Antimicrobial effect of honey produced by on some

common human pathogens

Apis mellifera
Osho, Aand Bello, O.O.
Department of Microbiology, Olabisi Onabanjo University,
P.M.B. 2002, Ago-Iwoye.
INTRODUCTIONANDLITERATUREREVIEW
The medicinal properties of honey have been reported and documented by beekeepers and medical practitioners [1, 2].
As a result of over-use and abuse of antibiotics, number of diseases, which seemto
evolve to become more virulent with each generation. Investigations into natural and potent antimicrobials seemed to
be the right step to take. The invasion of pathogenic organisms is on the rise. As a result, efforts are being made to
develop antimicrobial agents from natural sources for better therapeutic effects [3]. The therapies have drawn the
interest of both public and medical communities. Current research has focused on herbal and aromatherapy
products. However, a number of other products such as honey have shown therapeutic promise. Researches relating to
honey show that pure honey is bactericidal for many pathogenic organisms, including various gram-negative and
gram-positive bacteria [4-6]. Honey has been used in different diseases as as 2000 years [7]. The use
of honey as a medicine has continued into present-day medicine. It has been shown that natural unheated honey
has some broad-spectrum antibacterial activity when tested against pathogenic bacteria, oral bacteria as well as food
spoilage bacteria [8-10]. It is clear fromthese studies that bacteria are not uniformly affected by honey. Furthermore, it
has been shown that different honeys vary substantially in the potency of their antibacterial activity, which varies with
the plant source [9-12]. At present a number of honeys are sold with standardized levels of antibacterial activity. The
best known of these is NewZealandManuka honeyproducedfromthe Manuka bush, [11].
The oral streptococci play an important role in oral health. They are involved in dental plaque development and the
formation of dental caries. is an opportunistic pathogen of the oral cavity that may cause oral disease
in especially the immune compromised individual, the elderly and those individuals wearing dentures. There is
evidence that honey with a high antibacterial activity could be used to reduce dental plaque in the treatment of oral
there has been an increase in the
been
treating far ago
the
Leptospermumscoparium
Candida albicans
ABSTRACT
Antibacterial activities of the two honey samples, produced by the honeybee (Apis mellifera), were assayed using standard
well diffusion method. Both honey samples were tested at four concentrations (5%, 25%, 50% and 100% w/v) against
Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Bacillus subtilis and Escherichia coli, and mean
zones of inhibition were measured. Both honey samples used were effective in inhibiting the growth of the test organisms at 25-
100% concentrations (w/v). No honey sample was inhibitory at 5% concentration (w/v). The inhibitory effect of undiluted
concentration of the honey samples (100% w/v) on Escherichia coli was comparable to that of Amoxicillin (20.0 0.1 mm)
and Chloramphenicol (17.5 0.2 mm). Tetracycline had a very low inhibitory effect on the test organisms. In conclusion,
natural honey can be employed in treating some common diseases especially those caused by the tested microorganisms and
carries the potential of introducingnewtemplate intomodern medicine.
KEYWORDS: Antimicrobial, Honey, Human Pathogens
ASIAN J. EXP. BIOL. SCI. VOL 1(4) 2010:875-880
Society of Applied Sciences
ORIGINALARTICLE
875 ASIAN J. EXP. BIOL. SCI. VOL 1 (4) 2010
disease [13]. However little information is available with regards to effect of different honeys on putative oral
pathogens. Laboratory studies have shown that pure honey has significant antibacterial activities against the major
wound-infecting species [14, 15], especially those with the potential to develop antibiotic resistance such as
Methicillin-Resistant (MRSA), -haemolytic streptococci, Vancomycin
Resistant Enterococci (VRE) and species [16, 17]. The antibacterial activity of honey has also been
investigated and reported for its potential use in reducing food-borne pathogens [18]; preventing entry site of infection
[19]; for the treatment of colitis [20]. The application of honey to wounds of animals in veterinary environments has
also been noted [7]. This study was designed to evaluate antimicrobial activity of natural honey
against some pathogenic microorganisms, and whose
activitywas withselectedbroadspectrumantibiotics
Two honey samples produced by honeybee ( ) were evaluated. Honey sample Awas provided by local
apiarists fromSango, Ibadan, Oyo State while honey sample Bwas collected froma bee farmalso by a local apiarist in
Ijebu-Ode Township, Ogun State, South-Western, Nigeria. These were obtained by draining the honey after manually
uncapping the comb frames. These honey samples were aseptically collected in sterile screwed cups and kept in a cool
anddry place (at roomtemperature) overnight before theywere finallytransportedtothe laboratory.
Stocked cultures of and
used in this study were obtained fromthe Department of Microbiology, Olabisi Onabanjo University,
Ago Iwoye, Ogun State. The isolates were identified based on standard microbiological techniques, and sub-cultured
in nutrient agar slopes at 37 C for 24 hrs. Colonies of fresh cultures of the different microorganisms from overnight
growth were picked with sterile inoculating loop and suspended in 3-4 ml nutrient broth contained in sterile test tubes
andincubatedfor 2-3 hrs at 37 C. This was dilutedwithdistilledwater toset inoculumdensityused inthis study.
The agar diffusion technique (well diffusion method) was employed. The honey samples were first inoculated
separately on standard nutrient media with no test organisms so as to evaluate their possible contamination.
Thereafter, solidified nutrient agar plates were separately flooded with the liquid inoculums of the different test
organisms using the pour plate method. The plates were drained and allowed to dry at 37 C for 30 mins after which
four equidistant wells of 6 mmin diameter were punched using a sterile cork borer at different sites on the plates. 50 ul
of the different concentrations (5%, 25%, 50% and 100% w/v) of the honey samples were separately placed in the
different punched wells with 1 ml sterile syringe. The plates were allowed to stay for 15 mins for pre-diffusion to take
place followed by an overnight incubation that lasted for 24 hrs at 37 C. The resulting zones of inhibition were
measured with the use of a caliper and recorded. The Minimal Inhibitory Concentrations (MIC) of honey samples
were carried out using the modified method of Eloff [21] and reported as the least concentration that inhibited the
growth of the test organisms. Also, solidified plates which had been flooded with different test organisms were
allowed to dry at 37 Cfor 30 mins and conventional antibiotics (Amoxicilin, Tetracyclin and Chloramphenicol) disks
were placed on them. The plates were incubated at 37 C for 24 hrs. The diameters of the zones of inhibition were
measuredusing a caliper andalsorecorded.
The results of the susceptibility of the test organisms to the two honey samples were similar. All the test
organisms were sensitive to undiluted (100% w/v) and half-diluted (50% w/v) concentrations of both honey samples
but, however, no test organism was susceptible to the two honey samples at 5% w/v concentration (Figures 1 and 2).
There was no evidence of growth inhibition in the cases of and at
concentrations up to 25% w/v, yet it was confirmed that the honey samples still contained some antimicrobial
properties at that concentration as it was able to inhibit the growth of other test organisms at that same concentration.
Staphylococcus aureus
Pseudomonas
Apis mellifera
Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Bacillus subtilis
Escherichia coli
in vitro
Klebsiella pneumonia Pseudomonas aeruginosa
the produced by
, a particular species of honeybee in Nigeria,
compared
Apis
mellifera
MATERIALSANDMETHODS
Collectionof samples
Preparationof test organisms
Antimicrobial Assay
RESULTSANDDISCUSSION
0
0
0
0
0
0
Antimicrobial effect of honey produced by Apis mellifera ...............................................................Osho, A and Bello, O.O.
876 ASIAN J. EXP. BIOL. SCI. VOL 1 (4) 2010
The inhibitory effect of both honey samples was highest on and followed by
at all concentrations, except at 5%w/v concentration where no growth inhibition was observed in all cases (Figures 1
and 2). has always been employed in many microbiological evaluations of honey because of
its high sensitivity [16, 22]. The reason for this unusual sensitivity is not known. It may, however, be related to the
sensitivityof toacidic
environment of natural honey [22]. The minimal inhibitory concentration (MIC) of the honey samples can be seen in
Tables 1 and 2. The MICof both honey samples on and was 25%w/v, and 50%w/v as in the
cases of and This, however, indicated that both honey samples have less antimicrobial
effect against and compared with other test organisms. The reason for this is not clear.
The three selected standard antibiotics used were found to inhibit the growth of all the test organisms except
Tetracycline which was insensitive against and (Figure 3) It was also observable that
Tetracycline had little effect, generally, on growth inhibition of other test organisms that were found susceptible
comparedwithAmoxycilinandChloramphenicol (Figure 3).
Another area of interest is the ability of both honey samples to exert antimicrobial effects on and
which were resistant to Tetracycline, a broad-spectrum antibiotic, in this study. The low antibacterial activity of
Tetracyclin and its inability to inhibit the growth of and could be as a result of misuse and abuse of
drug, as Tetracycline is one of the common antibiotics that have been greatly abused. The pattern of inhibition of
growth of by the two honey samples was the same in some cases with those of the standard antibiotics. For
instance, undiluted concentration of honey sampleAgave a zone of inhibition of 20.0 0.1 mmwhich was exactly the
same as that of Amoxycilin (Figures 1 and 3), while honey sample Bgave a zone of clearance of 18.0 0.1 which was
just negligibly higher than 17.5 0.2 mmproduced by Chloramphenicol (Figures 2 and 3). These results suggest that
the honey samples used contained some bio-components whose antimicrobial activities against are highly
comparable with those of these two antibiotics. The results of this study are in line with a number of previous studies
that have been reported by
), lowprotein content and hydrogen peroxide [23, 24]. Also, Radwan [25] attributed the
antibacterial activity to specific chemicals in honey. The nature of these chemicals and the mechanisms of their action
are not fully understood even though Thin Layer Chromatography (TLC), Polyacrylamide Gel Electrophoresis
(PAGE) and High Performance Liquid Chromatography (HPLC) have confirmed the presence of fatty acids, lipids,
amylases and ascorbic acids in pure honey [26, 27]. It has been reported that honey contains lysozyme, a well known
antibacterial agent [28]. However, in another study no lysozyme activity was found [29]. The antibacterial flavonoid
pinocembrin is present in honey, but its concentration and contribution to honey's non-peroxide antibacterial activity
is small [30]. Nzeako and Hamdi [31] in their studies of six commercial honeys found that inhibition of
and didnot occur at honeyconcentrations less than40%(w/v).
Mogessie [32] reported that honey produced by sting-less bee ( ) was found to be
effective against some food pathogens of humans. Growth retardation and inhibition on
and were noted at 15% and 20% (w/v) honey concentrations, while a more marked growth retardation and
inhibition on and were observed at concentration of 10%(w/v) honey. This is, however, in contrast
to the findings of this study. Honey produced by honeybees ( in this present study could retard most of
the test organisms at a little higher concentration of 25%(w/v). Here, it could be explained that the source of nectars is
a contributory factor to the variation in the antimicrobial potential of the honeys used in this study as compared with
the previous studies. The flora source determines attributes of natural products such as honey making the composition
highly variable [33]. In conclusion, honey produced by has antimicrobial activities, and is effective
against both gram-positive and gram-negative bacteria. This is a demonstration of broad spectrum of activities of
honey. However, pharmacological standardization and clinical evaluation on the effect of honey are essential so as to
make honey one of the standard and official remedies that could be used to effectively combat some of the mutated
pathogens that have developed resistance against the abused conventional antibiotics. In light of the enormous
potentials for the application of honey within clinical environment, a continuing need exists for potent natural
Escherichia coli, Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
E. coli, S. aureus B. subtilis
K. pneumoniae P. aeruginosa.
K. pneumoniae P. aeruginosa
E. coli P. aeruginosa .
E. coli P. aeruginosa
E. coli P. aeruginosa
E. coli
E. coli
et al.
S. aureus, E.
coli P. aeruginosa
Tazmar mar Apis mellipodae
S. typhimuriumS. enteritidis
E. coli
B. cereus S. aureus
Apis mellirera)
Apis mellifera
different researchers in the past. Antimicrobial activity of honey is thought to be due to
some physicochemical properties such as high content of reducing sugars, high viscosity, high osmotic pressure, low
pH, lowwater activity (
w
Antimicrobial effect of honey produced by Apis mellifera ...............................................................Osho, A and Bello, O.O.
877 ASIAN J. EXP. BIOL. SCI. VOL 1 (4) 2010
Table 1 Minimal Inhibitory Concentration of honey sample A
Table 2 Minimal Inhibitory Concentration of honey sample B
+ = Zone of Inhibition Observed
-=No Zone of InhibitionObserved
+ = Zone of Inhibition Observed
-= No Zone of InhibitionObserved
Honey concentration (w/v) and minimal inhibitory concentration
Test Organism 5% 25% 50% 100%
Escherichia coli - + + +
Staphylococcus aureus - + + +
Klebsiella pneumonia - - + +
Pseudomonas aeruginosa - - + +
Bacillus subtilis - + + +
Honey concentration (w/v) and minimal inhibitory concentration
Test Organism 5% 25% 50% 100%
Escherichia coli - + + +
Staphylococcus aureus - + + +
Klebsiella pneumonia - - + +
Pseudomonas aeruginosa - - + +
Bacillus subtilis - + + +
Figure 1: Antimicrobial activity of honey
(sample A) on tested organisms
0
5
10
15
20
25
E. coli Staphylococcus
aureus
Klesiella
pneumonia
Pseudomonas
aeruginosa
Bacillus subtilis
Tested bacteria
m
e
a
n
d
i
a
m
e
t
e
r
o
f
I
n
h
i
b
i
t
i
o
n
z
o
n
e
(
m
m
)
5%
25%
50%
100%
Antimicrobial effect of honey produced by Apis mellifera ...............................................................Osho, A and Bello, O.O.
878 ASIAN J. EXP. BIOL. SCI. VOL 1 (4) 2010
p
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i
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i
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(
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)
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25%
50%
100%
Antimicrobial effect of honey produced by Apis mellifera ...............................................................Osho, A and Bello, O.O.
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Correspondence to Author: Dr. Adeleke Osho , Department of Microbiology, Olabisi Onabanjo University, P.M.B.
2002, Ago-Iwoye. E-mail: adelekeosho@yahoo.omPhone Number: +234 8034714411
Antimicrobial effect of honey produced by Apis mellifera ...............................................................Osho, A and Bello, O.O.
880 ASIAN J. EXP. BIOL. SCI. VOL 1 (4) 2010