Antibacterial activity of honey on pseudomonas species

1. ANTIBACTERIAL ACTIVITY OF HONEY ON
PSEUDOMONAS SPECIES
INTRODUCTION:
The use of traditional and herbal medicine to treat infections was
practiced since the origin of mankind, and it was the only option to treat
before the ear of antibiotics . A variety of plants and their extracts have
been used for the treatment requiring antimicrobial activity, and one of
the popular natural antimicrobial substances described in the ancient
medicine is honey.
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 . Though honey is used widely in traditional
medicine, its use in modern medicine is limited . 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 .
The high osmotic nature and naturally low pH (3.2–4.5), ability to
produce hydrogen peroxide, which plays a key role in the antimicrobial
activity of honey and phytochemical factors such as tetracycline
derivatives, peroxides, amylase, fatty acids, phenols, ascorbic acid,

2. terpenes, benzyl alcohol and benzoic acid are factors attributed honey to
have potent bactericidal and bacteriostatic activity against pathogenic
bacteria.
Currently, many researchers have reported the antibacterial activity
of honey and found that natural unheated honey has some broad-
spectrum antibacterial activity when tested against pathogenic bacteria,
oral bacteria as well as food spoilage bacteria. In most ancient cultures
honey has been used for both nutritional and medical purposes. The
belief that honey is a nutrient, a drug and an ointment has been carried
into our days, and thus, an alternative medicine branch, called
apitherapy, has been developed in recent years, offering treatments based
on honey and other bee products against many diseases including
bacterial infections. At present a number of honeys are sold with
standardized levels of antibacterial activity. TheLeptospermum
scoparium (L. scoparium) honey,the best known of the honeys, has been
reported to have an inhibitory effect on around 60 species of bacteria,
including aerobes and anaerobes, gram-positives and gram-negatives. It
was reported that Tualang honey has variable but broad-spectrum
activities against many different kinds of wound and enteric bacteria.
Unlike glucose oxidase, the antibacterial properties
fromLeptospermum spp. honeys are light- and heat-stable. Natural honey
of other sources can vary as much as 100-fold in the potency of their
antibacterial activities, which is due to hydrogen peroxide. In addition,

3. honey is hygroscopic, which means that it can draw moisture out of the
environment and dehydrate bacteria, and its high sugar content and low
level pH can also prevent the microbes from growth.
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, a Sumerian tablet writing, dating back to
2100-2000 BC, mentions honey's use as a drug and an ointment.
Aristotle (384-322 BC), when discussing different honeys, referred to
pale honey as being “good as a salve for sore eyes and wounds”. Manuka
honey has been reported to exhibit antimicrobial activity against
pathogenic bacteria such as Staphylococcus aureus (S. aureus)
and Helicobacter pylori (H. pylori) making this honey a promising
functional food for the treatment of wounds or stomach ulcers.
The honey has been used from ancient times as a method of
accelerating wound healing, and the potential of honey to assist with
wound healing has been demonstrated repeatedly. Honey is gaining
acceptance as an agent for the treatment of ulcers, bed sores and other
skin infections resulting from burns and wounds. The healing properties
of honey can be ascribed to the fact that it offers antibacterial activity,
maintains a moist wound environment that promotes healing, and has a
high viscosity which helps to provide a protective barrier to prevent
infection. There are many reports of honey being very effective as

4. dressing of wounds, burns, skin ulcers and inflammations; the
antibacterial properties of honey speed up the growth of new tissue to
heal the wound. The medihoney and manuka honey have been shown to
have in vivo activity and are suitable for the treatment of ulcers, infected
wounds and burns.
The honey, when applied topically, rapidly clears wound infection
to facilitate healing of deep surgical wounds with infection. The
application of honey can promote the healing in infected wounds that do
not respond to the conventional therapy, i.e., antibiotics and antiseptics,
including wounds infected with methicillin-resistant S. aureus .
Moreover, it can be used on skin grafts and infected skin graft donor
sites successfully.

5. POTENTIAL ANTIBACTERIAL AGENT :
The use of honey as a traditional remedy for microbial infections
dates back to ancient times. Research has been conducted on manuka (L.
scoparium) honey, which has been demonstrated to be effective against
several human pathogens, including Escherichia coli (E.
coli), Enterobacter aerogenes, Salmonella typhimurium, S. aureus.
Laboratory studies have revealed that the honey is effective against
methicillin-resistant S. aureus (MRSA), β-haemolytic streptococci and
vancomycin-resistant Enterococci (VRE). However, the newly identified
honeys may have advantages over or similarities with manuka honey due
to enhanced antimicrobial activity, local production (thus availability),
and greater selectivity against medically important organisms. The
coagulase-negative staphylococci are very similar to S. aureus in their
susceptibility to honey of similar antibacterial potency and more
susceptible than Pseudomonas aeruginosa (P. aeruginosa)
and Enterococcus species.
The disc diffusion method is mainly a qualitative test for detecting
the susceptibility of bacteria to antimicrobial substances; however, the
minimum inhibitory concentration (MIC) reflects the quantity needed for
bacterial inhibition. Following the in vitro methods, several bacteria
(mostly multidrug resistant; MDR) causing human infections that were
found susceptible to honey.

6. Antibacterial activity of honey against bacteria causing life-
threatening infection to humans.
Authors
Proteus spps. Bacterial strain Clinical importance
Agbagwa and Frank-
Peterside[2]
Serratia marcescens Septicemia, wound infections Molan[1]
Vibrio cholera Cholera Molan[1]
S. aureus Community acquired and
nosocomial infection
Taormina et al[3]
Chauhan et al[4]
Sherlock et al[5]
E. coli Urinary tract infection, diarrhea,
septicemia, wound infections
Chauhan et al[4]
Sherlock et al[5]
P. aeruginosa Wound infection, diabetic foot
ulcer, Urinary infections
Chauhan et al[4]
Sherlock et al[5]
Mullai and Menon[6]
S. maltophilia Pneumonia, urinary tract infection,
blood stream infection,
nosocomial infection
Tan et al[7]
A. baumannii Opportunistic pathogen infects
immunocompromised individuals
through open wounds, catheters
and breathing tubes
Tan et al[7]
A. schubertii Burn- wound infection Hassanein et al[8]
H.
paraphrohaemlyticus
Micrococcus luteus
Cellulosimicrobium
cellulans

7. MECHANISM OF ANTIMICROBIAL ACTIVITY OF HONEY
The antimicrobial activity of honey is highly complex and still
remains not fully recognized. To date, it has been established that several
components of this product play a crucial role for its antimicrobial
properties :
 high concentration of sugars (about 80% of weight of this product)
eliminates microorganisms, mainly bacteria that are sensitive to
high osmotic pressure and inhibit the development of more
osmotolerant microorganisms;
 low pH value—high concentration of organic acids (e.g., gluconic
acid). The pH of most honey types is in the range from 3.4 to 6.1,
which in combination with high osmotic pressure eliminates or
enables the development of most microorganisms;
 glucose oxidase—the enzyme, oxidoreductase that catalyzes the
oxidation of glucose to gluconic acid. The side product of this
reaction, hydrogen peroxide (H2O2), is a strong antimicrobial agent.
The detailed mechanism of this reaction is presented below (Figure
2). The enzyme is produced in honeybees’ salivary glands and
introduced to the collected nectar. It protects the ripening honey
against the development of pathogenic microorganisms.
Interestingly, the enzyme is present but not active in the mature
honey; this product is sufficiently protected with high osmotic

8. pressure and low acidity. When the honey is diluted, the enzyme
regains activity, which is extremely important for honeybees and
especially their larvae health. Honey is the most important
component of honeybees’ diet; however, before consumption, it is
diluted in water. The generated by the enzyme H2O2 is a major
antimicrobial defense factor for this diluted honey. Its production is
also crucial for antimicrobial potential of honey used for treatment
of skin and soft tissue infections, infected wounds or eradication
pathogenic bacteria located within upper respiratory tract
orHelicobacter pylori located in human stomach.
Honey produced from some botanical sources exhibits high
antimicrobial activity. Possibilities of application of this product for
treatment infections in clinical practice should be the subject of intensive
investigations in the near future. Except of high activity, the most
important advantages of this product are as follows:
 lack of side effects for patients (important drawback of antibiotics);
 low costs of therapies;
 low possibility of development of resistant strains—the cells of
pathogens are simultaneously affected with several factors, for

9. example, hydrogen peroxide, bee defensin-1, methylglyoxal or
other phytochemicals;
 the honey provides the body of the patient many health-promoting
components, for example, antioxidants, microelements, trace
elements and vitamins.
However, it has to be notice that several important problems would have
to be solved for more common application of honey for treatment
infections.
 only the honey classified as medical grade—with confirmed
antibacterial activity, free of pathogenic microorganisms and toxic
components could be used in medical applications;
 each batch of raw material (honey) would have to be tested for its
biological activity;
 the method of sterilization, safe for proteinaceous antibacterial
components of honey (glucose oxidase, bee defensin-1), would
have to be developed (gamma-irradiation sterilization seems to be
promising [48]);
 much more studies are necessary to check the in vivo effects of
treatment of infections;
 one method of determination of antimicrobial activity of honey
should be recommended, as a consequence comparison of activity
of the product tested in different laboratories would be easier

10. (based on our experience, we would recommend serial dilution
method for this purpose).
Summarizing we have no doubt that honey is an interesting and
promising alternative to classical antibiotics and should be more
seriously considered as therapeutic agents.

11. OBJECTIVE OF THE STUDY:
1. To determine the antibacterial activity of Honey by disk
diffusion method.
2. To determine the antibacterial activity of Honey by well
diffusion method.
REQUIRED MATERIALS:
a) Sample collection- Pathogen collected from hospital.
b) Glassware: 1) Conical flask
2) Petri plates
3) Measuring cylinder
4) Beaker
5) Test tube
c) Chemical: 1) Muller Hinton agar
2) Eosin methylene blue agar
3) Alcohol

12. d) Instruments: 1) Laminar air flow
2) Incubator
3) Autoclave
4) Hot Air oven
5) Weighing balance
e) Others: 1) Antibiotic disc
2) Sewage sample
3) Inoculating loop
4) Cotton plug
5) Spreader

13. METHODOLOGY:
1. Bacterial strains
Strains of Pseudomonas were isolated from swabs collected
fromDiseased paitient routinely submitted to the department of medical
microbiology and public health at the private Hospital . It was tested by
standard bacteriological techniques and pure cultures were kindly
supplied for this study. Only One strain of gram negative bacteria (
pseudomonas) were used.
2. Inoculums preparation
Bacterial strain was subcultured overnight at 37 °C in Nutrient
broth.
3. Antibacterial activity of Honey
The disk diffusion method is used to evaluate antimicrobial
activity of the honey.
a) Firstly the Honey were collected.
b) Then Filter paper discs loaded with 5 µg of honey was used .
c) 15 ml of Nutrient agar medium was poured into sterile Petri dishes.
d) Then using swab stick, take bacterial suspension and spread over
nutrient agar medium.

14. e) Then with the help of forcep put discs on medium .
f) Incubate it for 24 hours at 37 ̊ c.
The presence of inhibition zones were measured , recorded and
considered as indication for antibacterial activity.
The well diffusion method is also used to evaluate antibacterial
activity of the Honey.
a) Antibacterial activity of aqueous and solvent extracts was
determined by agar well diffusion method according to National
Committee for Clinical Laboratory Standards (NCCLS) .
b) Inoculum containing bacterial culture to be tested was spread on
nutrient agar plates with a sterile swab moistened with the bacterial
suspension.
c) Subsequently, wells of 8 mm diameter were punched into the agar
medium and filled with 100 μl of Honey and allowed to diffuse at
room temperature for 24h.
d) The plates were then incubated in the upright position at 37° for 24
h.

15. OBSERVATION :

21. RESULT:
The results of in vitro confirm high antimicrobial potential of
honey against given pathogen. Both methods : Disk diffusion method
and well method have shown inhibition zone against pseudomonas
species. This initial screening with agar well diffusion assay revealed
that honey exhibited antibacterial activity against pseudomonas species.
CONCLUSION:
We do not have doubts that honey is promising antimicrobial
agents and possibilities of their application in clinical medicine deserve
consideration.

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