multifactorial way, hence,
promoting honey resistant is minimal. Taken together, all these data indicates
that honey is an effective topical antibacterial agent as a wound dressing.
Also, It was found that high osmolarity of honey inhibits bacterial growth
because the glucose molecules tie up water molecules so the bacteria die of
dehydration. The osmolarity is useful in treatment of skin wounds because it
prevents the growth of bacteria and boosts the healing process [Bagdonov, 1984;
Chrifie et al., 1982]. A study made by Jenkins et al., 2011 showed that 10%
artificial honey (AH) (w/v) did not inhibit MRSA, while 10% manuka honey (MH)
did [Jenkins et al., 2011]. However, the artificial honey consisted of a solution of
four predominant sugars (glucose, fructose, maltose and sucrose) in which they
occur in the natural product. The differences between AH and MH is that AH not
containing all sugars which MH contains. Therefore, this could be more
comparable if the AH consisted of all sugars which natural honey has. However,
29
this emphasize that honey may content besides other compounds, which may
have antibacterial activity. On the other hand, AH and MH contained same
concentration (10%) sugar in this study, which showed different results. This
indicate that the sugar in honey has not antibacterial activity at all because if it
was sugar that have the antibacterial activity, might be the same result in both
artificial and natural honey. Therefore, it can be other compounds than sugar
that may have antibacterial activity.
In addition, hydrogen peroxide in honey has been assumed to be the main
compound responsible for the antibacterial activity [White et al., 1963; Weston,
2000; Brudzynski, 2006]. However, some organisms can stop the hydrogen
peroxide activity by producing catalase [White et al., 1963; Bang et al., 2003;
Turner 1983; Block 1977]. Hence, organisms with high cellular catalase activity
e.g. S. aureus requires longer exposure time (30-60 minutes) to 0.6% hydrogen
peroxide for a 108 reduction in cell counts, than organisms with lower catalase
activity e.g. Pseudomonas species which requires 15 minutes exposure [Schaeffer
et al., 1980]. Although, the concentration of hydrogen peroxide in honey is
approximately 1000 times less than the solution they use as an antiseptic
[Molan, 1992]. This low amount of hydrogen peroxide is intrinsically not enough
for an antibacterial activity, but these effects might be modulated by other
compounds. Therefore, our understanding for honeys antibacterial activity will
be limited if the focus solely is on hydrogen peroxide. However, current study
made by Sherlock et al., 2010 showed that other compounds in honey enhanced
the hydrogen peroxide antibacterial activity [Sherlock et al., 2010]. Another
study made by Vandenbroucke et al., 2010 demonstrated the antibacterial
activity of honey depends on combined interaction between methylglyoxal,
hydrogen peroxide and bee defensin-1 [Vandenbroucke et al.,2010].
Defensins are natural occurring peptide of the innate immune system. These
peptides have shown a wide range of antimicrobial activity. However, several
strains of MRSA have shown to be resistant to defensins [Midorikawa et al.,
2003]. A study has demonstrated that honey contains bee defensin-1 and was
effective in interaction with other contents of honey against MRSA. Defensins in
honey has not enough antibacterial activity itself, but it contributes to the
30
activity of honey against several bacteria where presences of other compounds
were required [Vandenbroucke et al., 2010].
Latest, it has been shown that honey down-regulated a protein called universal
stress protein A (UspA). UspA is discovered in many microorganisms including
MRSA. This protein is responsible for stress factors e.g. heat, starvation and
antimicrobial agent [Jenkins et al., 2011]. A removal of this protein would make
the bacteria more sensitive to stress and DNA-damaging agents. Cells treated
with honey lower the expression of UspA protein 16-fold, compared with
untreated cells [Jenkins et al., 2011]. This could shed light on the eradication and
limitation of bacteria from wounds. Additionally, Müller et al., 2013 examined
whether there was any synergy between rifampicin and Medihoney. Rifampicin
is a widely used antibiotic. However, Honey has been reported to have a
synergistic activity between rifampicin, whereas sugar solution failed to show
any synergy. They found an increased sensitivity against both S. aureus and
MRSA when honey combined with Rifampicin [Müller et al., 2013]. This study
emphasizes the potential of synergistic activity of honey between antibiotics in
the treatment of S. aureus on skin wound. Further research requires clarifying
synergy between honey and rifampicin in vivo. In addition, we suggest that
honey may contain unidentified compounds, which may down-regulate some
genes of MRSA. This may result in that MRSA become sensitive and susceptible
to rifampicin.
In the light of the results mentioned above, further investigation needed to
clarify whether antibiotics, that are now unable to treat MRSA, can be combined
with honey and make it efficient or not.
Moreover, agar dilution and broth dilution methods have been used to determine
the minimum inhibitory concentration (MIC) of honey. The MIC values are
decisive for the results, by being responsible to determine whether honey has
high or low antibacterial activity. The low MIC value indicates high bactericidal
activity of honey. However, there is a small variation between different MIC
values in different studies. This could be a result of their methods used. The most
common method to testing honey is broth dilution. Compared with agar dilution,
the MIC values determined by broth dilution were lower. This may indicate that
active compounds in agar may be slower than those in broth [Okeke et al., 2001;
31
Tan et al., 2009]. Although, in agar dilution method honey is incorporated
directly into the growth media, which could be useful to skin wound bacteria,
since the honey is in direct contact with bacteria.
32
Conclusion
The aim for this project was to investigate and discuss the antibacterial activity
of honey in respect to Staphylococcus aureus on open wounds. This project
provides insight into the mode of action of honey in the inhibition of methicillin
resistant Staphylococcus aureus (MRSA). In conclusion, honeys antibacterial
activity is multifactorial; meaning that the antibacterial effect cannot be solely
attributed to any single compound. All active compounds in honey may act in
synergy with each other and it is probably that a multitude of effects of honey
are due to more than one of its compounds. Taken together, all these active
factors indicate that honey is an effective topical antibacterial agent for wounds
infected with MRSA. In light of the enormous potential for application of honey in
treatment of skin wounds to prevent the growth of MRSA and enhance the
healing process, it is important to use honey as a whole product. However,
further investigations are required to clarify the precise mode of action of honey
as an antibacterial agent both in vitro and in vivo.
33
Perspective
This report has revealed the extent of antibacterial effect of honey, with focus on
S.aureus. However, it is still unknown, precisely how honey has an effect on S.
aureus in wounds. Further investigations in vitro and in vivo are required in
order to understand the specific molecular mechanisms of honey that effect
bacteria in wounds.
Rifampicin is now a useless drug against S. aureus. An experiment with
interaction between honey and rifampicin, would be able to show whether the
genes become up- or down regulated.
The aim of these experiments would be to make the useless antibiotical drugs
useful again by combining them with honey in future treatments.
Research with wound healing is important and minor results can have a major
impact in the future treatment of patients with wounds.
Properties of honey have demonstrated that certain types of honey, of which
many have been tested in the laboratory, are promising pharmacological agents
for antibacterial effects. However, clinical trials must be performed to completely
validate honey as a medicament in medical applications.
For an optimal healing effect of the wound, infected with S. aureus, another
clinically research could investigate different carbohydrate concentrations in
bacteria from wounds, for determination of the macrophages efficiency in
patients and potential computing significance graphs that present the
comparisons of the macrophages activity with carbohydrates and without
carbohydrates as control. This research would investigate honey’s composition
of carbohydrates, which among others consists of sugars, which further are
required by the macrophages and leucocytes in the wound healing process.
To prevent oxidative degradation of lipids in the cell membranes caused by free
radicals, vitamin E is necessary to act as an anti-oxidant and assist the wounds
healing. Free radicals, vitamin E and anti-oxidants are already determined in
minor concentrations in honey. Further research could investigate the difference
between neutral compounds as free radicals, vitamin E and anti-oxidants
34
extruded from honey with the same compounds but synthesized, on bacteria
from wounds and reflect the data by significance graphs.
Adherence of the S. aureus to the cell surface requires physicochemical,
hydrophobicity and the affinities of bacterial cell surface moieties that recognize
the surface membrane receptor proteins of the cell. Further research could show
the effect of honey on the S. aureus cell surface hydrophobicity by using twophase aqueous micelles systems
35
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