Abstract Background Honey has been shown to have wound healing properties which can be ascribed to its antimicrobial activity. The antimicrobial activity can be effective against a broad spectrum of bacterial species especially those of medical importance. It has also been shown that there is considerable variation in the antimicrobial potency of different types of honey, which is impossible to predict. With this in mind we tested the antimicrobial activity of honeys produced from plants grown in South Africa for their antibacterial properties on selected standard strains of oral micro-organisms. Methods The honeys used were produced from the blossoms of <it>Eucalyptus cladocalyx </it>(Bluegum) trees, an indigenous South African plant <it>Leucospermum cordifolium </it>(Pincushion), a mixture of wild heather shrubs, mainly <it>Erica </it>species (Fynbos) and a <it>Leptospermum scoparium </it>(Manuka) honey. Only pure honey which had not been heated was used. The honeys were tested for their antimicrobial properties with a broth dilution method. Results Although the honeys produced some inhibitory effect on the growth of the micro-organisms, no exceptionally high activity occurred in the South African honeys. The carbohydrate concentration plays a key role in the antimicrobial activity of the honeys above 25%. However, these honeys do contain other antimicrobial properties that are effective against certain bacterial species at concentrations well below the hypertonic sugar concentration. The yeast <it>C. albicans </it>was more resistant to the honeys than the bacteria. The species <it>S. anginosus </it>and <it>S. oralis </it>were more sensitive to the honeys than the other test bacteria. Conclusion The honeys produced from indigenous wild flowers from South Africa had no exceptionally high activity that could afford medical grade status.</p

A Simon

AI Root

D Steinberg

DJ Willix

HK English

HM Ericsson

IE Haffejee

JM Wilkinson

JW White

KL Allen

LM Bang

M Subrahmanyam

MA Mundo

Nicolaas J Basson

NJ Basson

OA Moore

PC Molan

PE Lusby

PJ Armon

R Bromfield

SE Efem

Sias R Grobler

VM French

English

PubMed

Springer - Publisher Connector

BioMed Central
BMC Complementary and 
Alternative Medicine
ssOpen AcceResearch article
Antimicrobial activity of two South African honeys produced from 
indigenous Leucospermum cordifolium and Erica species on selected 
micro-organisms
Nicolaas J Basson*† and Sias R Grobler†
Address: Oral and Dental Research Institute, Faculty of Dentistry, University of the Western Cape, Private Bag X1, Tygerberg 7505, South Africa
Email: Nicolaas J Basson* - njbasson@uwc.ac.za; Sias R Grobler - srgrobler@uwc.ac.za
* Corresponding author †Equal contributors
Abstract
Background: Honey has been shown to have wound healing properties which can be ascribed to
its antimicrobial activity. The antimicrobial activity can be effective against a broad spectrum of
bacterial species especially those of medical importance. It has also been shown that there is
considerable variation in the antimicrobial potency of different types of honey, which is impossible
to predict. With this in mind we tested the antimicrobial activity of honeys produced from plants
grown in South Africa for their antibacterial properties on selected standard strains of oral micro-
organisms.
Methods: The honeys used were produced from the blossoms of Eucalyptus cladocalyx (Bluegum)
trees, an indigenous South African plant Leucospermum cordifolium (Pincushion), a mixture of wild
heather shrubs, mainly Erica species (Fynbos) and a Leptospermum scoparium (Manuka) honey. Only
pure honey which had not been heated was used. The honeys were tested for their antimicrobial
properties with a broth dilution method.
Results: Although the honeys produced some inhibitory effect on the growth of the micro-
organisms, no exceptionally high activity occurred in the South African honeys. The carbohydrate
concentration plays a key role in the antimicrobial activity of the honeys above 25%. However,
these honeys do contain other antimicrobial properties that are effective against certain bacterial
species at concentrations well below the hypertonic sugar concentration. The yeast C. albicans was
more resistant to the honeys than the bacteria. The species S. anginosus and S. oralis were more
sensitive to the honeys than the other test bacteria.
Conclusion: The honeys produced from indigenous wild flowers from South Africa had no
exceptionally high activity that could afford medical grade status.
Background
The potential of honey to assist with wound healing has
been demonstrated repeatedly [1,2]. It has proven valua-
ble in the treatment of infantile gastroenteritis, infected
surgical wounds, burns, decubitus ulcers, skin crafting and
surgical debridement [2-9]. The healing properties of
honey can be ascribed to the fact that it offers antibacterial
activity, maintains a moist wound environment that pro-
motes healing and has a high viscosity which helps to pro-
vide a protective barrier to prevent infection [10].
Published: 15 July 2008
BMC Complementary and Alternative Medicine 2008, 8:41 doi:10.1186/1472-6882-8-41
Received: 17 March 2008
Accepted: 15 July 2008
This article is available from: http://www.biomedcentral.com/1472-6882/8/41
© 2008 Basson and Grobler; licensee BioMed Central Ltd. 
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 4
(page number not for citation purposes)
BMC Complementary and Alternative Medicine 2008, 8:41 http://www.biomedcentral.com/1472-6882/8/41It 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 [10-12]. It is also clear from these studies that
bacteria are not uniformly affected by honey. Further-
more, it has been shown that different honeys vary sub-
stantially in the potency of their antibacterial activity,
which varies with the plant source [10,11,13,14]. At
present a number of honeys are sold with standardized
levels of antibacterial activity. The best known of these is
New Zealand Manuka honey produced from the Manuka
bush, Leptospermum scoparium [14].
The oral streptococci play an important role in oral health.
They are involved in dental plaque development and the
formation of dental caries. Candida albicans is an oppor-
tunistic pathogen of the oral cavity that may cause oral
disease in especially the immune compromised individ-
ual, 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 treat-
ment of oral disease [15]. However little information is
available with regards to effect of different honeys on
putative oral pathogens.
The search for other honeys from different sources with
enhanced antibacterial activity continues. South Africa
has a large floral biodiversity with many unique plants
indigenous to the region. Honeys are produced from these
plants and sold commercially in South Africa. However
none of these have been tested for their antimicrobial
activity and possible use as medical grade honey. The aim
of the present study was to investigate the antimicrobial
activity of two South African honeys produced from indig-
enous plants unique to South Africa and two honeys pro-
duced from alien species against selected reference
cultures of potential oral pathogens.
Methods
The honey types used were as follows: a honey produced
from the blossoms of Leucospermum cordifolium, a wild
shrub unique to the Cape Peninsula of South Africa (Pin-
cushion honey); a honey produced from a mixture of
many heather shrubs but mainly from Erica species found
on the southern coastal parts of South Africa (Fynbos
honey); a honey produced from Eucalyptus cladocalyx trees
(Bluegum honey) an alien species originally from Aus-
tralia; a honey locally produced from the L. scoparium
plant (Manuka bush) that was obtained from New Zea-
land. Honey types were identified and their source noted
by the apiarists themselves. Only pure honey which had
not been heated was used. The honeys were stored in the
dark at room temperature. The honeys were tested for
their antimicrobial properties against a control solution
with sugar content similar to that of natural honey. The
control carbohydrate solution contained 39% weight/vol-
ume (w/v) d-fructose, 31% (w/v) d-glucose, 8% (w/v)
maltose, 3% (w/v) sucrose and 19% (w/v) water and was
kept refrigerated when not in use [16].
The minimal inhibitory concentrations (MICs) were
determined with a broth dilution method [17]. A 50%
stock solution of honey and of the sugar control solution
was prepared in double strength sterile Brain Heart Infu-
sion (BHI) broth (Oxoid). Two-fold dilutions of the stock
solutions were aseptically prepared in sterile BHI broth
(Oxoid) using a carry-over technique to give a final vol-
ume of 5 ml in each tube. This was repeated until a dilu-
tion range of 50%, 25%, 12.5%, 6.25% and 3.12% of
honey was obtained. After observing the range in which
inhibition of growth in the honey solutions occurred, a
closer dilution was prepared between dilutions of 25%
and 12.5%.
Phenol was used as a positive control. A 40% stock solu-
tion was prepared in double strength BHI broth from an
80% phenol solution (Merck). Two-fold dilutions of the
40% dilution were aseptically prepared in BHI as
described above.
The inocula used were prepared from overnight growth
cultures in BHI-broth. One hundred microlitre of a 1/100
dilution of the culture was used to inoculate 5 ml of the
test solutions. The tubes were incubated aerobically at 37
degrees C for 24 hours.
In view of the high risk of bacterial contamination from
the environment, each honey sample was tested for steril-
ity beforehand by inoculating 100 ml sterile BHI with 5
ml of the 50% stock solution. Furthermore, each growth
positive tube in the tests was checked microscopically to
confirm that the cell morphology corresponded with the
cell morphology of the inoculum.
The microbial species tested were Streptococcus mutans
(NCTC 10449), Streptococcus salivarius (NCTC 8618),
Streptococcus sanguis (NCTC7864), Streptococcus anginosus
(NCTC 10708), Streptococcus gordonii (NCTC 3165), Strep-
tococcus oralis (NCTC 11427), Streptococcus sobrinus (NCTC
10921), Candida albicans (NCPF 3118), Escherichia coli
(NCTC 9001) and Staphylococcus aureus (NCTC 8530).
The hydrogen peroxide concentration of each test dilution
was determined one hour after dilution using Peroxid-
Test strips (Merck). The pH of the undiluted honey, as
well as the pH of each dilution was determined with a
glass electrode. All tests were done in triplicate. The honey
solutions were freshly prepared for each assay.Page 2 of 4
(page number not for citation purposes)
BMC Complementary and Alternative Medicine 2008, 8:41 http://www.biomedcentral.com/1472-6882/8/41Results
The minimum inhibitory concentrations (MICs) of the
four natural honeys, including the control carbohydrate
solution, are shown in Table 1. No difference was
observed in the antimicrobial activity between the four
honeys for concentrations up to 21%. At these concentra-
tions, honey and the control solution had similar activity
towards all organisms except for two streptococcus spe-
cies. Both S. anginosus and S. oralis were inhibited by the
honeys at concentrations of 17% and 12.5% respectively
but grew up to a carbohydrate concentration of 21% (MIC
25%). At concentrations of 25%, the honeys and the con-
trol solution had variable activity towards the organisms
tested. Bluegum, Pincushion and Manuka honey inhib-
ited the growth of all test bacteria, but not the yeast. How-
ever, E. coli and S. salivarius grew in the Fynbos honey at
this concentration. The carbohydrate control solution
could not inhibit growth of S mutans, S. salivarius, E. coli
and C. albicans at a concentration of 25%. No growth was
observed at concentrations of 50% for the carbohydrate
control or for the honeys tested.
Hydrogen peroxide was detected only at dilutions of
6.25% and 3.12%, at a low concentration of 1 mg/liter.
The pH of the undiluted natural honeys was in the order
of ± 3.6. On dilution with the growth medium, the pH of
each of the dilutions increased from ± 5.25 for the 50%
dilution to ± 7.26 for the 3.12% dilution (Table 2).
Discussion
Our study clearly shows that honeys produced from two
indigenous South African flowers, the endemic L. cordifo-
lium (Pincushion) and Erica spp. (Fynbos) do not have
any exceptionally high broad-spectrum antimicrobial
activity. Furthermore, these honeys were not any different
from two alien species grown in South Africa. In previous
studies, very similar results were obtained with E. cladoca-
lyx (Bluegum) honey [12,18]. These honeys are commer-
cially produced on a large scale and are easily obtainable
off the shelf. However, on a very small scale, honeys are
produced from other floral sources. Considering the large
floral biodiversity in South Africa, a survey of these rare
honeys might uncover a honey with high levels of antimi-
crobial activity.
The poor activity of Manuka honey was disappointing as
previous reports have shown that Manuka honey may
have a very high antimicrobial activity against a number
of bacteria [13,19]. However, not all Manuka honey
exhibits antibacterial activity. High antimicrobial activity
is recorded only in Manuka honey produced from specific
localities, in particular, the East Cape region of the North
Island of New Zealand [20].
The results from this report confirm that honeys from dif-
ferent countries and regions have a wide variability in
their antimicrobial activity. It has been shown that the
antimicrobial activity of honey may range from concen-
trations lower than 3% to concentrations of 50% and
higher [10,13,19]. The honeys we tested had antimicro-
bial activity in the 17 – 25% range against the organisms
that were tested except for S. oralis which showed sensitiv-
ity at 12.5%. The bacteria S. aureus and E. coli, which were
included as reference cultures to previous reports, were
both inhibited in the 50 – 25% range. These species have
been shown to be sensitive at concentrations as low 1.8 –
11% for some honeys [13,21]. This indicates that our
honeys were of a poor antimicrobial quality.
Honey may inhibit bacterial growth due to a number of
different reasons. High sugar concentration (reduced
water activity), low pH, hydrogen peroxide generation,
proteinaceous compounds or other unidentified compo-
nents present in the honey may all provide antimicrobial
activity [11]. Low pH can be ruled out in this study
because the average pH of the honey samples at 21% was
6.47 which falls within the growth range of the micro-
organisms. Although hydrogen peroxide was produced in
our study, it only occurred at the very low dilutions and at
concentrations of ± 1 mg/litre. These concentrations are
Table 1: Minimum inhibitory concentrations (%) of different honeys towards standard strains of oral micro-organisms
Organisms E. cladocalyx 
(Bluegum)
Erica species 
(Fynbos)
L. cordifolium 
(Pincushion)
L. scoparium 
(Manuka)
Carbohydrate 
control
Phenol control
S. mutans (10449) 25 25 25 25 50 <2.5
S. salivarius (8618) 25 50 25 25 50 <2.5
S. sanguis (7864) 25 25 25 25 25 <2.5
S. anginosus 
(10708)
17 17 17 17 25 <2.5
S. gordonii (3165) 25 25 25 25 25 <2.5
S. oralis (11427) 12.5 12.5 12.5 12.5 25 <2.5
S. sobrinus (10921) 25 25 25 25 25 <2.5
C. albicans (3118) 50 50 50 50 50 <2.5
E. coli (9001) 25 50 25 25 50 <2.5
S. aureus (8530) 25 25 25 25 25 <2.5Page 3 of 4
(page number not for citation purposes)
BMC Complementary and Alternative Medicine 2008, 8:41 http://www.biomedcentral.com/1472-6882/8/41too low to have any bacteriostatic effect. Concentrations
of at least 10 mg/litre are required to inhibit bacterial
growth [22]. It is however possible that the hydrogen per-
oxide production was not at its peak at the time of testing.
It has been shown that hydrogen peroxide production can
peak at different times for different honeys. Some may
take as long as 24 hours [23]. If bacteriostatic concentra-
tions were obtained within the 24 hour incubation
period, lower MICs would probably have been attained.
The honey MICs were very similar to the carbohydrate
control solution which suggests that the main inhibitory
effect was probably as a result of the reduced water activ-
ity, the only exceptions being the bacteria S. anginosis and
S. oralis. Both these strains were sensitive to honey at con-
centrations lower than that observed for the carbohydrate
control, indicating that an unidentified component, that
is active against these organisms, may be present in the
honey.
Conclusion
We conclude that the honeys produced from the two
endemic wildflower plants from South Africa do not differ
from the alien species tested and show no exceptionally
high antimicrobial activity that could provide medical
grade status.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NJB participated in the design of the study, carried out the
microbiological analysis and drafted the manuscript. SRG
conceived the study, participated in its design, was
responsible for collection and identification of the honey
samples and assisted in drafting the manuscript. All
authors read and approved the final manuscript.
References
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12. Basson NJ, du Toit IJ, Grobler SR: Antibacterial action of honey
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13. Wilkinson JM, Cavanagh HM: Antibacterial activity of 13 honeys
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14. Allen KL, Molan PC, Reid GM: A survey of the antibacterial
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Pre-publication history
The pre-publication history for this paper can be accessed
here:
http://www.biomedcentral.com/1472-6882/8/41/prepub
Table 2: pH of the different honey solutions at each dilution
Honey type Dilution
50% 25% 21% 17% 12.50% 6.25% 3.12%
E. cladocalyx (Bluegum) 5.25 6.41 6.47 6.69 7.10 7.20 7.28
Erica species (Fynbos) 5.19 6.28 6.39 6.54 6.71 6.96 7.15
L. cordifolium (Pincushion) 5.23 6.40 6.52 6.70 6.90 7.30 7.38
L. scoparium (Manuka) 5.17 6.30 6.45 6.64 6.89 7.18 7.30Page 4 of 4
(page number not for citation purposes)

Antimicrobial activity of two South African honeys produced from indigenous  and  species on selected micro-organisms

Background: Honey has been shown to have wound healing properties which can be ascribed to
its antimicrobial activity. The antimicrobial activity can be effective against a broad spectrum of
bacterial species especially those of medical importance. It has also been shown that there is
considerable variation in the antimicrobial potency of different types of honey, which is impossible
to predict. With this in mind we tested the antimicrobial activity of honeys produced from plants
grown in South Africa for their antibacterial properties on selected standard strains of oral microorganisms.
Methods: The honeys used were produced from the blossoms of Eucalyptus cladocalyx (Bluegum)
trees, an indigenous South African plant Leucospermum cordifolium (Pincushion), a mixture of wild
heather shrubs, mainly Erica species (Fynbos) and a Leptospermum scoparium (Manuka) honey. Only
pure honey which had not been heated was used. The honeys were tested for their antimicrobial
properties with a broth dilution method.
Results: Although the honeys produced some inhibitory effect on the growth of the microorganisms,
no exceptionally high activity occurred in the South African honeys. The carbohydrate
concentration plays a key role in the antimicrobial activity of the honeys above 25%. However,
these honeys do contain other antimicrobial properties that are effective against certain bacterial
species at concentrations well below the hypertonic sugar concentration. The yeast C. albicans was
more resistant to the honeys than the bacteria. The species S. anginosus and S. oralis were more
sensitive to the honeys than the other test bacteria.
Conclusion: The honeys produced from indigenous wild flowers from South Africa had no
exceptionally high activity that could afford medical grade status.Web of Scienc

Basson, Nicolaas

Grobler, Sias Renier

University of the Western Cape Research Repository

BioMed CentralBMC Complementary and Alternative MedicinessOpen AcceResearch articleAntimicrobial activity of two South African honeys produced from indigenous Leucospermum cordifolium and Erica species on selected micro-organismsNicolaas J Basson*† and Sias R Grobler†Address: Oral and Dental Research Institute, Faculty of Dentistry, University of the Western Cape, Private Bag X1, Tygerberg 7505, South AfricaEmail: Nicolaas J Basson* - njbasson@uwc.ac.za; Sias R Grobler - srgrobler@uwc.ac.za* Corresponding author †Equal contributorsAbstractBackground: Honey has been shown to have wound healing properties which can be ascribed toits antimicrobial activity. The antimicrobial activity can be effective against a broad spectrum ofbacterial species especially those of medical importance. It has also been shown that there isconsiderable variation in the antimicrobial potency of different types of honey, which is impossibleto predict. With this in mind we tested the antimicrobial activity of honeys produced from plantsgrown in South Africa for their antibacterial properties on selected standard strains of oral micro-organisms.Methods: The honeys used were produced from the blossoms of Eucalyptus cladocalyx (Bluegum)trees, an indigenous South African plant Leucospermum cordifolium (Pincushion), a mixture of wildheather shrubs, mainly Erica species (Fynbos) and a Leptospermum scoparium (Manuka) honey. Onlypure honey which had not been heated was used. The honeys were tested for their antimicrobialproperties with a broth dilution method.Results: Although the honeys produced some inhibitory effect on the growth of the micro-organisms, no exceptionally high activity occurred in the South African honeys. The carbohydrateconcentration plays a key role in the antimicrobial activity of the honeys above 25%. However,these honeys do contain other antimicrobial properties that are effective against certain bacterialspecies at concentrations well below the hypertonic sugar concentration. The yeast C. albicans wasmore resistant to the honeys than the bacteria. The species S. anginosus and S. oralis were moresensitive to the honeys than the other test bacteria.Conclusion: The honeys produced from indigenous wild flowers from South Africa had noexceptionally high activity that could afford medical grade status.BackgroundThe potential of honey to assist with wound healing hasbeen demonstrated repeatedly [1,2]. It has proven valua-ble in the treatment of infantile gastroenteritis, infectedsurgical wounds, burns, decubitus ulcers, skin crafting andsurgical debridement [2-9]. The healing properties ofhoney can be ascribed to the fact that it offers antibacterialactivity, maintains a moist wound environment that pro-motes healing and has a high viscosity which helps to pro-vide a protective barrier to prevent infection [10].Published: 15 July 2008BMC Complementary and Alternative Medicine 2008, 8:41 doi:10.1186/1472-6882-8-41Received: 17 March 2008Accepted: 15 July 2008This article is available from: http://www.biomedcentral.com/1472-6882/8/41© 2008 Basson and Grobler; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 4(page number not for citation purposes)BMC Complementary and Alternative Medicine 2008, 8:41 http://www.biomedcentral.com/1472-6882/8/41It has been shown that natural unheated honey has somebroad-spectrum antibacterial activity when tested againstpathogenic bacteria, oral bacteria as well as food spoilagebacteria [10-12]. It is also clear from these studies thatbacteria are not uniformly affected by honey. Further-more, it has been shown that different honeys vary sub-stantially in the potency of their antibacterial activity,which varies with the plant source [10,11,13,14]. Atpresent a number of honeys are sold with standardizedlevels of antibacterial activity. The best known of these isNew Zealand Manuka honey produced from the Manukabush, Leptospermum scoparium [14].The oral streptococci play an important role in oral health.They are involved in dental plaque development and theformation of dental caries. Candida albicans is an oppor-tunistic pathogen of the oral cavity that may cause oraldisease in especially the immune compromised individ-ual, the elderly and those individuals wearing dentures.There is evidence that honey with a high antibacterialactivity could be used to reduce dental plaque in the treat-ment of oral disease [15]. However little information isavailable with regards to effect of different honeys onputative oral pathogens.The search for other honeys from different sources withenhanced antibacterial activity continues. South Africahas a large floral biodiversity with many unique plantsindigenous to the region. Honeys are produced from theseplants and sold commercially in South Africa. Howevernone of these have been tested for their antimicrobialactivity and possible use as medical grade honey. The aimof the present study was to investigate the antimicrobialactivity of two South African honeys produced from indig-enous plants unique to South Africa and two honeys pro-duced from alien species against selected referencecultures of potential oral pathogens.MethodsThe honey types used were as follows: a honey producedfrom the blossoms of Leucospermum cordifolium, a wildshrub unique to the Cape Peninsula of South Africa (Pin-cushion honey); a honey produced from a mixture ofmany heather shrubs but mainly from Erica species foundon the southern coastal parts of South Africa (Fynboshoney); a honey produced from Eucalyptus cladocalyx trees(Bluegum honey) an alien species originally from Aus-tralia; a honey locally produced from the L. scopariumplant (Manuka bush) that was obtained from New Zea-land. Honey types were identified and their source notedby the apiarists themselves. Only pure honey which hadnot been heated was used. The honeys were stored in thedark at room temperature. The honeys were tested fortheir antimicrobial properties against a control solutionwith sugar content similar to that of natural honey. Thecontrol carbohydrate solution contained 39% weight/vol-ume (w/v) d-fructose, 31% (w/v) d-glucose, 8% (w/v)maltose, 3% (w/v) sucrose and 19% (w/v) water and waskept refrigerated when not in use [16].The minimal inhibitory concentrations (MICs) weredetermined with a broth dilution method [17]. A 50%stock solution of honey and of the sugar control solutionwas prepared in double strength sterile Brain Heart Infu-sion (BHI) broth (Oxoid). Two-fold dilutions of the stocksolutions were aseptically prepared in sterile BHI broth(Oxoid) using a carry-over technique to give a final vol-ume of 5 ml in each tube. This was repeated until a dilu-tion range of 50%, 25%, 12.5%, 6.25% and 3.12% ofhoney was obtained. After observing the range in whichinhibition of growth in the honey solutions occurred, acloser dilution was prepared between dilutions of 25%and 12.5%.Phenol was used as a positive control. A 40% stock solu-tion was prepared in double strength BHI broth from an80% phenol solution (Merck). Two-fold dilutions of the40% dilution were aseptically prepared in BHI asdescribed above.The inocula used were prepared from overnight growthcultures in BHI-broth. One hundred microlitre of a 1/100dilution of the culture was used to inoculate 5 ml of thetest solutions. The tubes were incubated aerobically at 37degrees C for 24 hours.In view of the high risk of bacterial contamination fromthe environment, each honey sample was tested for steril-ity beforehand by inoculating 100 ml sterile BHI with 5ml of the 50% stock solution. Furthermore, each growthpositive tube in the tests was checked microscopically toconfirm that the cell morphology corresponded with thecell morphology of the inoculum.The microbial species tested were Streptococcus mutans(NCTC 10449), Streptococcus salivarius (NCTC 8618),Streptococcus sanguis (NCTC7864), Streptococcus anginosus(NCTC 10708), Streptococcus gordonii (NCTC 3165), Strep-tococcus oralis (NCTC 11427), Streptococcus sobrinus (NCTC10921), Candida albicans (NCPF 3118), Escherichia coli(NCTC 9001) and Staphylococcus aureus (NCTC 8530).The hydrogen peroxide concentration of each test dilutionwas determined one hour after dilution using Peroxid-Test strips (Merck). The pH of the undiluted honey, aswell as the pH of each dilution was determined with aglass electrode. All tests were done in triplicate. The honeysolutions were freshly prepared for each assay.Page 2 of 4(page number not for citation purposes)BMC Complementary and Alternative Medicine 2008, 8:41 http://www.biomedcentral.com/1472-6882/8/41ResultsThe minimum inhibitory concentrations (MICs) of thefour natural honeys, including the control carbohydratesolution, are shown in Table 1. No difference wasobserved in the antimicrobial activity between the fourhoneys for concentrations up to 21%. At these concentra-tions, honey and the control solution had similar activitytowards all organisms except for two streptococcus spe-cies. Both S. anginosus and S. oralis were inhibited by thehoneys at concentrations of 17% and 12.5% respectivelybut grew up to a carbohydrate concentration of 21% (MIC25%). At concentrations of 25%, the honeys and the con-trol solution had variable activity towards the organismstested. Bluegum, Pincushion and Manuka honey inhib-ited the growth of all test bacteria, but not the yeast. How-ever, E. coli and S. salivarius grew in the Fynbos honey atthis concentration. The carbohydrate control solutioncould not inhibit growth of S mutans, S. salivarius, E. coliand C. albicans at a concentration of 25%. No growth wasobserved at concentrations of 50% for the carbohydratecontrol or for the honeys tested.Hydrogen peroxide was detected only at dilutions of6.25% and 3.12%, at a low concentration of 1 mg/liter.The pH of the undiluted natural honeys was in the orderof ± 3.6. On dilution with the growth medium, the pH ofeach of the dilutions increased from ± 5.25 for the 50%dilution to ± 7.26 for the 3.12% dilution (Table 2).DiscussionOur study clearly shows that honeys produced from twoindigenous South African flowers, the endemic L. cordifo-lium (Pincushion) and Erica spp. (Fynbos) do not haveany exceptionally high broad-spectrum antimicrobialactivity. Furthermore, these honeys were not any differentfrom two alien species grown in South Africa. In previousstudies, very similar results were obtained with E. cladoca-lyx (Bluegum) honey [12,18]. These honeys are commer-cially produced on a large scale and are easily obtainableoff the shelf. However, on a very small scale, honeys areproduced from other floral sources. Considering the largefloral biodiversity in South Africa, a survey of these rarehoneys might uncover a honey with high levels of antimi-crobial activity.The poor activity of Manuka honey was disappointing asprevious reports have shown that Manuka honey mayhave a very high antimicrobial activity against a numberof bacteria [13,19]. However, not all Manuka honeyexhibits antibacterial activity. High antimicrobial activityis recorded only in Manuka honey produced from specificlocalities, in particular, the East Cape region of the NorthIsland of New Zealand [20].The results from this report confirm that honeys from dif-ferent countries and regions have a wide variability intheir antimicrobial activity. It has been shown that theantimicrobial activity of honey may range from concen-trations lower than 3% to concentrations of 50% andhigher [10,13,19]. The honeys we tested had antimicro-bial activity in the 17 – 25% range against the organismsthat were tested except for S. oralis which showed sensitiv-ity at 12.5%. The bacteria S. aureus and E. coli, which wereincluded as reference cultures to previous reports, wereboth inhibited in the 50 – 25% range. These species havebeen shown to be sensitive at concentrations as low 1.8 –11% for some honeys [13,21]. This indicates that ourhoneys were of a poor antimicrobial quality.Honey may inhibit bacterial growth due to a number ofdifferent reasons. High sugar concentration (reducedwater activity), low pH, hydrogen peroxide generation,proteinaceous compounds or other unidentified compo-nents present in the honey may all provide antimicrobialactivity [11]. Low pH can be ruled out in this studybecause the average pH of the honey samples at 21% was6.47 which falls within the growth range of the micro-organisms. Although hydrogen peroxide was produced inour study, it only occurred at the very low dilutions and atconcentrations of ± 1 mg/litre. These concentrations areTable 1: Minimum inhibitory concentrations (%) of different honeys towards standard strains of oral micro-organismsOrganisms E. cladocalyx (Bluegum)Erica species (Fynbos)L. cordifolium (Pincushion)L. scoparium (Manuka)Carbohydrate controlPhenol controlS. mutans (10449) 25 25 25 25 50 <2.5S. salivarius (8618) 25 50 25 25 50 <2.5S. sanguis (7864) 25 25 25 25 25 <2.5S. anginosus (10708)17 17 17 17 25 <2.5S. gordonii (3165) 25 25 25 25 25 <2.5S. oralis (11427) 12.5 12.5 12.5 12.5 25 <2.5S. sobrinus (10921) 25 25 25 25 25 <2.5C. albicans (3118) 50 50 50 50 50 <2.5E. coli (9001) 25 50 25 25 50 <2.5S. aureus (8530) 25 25 25 25 25 <2.5Page 3 of 4(page number not for citation purposes)BMC Complementary and Alternative Medicine 2008, 8:41 http://www.biomedcentral.com/1472-6882/8/41too low to have any bacteriostatic effect. Concentrationsof at least 10 mg/litre are required to inhibit bacterialgrowth [22]. It is however possible that the hydrogen per-oxide production was not at its peak at the time of testing.It has been shown that hydrogen peroxide production canpeak at different times for different honeys. Some maytake as long as 24 hours [23]. If bacteriostatic concentra-tions were obtained within the 24 hour incubationperiod, lower MICs would probably have been attained.The honey MICs were very similar to the carbohydratecontrol solution which suggests that the main inhibitoryeffect was probably as a result of the reduced water activ-ity, the only exceptions being the bacteria S. anginosis andS. oralis. Both these strains were sensitive to honey at con-centrations lower than that observed for the carbohydratecontrol, indicating that an unidentified component, thatis active against these organisms, may be present in thehoney.ConclusionWe conclude that the honeys produced from the twoendemic wildflower plants from South Africa do not differfrom the alien species tested and show no exceptionallyhigh antimicrobial activity that could provide medicalgrade status.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsNJB participated in the design of the study, carried out themicrobiological analysis and drafted the manuscript. SRGconceived the study, participated in its design, wasresponsible for collection and identification of the honeysamples and assisted in drafting the manuscript. Allauthors read and approved the final manuscript.References1. Molan PC: The evidence supporting the use of honey as awound dressing. Int J Low Extrem Wounds 2006, 5:40-54.2. Simon A, Traynor K, Santos K, Blaser G, Bode U, Molan P: MedicalHoney for Wound Care - Still the 'Latest Resort'? Evid BasedComplement Alternat Med 2008, Epub 2008 Jan7:.3. Armon PJ: The use of honey in the treatment of infectedwounds. Trop Doct 1980, 10:91.4. Bromfield R: Honey for decubitus ulcers. Jama 1973, 224:905.5. Haffejee IE, Moosa A: Honey in the treatment of infantile gas-troenteritis. Br Med J (Clin Res Ed) 1985, 290:1866-1867.6. Subrahmanyam M: Topical application of honey in treatment ofburns. Br J Surg 1991, 78:497-498.7. Efem SE: Clinical observations on the wound healing proper-ties of honey. Br J Surg 1988, 75:679-681.8. Moore OA, Smith LA, Campbell F, Seers K, McQuay HJ, Moore RA:Systematic review of the use of honey as a wound dressing.BMC Complement Altern Med 2001, 1:2.9. Simon A, Sofka K, Wiszniewsky G, Blaser G, Bode U, Fleischhack G:Wound care with antibacterial honey (Medihoney) in pediat-ric hematology-oncology. Support Care Cancer 2006, 14:91-97.10. Lusby PE, Coombes AL, Wilkinson JM: Bactericidal activity of dif-ferent honeys against pathogenic bacteria. Arch Med Res 2005,36:464-467.11. Mundo MA, Padilla-Zakour OI, Worobo RW: Growth inhibition offoodborne pathogens and food spoilage organisms by selectraw honeys. Int J Food Microbiol 2004, 97:1-8.12. Basson NJ, du Toit IJ, Grobler SR: Antibacterial action of honeyon oral streptococci. J Dent Assoc S Afr 1994, 49:339-341.13. Wilkinson JM, Cavanagh HM: Antibacterial activity of 13 honeysagainst Escherichia coli and Pseudomonas aeruginosa. J MedFood 2005, 8:100-103.14. Allen KL, Molan PC, Reid GM: A survey of the antibacterialactivity of some New Zealand honeys. J Pharm Pharmacol 1991,43:817-822.15. English HK, Pack AR, Molan PC: The effects of manuka honey onplaque and gingivitis: a pilot study. J Int Acad Periodontol 2004,6:63-67.16. Root AI: ABC and XYZ of bee culture. Ohio, Root PublishingCompany; 1983. 17. Ericsson HM, Sherris JC: Antibiotic sensitivity testing. Report ofan international collaborative study. Acta Pathol Microbiol Scand[B] Microbiol Immunol 1971, 217:Suppl 217:1+.18. Steinberg D, Kaine G, Gedalia I: Antibacterial effect of propolisand honey on oral bacteria. Am J Dent 1996, 9:236-239.19. French VM, Cooper RA, Molan PC: The antibacterial activity ofhoney against coagulase-negative staphylococci. J AntimicrobChemother 2005, 56:228-231.20. Molan PC: The antibacterial properties of honey. Chemistry inNew Zealand 1995, July:10-14.21. Willix DJ, Molan PC, Harfoot CG: A comparison of the sensitiv-ity of wound-infecting species of bacteria to the antibacterialactivity of manuka honey and other honey. J Appl Bacteriol1992, 73:388-394.22. White JW, Subers MH, Schepartz AL: The identification of inhib-ine, the antibacterial factor in honey, as hydrogen peroxideand its origen in a honey glucose-oxidase system. Biochemicaand Biophysica Acta 1963, 73:57-70.23. Bang LM, Buntting C, Molan P: The effect of dilution on the rateof hydrogen peroxide production in honey and its implica-tions for wound healing. J Altern Complement Med 2003,9:267-273.Pre-publication historyThe pre-publication history for this paper can be accessedhere:http://www.biomedcentral.com/1472-6882/8/41/prepubTable 2: pH of the different honey solutions at each dilutionHoney type Dilution50% 25% 21% 17% 12.50% 6.25% 3.12%E. cladocalyx (Bluegum) 5.25 6.41 6.47 6.69 7.10 7.20 7.28Erica species (Fynbos) 5.19 6.28 6.39 6.54 6.71 6.96 7.15L. cordifolium (Pincushion) 5.23 6.40 6.52 6.70 6.90 7.30 7.38L. scoparium (Manuka) 5.17 6.30 6.45 6.64 6.89 7.18 7.30Page 4 of 4(page number not for citation purposes)

Antimicrobial activity of two South African honeys produced from indigenous Leucospermum cordifolium and Erica species on selected micro-organisms

Crossref

Abstract Background Honey has been shown to have wound healing properties which can be ascribed to its antimicrobial activity. The antimicrobial activity can be effective against a broad spectrum of bacterial species especially those of medical importance. It has also been shown that there is considerable variation in the antimicrobial potency of different types of honey, which is impossible to predict. With this in mind we tested the antimicrobial activity of honeys produced from plants grown in South Africa for their antibacterial properties on selected standard strains of oral micro-organisms. Methods The honeys used were produced from the blossoms of Eucalyptus cladocalyx (Bluegum) trees, an indigenous South African plant Leucospermum cordifolium (Pincushion), a mixture of wild heather shrubs, mainly Erica species (Fynbos) and a Leptospermum scoparium (Manuka) honey. Only pure honey which had not been heated was used. The honeys were tested for their antimicrobial properties with a broth dilution method. Results Although the honeys produced some inhibitory effect on the growth of the micro-organisms, no exceptionally high activity occurred in the South African honeys. The carbohydrate concentration plays a key role in the antimicrobial activity of the honeys above 25%. However, these honeys do contain other antimicrobial properties that are effective against certain bacterial species at concentrations well below the hypertonic sugar concentration. The yeast C. albicans was more resistant to the honeys than the bacteria. The species S. anginosus and S. oralis were more sensitive to the honeys than the other test bacteria. Conclusion The honeys produced from indigenous wild flowers from South Africa had no exceptionally high activity that could afford medical grade status.</p

Grobler Sias R

Basson Nicolaas J

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BMC Complementary and Alternative Medicine

Antimicrobial activity of two South African honeys produced from indigenous <it>Leucospermum cordifolium </it>and <it>Erica </it>species on selected micro-organisms

ABC and XYZ of bee culture.

AL: The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origen in a honey glucose-oxidase system. Biochemica and Biophysica Acta

Cavanagh HM: Antibacterial activity of 13 honeys against Escherichia coli and Pseudomonas aeruginosa.

Clinical observations on the wound healing properties of honey.

du Toit IJ, Grobler SR: Antibacterial action of honey on oral streptococci.

Fleischhack G: Wound care with antibacterial honey (Medihoney) in pediatric hematology-oncology. Support Care Cancer

Gedalia I: Antibacterial effect of propolis and honey on oral bacteria.

GM: A survey of the antibacterial activity of some New Zealand honeys.

Harfoot CG: A comparison of the sensitivity of wound-infecting species of bacteria to the antibacterial activity of manuka honey and other honey.

Honey for decubitus ulcers. Jama

JM: Bactericidal activity of different honeys against pathogenic bacteria. Arch Med Res

Medical Honey for Wound Care - Still the 'Latest Resort'? Evid Based Complement Alternat Med

Molan PC: The antibacterial activity of honey against coagulase-negative staphylococci.

Molan PC: The effects of manuka honey on plaque and gingivitis: a pilot study.

Moosa A: Honey in the treatment of infantile gastroenteritis.

RA: Systematic review of the use of honey as a wound dressing.

Sherris JC: Antibiotic sensitivity testing. Report of an international collaborative study.

The antibacterial properties of honey. Chemistry in New Zealand

The effect of dilution on the rate of hydrogen peroxide production in honey and its implications for wound healing.

The evidence supporting the use of honey as a wound dressing. Int J Low Extrem Wounds

The use of honey in the treatment of infected wounds. Trop Doct

Topical application of honey in treatment of burns.

Worobo RW: Growth inhibition of foodborne pathogens and food spoilage organisms by select raw honeys.

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Antimicrobial activity of two South African honeys produced from indigenous Leucospermum cordifolium and Erica species on selected micro-organisms

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