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The Antibacterial Activity of Honey Derived from
Australian Flora
Julie Irish, Shona Blair, Dee A. Carter*
School of Molecular Bioscience, University of Sydney, Camperdown, Australia
Abstract
Chronic wound infections and antibiotic resistance are driving interest in antimicrobial treatments that have generally been
considered complementary, including antimicrobially active honey. Australia has unique native flora and produces honey
with a wide range of different physicochemical properties. In this study we surveyed 477 honey samples, derived from
native and exotic plants from various regions of Australia, for their antibacterial activity using an established screening
protocol. A level of activity considered potentially therapeutically useful was found in 274 (57%) of the honey samples, with
exceptional activity seen in samples derived from marri (Corymbia calophylla), jarrah (Eucalyptus marginata) and jellybush
(Leptospermum polygalifolium). In most cases the antibacterial activity was attributable to hydrogen peroxide produced by
the bee-derived enzyme glucose oxidase. Non-hydrogen peroxide activity was detected in 80 (16.8%) samples, and was
most consistently seen in honey produced from Leptospermum spp. Testing over time found the hydrogen peroxide-
dependent activity in honey decreased, in some cases by 100%, and this activity was more stable at 4uC than at 25uC. In
contrast, the non-hydrogen peroxide activity of Leptospermum honey samples increased, and this was greatest in samples
stored at 25uC. The stability of non-peroxide activity from other honeys was more variable, suggesting this activity may have
a different cause. We conclude that many Australian honeys have clinical potential, and that further studies into the
composition and stability of their active constituents are warranted.
Citation: Irish J, Blair S, Carter DA (2011) The Antibacterial Activity of Honey Derived from Australian Flora. PLoS ONE 6(3): e18229. doi:10.1371/
journal.pone.0018229
Editor: Michael Otto, National Institutes of Health, United States of America
Received December 8, 2010; Accepted February 28, 2011; Published March 28, 2011
Copyright: ß2011 Irish et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was financially supported by a Rural Industries Research and Development Corporation grant (Project No. US-128A), and an Australian
Postgraduate Award to JI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: dee.carter@sydney.edu.au
Introduction
The use of honey as a wound dressing is gaining acceptance in
modern medicine as a result of its antimicrobial activity and
wound healing properties. In particular, certain types of honey
exhibit broad-spectrum antimicrobial activity and are effective
against antibiotic resistant bacterial pathogens [1,2,3,4,5]. Honey-
based wound care products have been registered with medical
regulatory authorities as wound care agents in Australia, Canada,
the European Union, Hong Kong, New Zealand and the USA. In
most instances these products use manuka honey from New
Zealand or the equivalent honey produced from other Leptospermum
species in Australia.
Honey has several properties that contribute to its antimicrobial
activity. In most honeys, low pH and high osmolarity are
combined with the enzymatic production of hydrogen peroxide
that exerts an antimicrobial effect [6,7]. Phytochemical compo-
nents derived from the floral source of the honey can confer
additional activity that is stable in the presence of catalase, an
enzyme that destroys hydrogen peroxide [8]. This non-peroxide
activity was first identified in manuka (Leptospermum scoparium)
honey from New Zealand where it is often marketed as the Unique
Manuka Factor (UMFH).
Variations in the type and level of antimicrobial activity in
honey are associated with their floral source. However, while some
floral sources appear to be associated with particular levels of
hydrogen peroxide activity, variation in this activity among honeys
from within the same floral species has also been observed
[9,10,11]. This may be due to the geographical location of the
floral source and the prevailing environmental conditions, which
affect the physiology of the floral species [12], or to bee-related
factors such as age or colony health, which may affect the
production or activity of glucose oxidase (the enzyme responsible
for hydrogen peroxide production in honey) [13,14,15,16]. The
precise mechanisms determining the level of this type of activity
are yet to be elucidated.
Honeys with non-peroxide antimicrobial activity are more
closely associated with floral source, being generally derived from
Leptospermum species [8,9], although this type of activity has also
been found in a small number of non-Leptospermum honeys
[9,17,18,19]. In a clinical setting where honey is used as a topical
antimicrobial and wound dressing, non-peroxide activity may be
advantageous as it is not destroyed by catalase present in body
fluids, and is unaffected by gamma irradiation [20], allowing these
honeys to be sterilized for medicinal use. The compound primarily
responsible for non-peroxide activity in New Zealand manuka
honey has recently been identified as methylglyoxal (MG) [21,22],
which is derived from dihydroxyacetone, a compound present in
high levels in manuka nectar [23]. The reasons for varying
dihydroxyacetone levels in different plants are not yet understood.
An agar well diffusion method to determine the antibacterial
activity of honey with reference to phenol [9] has become the de
facto standard in medical honey testing, and is used commercially
to assign a UMFHvalue to medicinal honeys. This method is a
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simple and rapid way to screen large numbers of honey samples
for antibacterial activity; however, it does not discriminate
between individual antibacterial factors and their relative
contributions to overall antibacterial activity. Using this method,
Allen et al. [9] conducted a survey of 345 New Zealand honeys and
found wide variation in hydrogen peroxide-dependent antibacte-
rial activity, both within and among floral sources. Non-peroxide
activity was identified in a significant proportion of samples of
manuka (L. scoparium) and Viper’s bugloss (Echium vulgare) honeys. A
survey of 179 non-manuka New Zealand honeys by Brady et al.
[10] also found wide variation in hydrogen peroxide-dependent
activity, and non-peroxide activity was not detected in any
samples. The only study using the phenol equivalence method
conducted outside New Zealand is a small survey of 30 Portuguese
honeys from several floral sources [17]. This study revealed low
levels of hydrogen peroxide-dependent activity in all samples, and
low levels of non-peroxide activity in six samples, primarily from
Lavandula species.
Australia is home to diverse and unique floral resources that are
exploited by the beekeeping industry. No published data exist on
the antimicrobial activity of most Australian honey, and the
benefits of this knowledge to both the apiary industry and the
health care sector are clear. Therefore, the aim of this study was to
survey a wide range of Australian honey sourced from different
native and exotic flora for antimicrobial activity. Honey samples
were tested for their levels of total antibacterial activity and non-
peroxide activity, and correlations were investigated between the
type and level of antimicrobial activity and the floral source of the
honey, its region of origin and the age of the honey sample. Over
half of the honey samples tested had antibacterial activity in the
range considered to be therapeutically useful. Exceptional
hydrogen peroxide-dependent antibacterial activity was found in
honey derived from Eucalyptus marginata (jarrah) and Corymbia
calophylla (marri) from Western Australia, and very highly active
non-peroxide honeys were produced from Leptospermum species,
particularly L. polygalifolium, growing in the coastal New South
Wales-Queensland border region. Although floral source and
region were clearly important in the production of active honey,
the level of activity varied widely among samples and changed
during storage.
Materials and Methods
Honey samples
A total of 477 honey samples were received from beekeepers
and honey companies throughout Australia between March 2005
and June 2007. A map indicating the location of the honey
samples is shown in Figure 1. Each sample was assigned a unique
reference number and details provided by the beekeepers were
entered into a database (see Table S1). Honey samples were stored
in glass or plastic containers at room temperature in the dark.
Comvita Wound Care 18+honey (Comvita New Zealand Ltd.,
Paengaroa, New Zealand), a pure manuka honey from New
Zealand with non-peroxide antibacterial activity equivalent to at
least 18% phenol was used as a positive control. This honey is
commercially available as a wound dressing and is registered with
appropriate regulatory bodies in Australia, New Zealand, the USA
and the EU.
Identification of the floral source of the honey was performed by
the beekeepers based on the availability of flora for nectar foraging,
location of the apiary and organoleptic characteristics of the honey.
Where beekeepers supplied only the common name of the floral
source, the scientific name was determined from the Australian
Plant Common Name Database [24], Australian Plant Name Index
[25] and/or floral distribution maps [26,27,28,29,30], where
possible.
Phenol equivalence assay for antibacterial activity
Antibacterial activity of honey samples with reference to phenol
was determined as described by Allen et al. [9]. An 18 h culture of
Staphylococcus aureus ATCC 9144 (Oxoid, Hampshire, UK) grown
in tryptone soya broth (TSB; Oxoid) was adjusted to an
absorbance of 0.5 at 540 nm. Large assay plates (2456245 mm;
Corning Inc., Corning, NY, USA) were prepared with 150 ml of
nutrient agar (Becton, Dickinson and Company, Sparks, MD,
USA) that had been seeded with 100 ml of the prepared S. aureus
culture. Plates were stored inverted at 4uC for use the next day,
when 64 wells were cut into the agar with a sterile 8 mm diameter
cork borer, over a 25 mm grid. Each well was numbered, in
duplicate, using a quasi-Latin square that enabled the duplicate
samples to be placed randomly on the plate.
Honey samples were prepared freshly for each assay by adding
10 ml of sterile deionised water to 10 g of well-mixed honey. One
ml of each honey solution was mixed with 1 ml of sterile deionised
water for total activity testing, or 1 ml of a freshly prepared
5600 U/ml catalase solution (Sigma, St Louis, MO, USA) for non-
peroxide activity testing. A 100 ml aliquot of each solution was
placed in wells of the assay plate, in duplicate.
Phenol (BDH, VWR International Ltd., Poole, UK) standards
of 2%, 3%, 4%, 5%, 6%, and 7% were freshly prepared every four
weeks in sterile deionised water and stored at 4uC. Aliquots of
100 ml of each solution were placed in duplicate wells of the assay
plate. Negative controls of sterile deionised water and catalase
solution were included in duplicate wells of each assay plate.
Comvita Wound Care 18+honey was prepared as for other honey
samples for use as a positive control. The plates were incubated at
37uC for 18 h.
The diameter of each zone of inhibition was measured in two
directions at right angles to each other using Vernier callipers. The
mean diameter of the zone of inhibition around each well was
calculated and squared, and a standard curve was generated of
phenol concentration against the mean squared diameter of the
zone of inhibition. The activity of each diluted honey sample was
calculated using the standard curve. To account for the dilution
and density of honey, this figure was multiplied by 4.69 (based on a
mean honey density of 1.35 g/ml, as determined by [31]), and the
activity of the honey was then expressed as the equivalent phenol
concentration (% w/v) [9,31]. Each honey sample was tested on at
least two separate occasions, and the mean phenol equivalence
was used in further analysis.
The effect of sample age on antibacterial activity
A subset of 20 honey samples (10 with hydrogen peroxide
activity only and 10 with non-peroxide activity) were selected for
retesting following storage of aliquots in the dark at 4uC and at
25uC for 8 to 22 months after the first test. Honey samples were re-
tested in duplicate on two separate occasions, and the mean
phenol equivalence was used in further analysis.
Data analysis
The data consisted of four categorical variables (floral source,
floral origin (native, exotic, or mixed), region, sample age), and two
main response variables (total activity and non-peroxide activity).
All data were analysed qualitatively, with the exception of the
change in antibacterial activity over time. Statistical analysis of
change in activity over time was performed with Minitab 14
statistical software (Minitab Inc. Pennsylvania, USA), using the
Wilcoxon signed ranks test. To aid statistical analysis, honeys with
Antibacterial Honey from Australian Plants
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antibacterial activity below the limit of detection of the assay
(approximately 5% phenol equivalent) were assigned a value of 5,
although these values are reported as ,5 where appropriate.
Results
Reproducibility of the phenol equivalence assay
Comvita Wound Care 18+honey was used as a positive control
to monitor the reproducibility of the phenol equivalence assay.
This commercially available product is standardised such that its
non-peroxide activity is at least 18% (w/v) phenol equivalent.
Over the course of this study, the mean total activity of this honey
was 17.960.9% phenol equivalent, and the mean non-peroxide
activity was 17.361% phenol equivalent. Day to day variation in
activity was within 62% phenol equivalent of the specified 18%.
This range was exceeded on only one occasion and all honey
samples in that plate were retested. Replicate tests of individual
honey samples were also within the range of 62% phenol
equivalent.
Total antibacterial activity of honey samples
Antibacterial activity equivalent to at least 10% (w/v) phenol
should provide therapeutic benefits as an antimicrobial [32]. The
antibacterial activity of the 477 honey samples was therefore
divided into categories of undetectable activity (,5% phenol
equivalent), low activity (5–10% phenol equivalent), potentially
therapeutically beneficial activity (10–20% phenol equivalent) and
high activity (.20% phenol equivalent).
The total antibacterial activity (encompassing both hydrogen
peroxide-dependent and non-peroxide activity) of the 477 honey
samples is shown in Figure 2. The average total activity was
10.669.5% phenol equivalent (range: ,5–34.3; median: 13).
Detectable antibacterial activity was found in 286 (60%) of the
honey samples, with an average total activity of 17.865% phenol
equivalent (range: 7.4–34.3; median: 17.1). A total of 274 (57%) of
the honey samples had activity of $10% phenol equivalent and
could be considered to be therapeutically useful.
The 477 honey samples were derived from 142 different floral
sources, including combinations of known flora, as well as
unspecified mixed flora. The majority of honey samples (372
samples = 78%) were derived from native Australian flora; 80
samples (16.8%) were of mixed origin and were likely to contain
native floral species; and 25 samples (5.2%) were derived from
exotic floral species. Table 1 shows the median antibacterial
activity of honeys from floral sources with three or more samples,
ranked by median total activity (for activity of all samples see
Table S1).
Honey with non-peroxide antibacterial activity
Non-peroxide activity was detected in 80 honey samples
(16.8%), with a mean of 15.664.7% phenol equivalent (range:
8.1–25.9; median: 15.4). A summary of these honeys is shown in
Table 2, and a map indicating their floral source and region of
origin is shown in Figure 1. Samples that were derived from
Leptospermum floral species or contained Leptospermum as part of a
mixed floral source comprised 77.5% of honey samples with
detectable non-peroxide activity (mean non-peroxide activity of
Leptospermum-containing honeys: 17.264.1% phenol equivalent;
range: 9.8–25.9; median: 16.4). Eighteen (22.5%) of the honey
samples derived from flora other than Leptospermum also exhibited
non-peroxide activity (average non-peroxide activity of non-
Leptospermum honeys: 10.161.7% phenol equivalent; range: 8.1–
15.9; median: 10). Non-peroxide activity in Leptospermum-contain-
ing honeys generally comprised a higher proportion of the total
antibacterial activity (up to 100%) than in non-Leptospermum
honeys.
The non-peroxide antibacterial activity of honey derived from
single Leptospermum species is shown in Table 3. Non-peroxide
Figure 1. Location and activity of honey samples. a) Samples from west Australia (WA = Western Australia); b) Samples from east Australia and
Tasmania (QLD = Queensland; NSW = New South Wales; SA = South Australia; VIC = Victoria; TAS = Tasmania). Numbers indicate floral source of the
honey samples with non-hydrogen peroxide activity: 1. Leptospermum spp. alone; 2. Leptospermum spp. in mixed flora; 3. Unspecified flora; 4.
Melaleuca and brush box; 5. Spotted gum; 6. Forest red gum; 7. Clover; 8. Wild flowers; 9. Messmate stringybark; 10. Orchard; 11. Coastal Moort; 12.
Melaleuca alone.
doi:10.1371/journal.pone.0018229.g001
Antibacterial Honey from Australian Plants
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activity was evident in honey from L. polygalifolium,L. liversidgei,L.
laevigatum and some unspecified species. These honeys were
collected primarily in the Northern Rivers region of New South
Wales and the adjacent Southeast Coast region of Queensland,
with one sample from the Capricornia region of Queensland
(Figure 1). Leptospermum honeys collected from other states and
regions did not exhibit non-peroxide activity.
The effect of sample age and storage temperature on
antibacterial activity
The majority of honey samples were collected from hives
between 2001 and 2007, and tested between 2006 and 2007. No
collection date was specified for 66 samples, and one sample was
collected in 1978. Scatter plots of antibacterial activity vs. sample
age for all honeys of known age showed no correlation between
antibacterial activity and age of the honey sample (r
2
= 0.0062 for
total antibacterial activity; r
2
= 0.0072 for non-peroxide activity).
Aliquots of a subset of 20 honey samples (10 samples with
hydrogen peroxide-dependent activity only and 10 samples with
non-peroxide activity) were stored in the dark at 25uCand4uC,
and were re-tested between 8 and 22 months after first testing
(Table 4; see Table S2 for the full dataset). Repeat testing found
the median total antibacterial activity of honeys exhibiting only
hydrogen peroxide-dependent activity significantly decreased
over time at both 25uCand4uC (Wilcoxon signed ranks test
P,0.01). This loss of activity was significantly greater after
storage at 25uC compared to storage at 4uC (Wilcoxon signed
ranks test P,0.01). All honeys exhibiting only hydrogen
peroxide-dependent activity decreased in activity, with an
average of loss of 9.5% phenol equivalent, and two samples lost
all detectable activity after storage at 25uC. For the 10 samples
exhibiting non-peroxide activity, the median total and non-
peroxide activity did not change significantly over time at either
storage temperature (Wilcoxon signed ranks test P.0.05).
However, among these it appeared that the three honey samples
derived from pure L. polygalifolium all increased in activity,
particularly those stored at 25uC(+16 to +34% change in total
activity and +13 to +37% change in non-peroxide activity), while
the five samples that were from sources excluding L. polygalifolium
showed only very minor increases or decreased in activity during
storage (–4 to –34% change in total activity and +2to–16%
change in non-peroxide activity at 25uC).
Discussion
The integration of honey into modern medicine as a therapeutic
agent requires that medicinal honey products exhibit a high level of
antimicrobial activity that is consistent and standardised, as with
any other medicinal product. It is therefore of critical importance to
the apicultural, horticultural and medical industries to identify floral
species that give rise to honey with consistently high activity. This
study is the first to provide a broad overview of the antibacterial
activity of Australian honey from a wide variety of floral sources.
Results show that these honeys exhibit a wide range of antibacterial
activity, and the majority have potential for therapeutic use.
Honey derived from certain Australian flora possesses
exceptional antibacterial activity
Honey with non-peroxide activity is highly sought after in the
medicinal honey market due to its potential clinical advantages. This
study demonstrates that the prevalence of non-peroxide activity
among Australian honey samples, and the level of this activity,
exceeds that reported in honey from other countries. Non-peroxide
activity was identified in 70.6% of Australian Leptospermum honey
samples tested, with a median of 16.7% phenol equivalent (Table 2).
The methylglyoxal (MG) content of Australian Leptospermum
honeys has not yet been investigated, and it is possible that this
compound is present in similar or higher levels than in manuka
honey. Non-peroxide activity was strongly associated with Leptos-
permum honeys collected from the Northern Rivers region of New
South Wales and the adjacent Southeast Coast region of Queens-
land (Figure 1), indicating that these regions are a potentially
valuable source of therapeutically beneficial honey. Among the
Leptospermum species, L. polygalifolium (jellybush) produced honey that
was particularly high in activity (Table 3). Although L. scoparium
(manuka) is the primary source of honey with non-peroxide activity
in New Zealand, none of the 11 samples of L. scoparium honey from
Australia had detectable non-peroxide activity. These findings
suggest that environmental conditions in different regions play a role
in the relationship between floral source and non-peroxide
antibacterial activity, or alternatively that different regions contain
as yet uncharacterised subspecies of Leptospermum that are respon-
sible for providing honeys with non-peroxide activity. In New
Zealand, different concentrations of phenolic compounds, including
MG, are found in L. scoparium honeys collected from different
regions, with the potential to affect antibacterial activity [33].
Figure 2. Total antibacterial activity of Australian honey samples. Graph shows combined peroxide and non-peroxide dependent activity in
477 honey samples collected from Australian floral sources, divided into increments of (w/v) phenol equivalent.
doi:10.1371/journal.pone.0018229.g002
Antibacterial Honey from Australian Plants
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Further botanical and genetic studies of Australian Leptospermum
species are required to elucidate these differences, and may inform
studies aimed at cultivating particular plant species in productive
regions for highly active medicinal honey.
Exceptionally high activity was also seen in hydrogen peroxide-
dependent honeys derived from marri (C. calophylla; median
activity 25.7, maximum 29.7) and jarrah (E. marginata; median
activity 25.1, maximum 31.4) from Western Australia. To our
Table 1. Total antibacterial activity of honey samples from floral sources with a sample size $3, ranked by median activity.
Floral source: Common name (Scientific name) No. samples
No. (%) with
detectable activity
1
Total activity
1
Range Median
Marri (Corymbia calophylla) 8 7 (88) ,5–29.7 25.7
Jarrah (Eucalyptus marginata) 19 18 (95) ,5–31.4 25.1
Jelly bush and heath flora (Leptospermum polygalifolium and unknown species) 3 3 (100) 17.3–19.9 19.8
Spotted gum (Corymbia maculata) 4 4 (100) 14.7–25.1 18.9
Tea tree and paperbark (Leptospermum semibaccatum and Melaleuca nodosa) 4 4 (100) 18.1–19.6 18.8
Jelly bush (L. polygalifolium) 29 28 (97) ,5–26.2 17.9
Jelly bush, tea tree (Leptospermum sp.) 14 12 (86) ,5–25.8 17.8
Mixed flora, Sydney metropolitan region 32 25 (78) ,5–29.8 15.9
Lemon-scented tea tree (Leptospermum liversidgei) 5 5 (100) 14.0–24.5 15.7
Red stringybark (Eucalyptus macrorhyncha) 9 5 (56) ,5–26.1 15.3
Crow’s ash and jelly bush (Guioa semiglauca and L. polygalifolium) 3 2 (67) ,5–19.4 15.2
Banksia (Banksia sp.) 25 22 (88) ,5–24.1 15.0
Jelly bush mix (L. polygalifolium and Leptospermum speciosum 3 3 (100) 14.2–14.7 14.6
Clover (Trifolium repens) 3 2 (67) ,5–16.3 14.3
Manuka (Leptospermum scoparium) 11 9 (82) ,5–16.3 13.1
Paperbark, tea tree (Melaleuca sp.) 22 18 (82) ,5–19.6 12.8
Mugga ironbark (Eucalyptus sideroxylon) 3 3 (100) 9.7–12.3 11.7
Mixed wildflowers, Tasmania 5 4 (80) ,5–16.1 11.6
Feather bush (Micromyrtus ciliata) 3 2 (67) ,5–13.6 11.5
Other mixed or unknown flora 35 19 (54) ,5–24.6 9.9
Messmate stringybark (Eucalyptus obliqua) 5 3 (60) ,5–15.2 9.8
Snow gum (Eucalyptus pauciflora) 3 2 (67) ,5–10.5 8.7
Tea tree and paperbark (Leptospermum laevigatum and Melaleuca nodosa) 4 2 (50) ,5–16.3 7.7
Tea tree, paperbark (Melaleuca quinquenervia) 3 2 (67) ,5–21.9 7.4
Paterson’s curse, Salvation Jane (Echium plantagineum) 4 2 (50) ,5–15.6 6.3
Leatherwood (Eucryphia lucida) 11 4 (36) ,5–17.5 ,5
Wandoo (Eucalyptus wandoo) 7 2 (29) ,5–18.7 ,5
Lemon-scented tea tree and pink bloodwood (Leptospermum liversidgei and
Corymbia intermedia)
17 3 (18) ,5–14.6 ,5
Eucalyptus (Eucalyptus sp.) 15 5 (33) ,5–24.9 ,5
Parrot bush (Dryandra sessilis) 3 1 (33) ,5–21.0 ,5
Coastal tea tree (Leptospermum laevigatum) 4 1 (25) ,5–21.4 ,5
Mixed rainforest flora, Queensland 3 1 (33) ,5–16.2 ,5
Blue gum (Eucalyptus globulus) 3 1 (33) ,5–15.3 ,5
Yellow box (Eucalyptus melliodora) 4 1 (25) ,5–12.7 ,5
Saw banksia (Banksia serrata) 4 0 (0) ,5,5
Coriander (Coriandrum sativum) 3 0 (0) ,5,5
Heather bush (Thryptomene micrantha) 3 0 (0) ,5,5
Tea tree and yellow box (Leptospermum sp. and E. melliodora) 3 0 (0) ,5,5
Macadamia (Macadamia integrifolia) 3 0 (0) ,5,5
Red mallee (Eucalyptus oleosa) 4 0 (0) ,5,5
Powderbark (Eucalyptus accedens) 3 0 (0) ,5,5
1. Activity calculated as % (w/v) phenol equivalent
doi:10.1371/journal.pone.0018229.t001
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knowledge these are the most potent antibacterial honeys yet
reported. Very high activity was also seen in 22% of honeys from
the Sydney metropolitan region, indicating that highly active
honey may be obtained from a number of different environments.
Although there is a focus in the literature on the antimicrobial
activity of Leptospermum honey, many in vitro studies investigating
the antimicrobial activity of honey have found that manuka honey
and honey with similar levels of hydrogen peroxide activity are
equally effective against bacterial pathogens [2,3,4,5,34,35,36].
Honeys with hydrogen peroxide-dependent activity are more
effective than manuka honey at inhibiting dermatophyte fungi [37]
and species of the yeast Candida [38], indicating that these honeys
may be more broad spectrum and valuable as antifungal agents
than manuka honey.
Antibacterial activity is highly variable
The antibacterial activity of the Australian honey samples tested
exhibited a distinctly bimodal distribution (Figure 2), with peaks at
0–5% and 15–20% (w/v) phenol equivalent. This suggests that the
antibacterial activity in fresh honey is largely ‘‘all-or-nothing’’,
although what governs this is not known as there was substantial
variation in activity both among and within floral sources. Of the
41 floral sources represented by three or more honey samples, only
6 produced uniformly active honey, and none of the honeys with
Table 2. Honey samples exhibiting non-peroxide antibacterial activity.
Floral source No. samples tested
No. (%) samples with non-
peroxide activity
Mean non-peroxide activity ±SD*
(mean % of total activity ±SD)
Leptospermum spp. alone 68 48 (71) 17.964.2 (94.966.4)
Leptospermum spp. in mixed flora 44 14 (32) 14.762.6 (85.8611.8)
Tasmanian wildflowers 5 3 (60) 12.762.7 (97.262.6)
Forest red gum 2 1 (50) 11.261.1 (46.567.7)
Melaleuca and brush box 2 1 (50) 10.560.7 (51.867.2)
Spotted gum 4 3 (75) 10.160.3 (51.1614.3)
Melaleuca alone 26 1 (4) 9.760.9 (66.862.2)
Unspecified flora 72 5 (7) 9.260.9 (78.4618.3)
Clover 3 1 (33) 9.260.1 (64.063.7)
Orchard 2 1 (50) 9.160.2 (28.461.4)
Messmate stringybark 6 1 (17) 9.060.4 (59.260)
Coastal moort 1 1 (100) 8.860.3 (67.4611.6)
*Calculated as % (w/v) phenol equivalent for samples within a floral source with non-peroxide activity.
doi:10.1371/journal.pone.0018229.t002
Table 3. Non-peroxide antibacterial activity and region of origin of honey derived from single Leptospermum species.
Leptospermum
species Region*
No. samples
tested
No. (%) samples with non-
peroxide activity
Mean non-peroxide
activity ±SD
L. polygalifolium Northern Rivers NSW 28 27 (96) 18.963.9
Capricornia QLD 1 1 (100) 21.1
L. liversidgei Northern Rivers NSW 5 5 (100) 16.164.4
L. laevigatum Northern Rivers NSW 1 1 (100) 19.7
Central VIC 2 0 (0) ,5
Hunter NSW 1 0 (0) ,5
L. scoparium Southeast Huon, Channel and Lower
Derwent Valley TAS
1 0 (0) ,5
Northeast and Flinders Island TAS 10 0 (0) ,5
L. flavescens Illawarra NSW 1 0 (0) ,5
L. continentale Central VIC 2 0 (0) ,5
Unspecified Leptospermum sp. Northern Rivers NSW 6 5 (83) 16.265.1
Southeast Coast QLD 4 4 (100) 19.565.4
Illawarra NSW 1 0 (0) ,5
Metropolitan NSW 1 0 (0) ,5
Northern Tablelands NSW 1 0 (0) ,5
Murraylands SA 1 0 (0) ,5
*NSW: New South Wales; QLD: Queensland; SA: South Australia; TAS: Tasmania; VIC: Victoria; see Figure 1 for map locations.
doi:10.1371/journal.pone.0018229.t003
Antibacterial Honey from Australian Plants
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more than 10 samples were consistently active (Table 1). At the
other end of the scale, few of the multiply sampled floral sources
produced uniformly inactive honey (Table 1). Plant-derived factors
that contribute to the antimicrobial activity in honey may be
influenced by local environmental conditions such as climate,
water and nutrient availability [12], and entomological factors
may also contribute to activity [39]. The complex interplay of
plant species, plant physiology, growth conditions, seasonal
variations and bee physiology make it difficult to predict whether
or not a given honey sample is likely to have antimicrobial activity.
A remarkable finding of the current study was that even honeys
produced in one location at one time could vary in activity. In one
example, 22 Banksia honey samples obtained following a single
flowering event were tested, with each honey sample collected
from a separate hive in the same apiary (samples B11–B32; Table
S1). Total antibacterial activity among 21 of these samples ranged
from 11.4 to 19.2% phenol equivalent, and one sample had no
detectable activity. Similarly, 18 Melaleuca honey samples that had
been collected from separate hives in a single apiary included four
inactive samples, with the remainder ranging in total activity from
10.8 to 14.3% phenol equivalent (samples T11–T28; Table S1).
This suggests that entomological differences can have a substantial
role in the activity of honey, even more so than the floral source.
The health of individual bee colonies and the age of foraging
workers may affect foraging activity or the secretion of enzymes
responsible for antibacterial activity, including glucose oxidase
[13,14,15,16]. In addition, since truly monofloral honeys are often
practically impossible to obtain, different foraging preferences
among colonies may result in honey produced from the nectar of
numerous floral species [40], thereby altering the overall activity.
Floral sources of honey are primarily identified as the dominant
species in flower at the time, and mixed floral sources may have
been more prevalent than was reported by beekeepers. This is of
particular interest for non-Leptospermum honeys exhibiting non-
peroxide activity, as there is the possibility that they contain some
nectar from Leptospermum species. This was considered unlikely in
the current study, however, since most were from regions where
Leptospermum is either not present or would not be in flower when
the bees were foraging. It is also possible that Leptospermum honey
with non-peroxide activity that was collected in the Northern
Rivers or Southeast Coast regions may contain nectar from L.
polygalifolium, even if beekeepers identified the dominant floral
source as a different Leptospermum species. A more detailed
investigation of the floral sources of these honeys, perhaps using
pollen analysis, is warranted.
Non-peroxide activity was identified in 18 honey samples not
derived from Leptospermum flora (Table 2; Figure 1), including the
majority of honeys derived from spotted gum and Tasmanian
wildflowers (3/4 and 3/5 of the honeys sampled, respectively). On
the whole, however, this activity was sporadic, with no clear link to
Table 4. Change in antibacterial activity of honey samples following storage.
Floral source: Common name (Scientific
name) [age at 1
st
assay in months] Activity pre-storage
1
Months in
storage
% Change in activity
post-storage at 256C
% Change in activity
post-storage at 46C
Total Non-peroxide Total Non-peroxide Total
Non-
peroxide
Red stringybark (Eucalyptus macrorhyncha) [10] 26.1 ,5 17 -34 0 -20 0
Mixed urban flora [11] 17.0 ,5 16 -28 0 -22 0
Viper’s bugloss and lucerne (Echium vulgare and
Medicago sativa) [22]
17.2 ,5 17 -41 0 -28 0
Grey ironbark (Eucalyptus paniculata) [1] 15.6 ,5 16 -100 0 -14 0
Forest red gum (Eucalyptus tereticornis) [5] 18.3 ,5 16 -26 0 -27 0
Turpentine (Syncarpia glomulifera) [42] 24.7 ,5 22 -100 0 -43 0
Bloodwood (Corymbia gummifera) [3] 23.3 ,5 16 -45 0 -10 0
Avocado (Persea americana) [3] 21.8 ,5 16 -42 0 -22 0
Mixed urban flora [45] 24.6 ,5 23 -33 0 -5 0
Red stringybark (Eucalyptus macrorhyncha) [42] 24.6 ,5 22 -48 0 -42 0
Jelly bush (Leptospermum polygalifolium)[,1] 15.9 15.3 18 +35 +37 +7+10
Jelly bush (L. polygalifolium) [46] 17.2 17.1 17 +29 +28 +8+5
Jelly bush (L. polygalifolium) [54] 23.4 23.4 9 +16 +13 +9+9
Jelly bush and crow’s ash (L. polygalifolium and
Guioa semiglauca)[46]
19.4 13.3 21 -12 +23 -24 +3
Jelly bush and tea tree (L. polygalifolium and
Leptospermum whitei)[9]
13.9 13.2 20 -12 -12 0 -4
Clover (Trifolium repens) [9] 14.3 9.2 23 -34 +2 -37 -3
Mixed flora [3] 9.9 8.5 21 -4 +1 -13 -2
Paperbark and brush box (Melaleuca sp. and
Lophostemon confertus)[7]
20.8 10.5 21 -15 -11 -4 -6
Lemon-scented tea tree (Leptospermum liversidgei) [8] 14.6 13.4 21 -21 -16 -5 -7
Lemon-scented tea tree (L. liversidgei) [17] 24.5 23.6 11 -9 -12 +1+1
Determined as (w/v) phenol equivalent.
doi:10.1371/journal.pone.0018229.t004
Antibacterial Honey from Australian Plants
PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e18229
a particular floral source or geographic region. Tests on the
stability of honey following storage found the samples with non-
peroxide activity derived from clover, mixed flora and paperbark/
brush box, as well as samples from L. liversidgei, either remained
relatively stable or declined in activity over time, while the three
honey samples derived from only L. polygalifolium increased in
activity (Table 4). Many beekeepers find that non-peroxide activity
increases over time [41], which may correspond to an increase in
Maillard reaction products including MG [23,33,42]. The fact
that this did not happen in non-peroxide honeys that were derived
from plants other than L. polygalifolium suggests that at least some of
the activity in these honeys is due to antimicrobial compounds
other than MG. Bee defensin-1 and other peptides, along with
various phenolics, have been found in different honey samples and
have been proposed to convey antimicrobial effects [19,39,43,44].
Whether any of these occur in the Australian non-peroxide honeys
remains to be determined.
The stability of the antibacterial activity of honey over time has
implications for the shelf life of medicinal honey products. In the
case of hydrogen peroxide-dependent honeys this is likely to be
due to the instability of glucose oxidase, the enzyme responsible for
hydrogen peroxide production, which is influenced by various
factors including pH and exposure to light [45]. Enzyme stability is
often affected by temperature, and the loss in activity was
mitigated to some extent by storage at 4uC (Table 4). Extra care
in the handling and storage of honeys with hydrogen peroxide-
dependent activity may therefore be necessary if these are to be
used in the clinical setting. Regardless of the reason behind any
change in activity, honeys that are used in laboratory tests over
prolonged periods should be tested regularly to ensure that the
level of activity has remained constant. Degradation of activity
over time does not preclude the use of honey as an antimicrobial
agent, since all medicinal products have a shelf life and many
require refrigeration. However, a greater understanding of the
time frame and the storage conditions that affect loss of activity are
vital in producing a standardised medicinal product.
Conclusions
This study has provided a broad overview of the antibacterial
activity of Australian honey and shown that many honeys have
potential for therapeutic use as antibacterial agents. Jarrah and
marri honeys have exceptional levels of hydrogen peroxide-
dependent activity, and non-peroxide activity in Australian
Leptospermum honeys is comparable to that found in New Zealand
manuka honey. These findings indicate that there is an
opportunity for Australian apiarists to share in the lucrative
medicinal honey market. However, the factors affecting antibac-
terial activity in honey are complex, numerous, and not solely
dependent on the floral source. This prevents generic statements
being made regarding the activity of honey derived from a given
floral source, and indicates the need to test individual batches of
honey for their level of antibacterial activity before they are
designated as therapeutic products.
Supporting Information
Supporting Table S1 Complete list of honey samples included
in survey including floral source, geographic region and
antibacterial activity (total and non-peroxide) for each sample.
(XLS)
Supporting Table S2 Change in antibacterial activity of honey
samples following storage at 25uC and 4uC (complete data set).
(DOCX)
Acknowledgments
We thank the many beekeepers who supplied honey samples for the survey,
and Comvita, Paengaroa, New Zealand, for the supply of Wound Care
18+manuka honey.
Author Contributions
Conceived and designed the experiments: JI SB DAC. Performed the
experiments: JI. Analyzed the data: JI SB DAC. Contributed reagents/
materials/analysis tools: JI SB DAC. Wrote the paper: JI SB DAC.
References
1. Blair SE, Cokcetin NN, Harry EJ, Carter DA (2009) The unusual antibacterial
activity of medical-grade Leptospermum honey: antibacterial spectrum, resistance
and transcriptome analysis. Eur J Clin Microbiol Infect Dis 28: 1199–1208.
2. Cooper RA, Wigley P, Burton NF (2000) Susceptibility of multiresistant strains
of Burkholderia cepacia to honey. Lett Appl Microbiol 31: 20–24.
3. Cooper RA, Halas E, Molan PC (2002) The efficacy of honey in inhibiting strains of
Pseudomonas aeruginosa from infected burns. J Burn Care Rehabil 23: 366–370.
4. Cooper RA, Molan PC, Harding KG (2002) The sensitivity to honey of Gram-
positive cocci of clinical significance isolated from wounds. J Appl Microbiol 93:
857–863.
5. French VM, Cooper RA, Molan PC (2005) The antibacterial activity of
honey against coagulase-negative staphylococci. J Antimicrob Chemother 56:
228–231.
6. Bang LM, Buntting C, Molan P (2003) The effect of dilution on the rate of
hydrogen peroxide production in honey and its implications for wound healing.
J Altern Complement Med 9: 267–273.
7. White JW, Subers MH, Schepartz AI (1963) The identification of inhibine, the
antibacterial factor in honey, as hydrogen peroxide and its origin in a honey
glucose-oxidase system. Biochim Biophys Acta 73: 57–70.
8. Molan PC, Russell KM (1988) Non-peroxide antibacterial activity in some New
Zealand honeys. J Apic Res 27: 62–67.
9. Allen KL, Molan PC, Reid GM (1991) A survey of the antibacterial activity of
some New Zealand honeys. J Pharm Pharmacol 43: 817–822.
10. Brady N, Molan P, Bang L (2004) A survey of non-manuka New Zealand honeys
for antibacterial and antifungal activities. J Apic Res 43: 47–52.
11. Molan PC, Smith IM, Reid GM (1988) A comparison of the antibacterial
activity of some New Zealand honeys. J Apic Res 27: 252–256.
12. Price JN, Morgan JW (2006) Variability of plant fitness influences range
expansion of Leptospermum scoparium. Ecography 29: 623–631.
13. Ohashi K, Natori S, Kubo T (1999) Expression of amylase and glucose oxidase
in the hypopharyngeal gland with an age-dependent role change of the worker
honeybee (Apis mellifera L.). Eu J Biochem 265: 127–133.
14. Janmaat AF, Winston ML, Ydenberg RC (2000) Condition-dependent response
to changes in pollen stores by honey bee (Apis mellifera) colonies with different
parasitic loads. Behav Ecol Sociobiol 47: 171–179.
15. Mattila HR, Otis GW (2006) Effects of pollen availability and Nosema infection
during the spring on division of labor and survival of worker honey bees
(Hymenoptera: Apidae). Environ Entomol 35: 708–717.
16. Yang X, Cox-Foster DL (2005) Impact of an ectoparasite on the immunity and
pathology of an invertebrate: evidence for host immunosuppression and viral
amplification. Proc Natl Acad Science USA 102: 7470–7475.
17. Henriques A, Burton NF, Cooper RA (2005) Antibacterial activity of selected
Portuguese honeys. J Apic Res 44: 119–123.
18. Cabrera L, Cespedes E, Nava R, de Rodriguez GO (2006) Non-peroxide
antibacterial activity in Zulia honeys. Revista Cientifica-Facultad De Ciencias
Veterinarias 16: 556–563.
19. Mundo M, Padilla-Zakour O, Worobo R (2004) Growth inhibition of foodborne
pathogens and food spoilage organisms by select raw honeys. Int J Food
Microbiol 97: 1–8.
20. Molan PC, Allen KL (1996) The effect of gamma-irradiation on the antibacterial
activity of honey. J Pharm Pharmacol 48: 1206–1209.
21. Adams CJ, Boult CH, Deadman BJ, Farr JM, Grainger MNC, et al. (2008)
Isolation by HPLC and characterisation of the bioactive fraction of New Zealand
manuka (Leptospermum scoparium) honey. Carbohydrate Res 343: 651–659.
22. Mavric E, Wittmann S, Barth G, Henle T (2008) Identification and
quantification of methylglyoxal as the dominant antibacterial constituent of
Manuka (Leptospermum scoparium) honeys from New Zealand. Mol Nutr Food Res
52: 483–489.
Antibacterial Honey from Australian Plants
PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e18229
23. Adams CJ, Manley-Harris M, Molan PC (2009) The origin of methylglyoxal in
New Zealand manuka (Leptospermum scoparium) honey. Carbohydrate Res 344:
1050–1053.
24. Australian National Botanic Gardens (2008) Australian Plant Common Name
Database. Canberra: Department of the Environment, Water, Heritage and the
Arts, http://www.anbg.gov.au/common.names; accessed throughout 2006-
2008.
25. Centre for Plant Biodiversity Research (2008) Australian Plant Name Index,
IBIS database. Canberra: Centre for Plant Biodiversity Research, Australian
Government, http://www.cpbr.gov.au/cgi-bin/apni; accessed throughout
2006-2008.
26. Brooker MIH, Kleinig DA (2001) Field Guide to Eucalypts, Vol. 2 - South-
western and Southern Australia. Melbourne, Australia: Bloomings Books.
27. Brooker MIH, Kleinig DA (2004) Field Guide to Eucalypts, Vol. 3 - Northern
Australia. Melbourne, Australia: Bloomings Books.
28. Brooker MIH, Kleinig DA (2006) Field Guide to Eucalypts, Vol. 1 - South-
eastern Australia. Melbourne, Australia: Bloomings Books.
29. Clemson A (1985) Honey and pollen flora. Melbourne: Inkata Press.
30. Centre for Plant Biodiversity Research (2008) Australia’s Virtual Herbarium
(map output). Council of Heads of Australian Herbaria: http://www.cpbr.gov.
au/cgi-bin/avh.cgi; accessed throughout 2008.
31. Allen KL, Molan PC, Reid GM (1991) The variability of the antibacterial
activity of honey. Apiacta 26: 114–121.
32. Cooper RA, Jenkins L (2009) A comparison between medical grade honey and
table honeys in relation to antimicrobial efficacy. Feb 12, 2009: http://www.
woundsresearchcom/content/a-comparison-be-tween-medicalgrade-honey-
and-table-honeys-relation-antimicrobial-efficacy; accessed 16 Feb 2011.
33. Stephens JM, Schlothauer RC, Morris BD, Yang D, Fearnley L, et al. (2010)
Phenolic compounds and methylglyoxal in some New Zealand manuka and
kanuka honeys. Food Chem 120: 78–86.
34. Allen KL, Molan PC (1997) The sensitivity of mastitis-causing bacteria to the
antibacterial activity of honey. N Z J Agric Res 40: 537–540.
35. Cooper RA, Molan PC (1999) The use of honey as an antiseptic in managing
Pseudomonas infection. J Wound Care 8: 161–164.
36. Cooper RA, Molan PC, Harding KG (1999) Antibacterial activity of honey
against strains of Staphylococcus aureus from infected wounds. J R Soc Med 92:
283–285.
37. Brady NF, Molan PC, Harfoot CG (1996) The sensitivity of dermatophytes to
the antimicrobial activity of manuka honey and other honey. Pharm Sci 2:
471–473.
38. Irish J, Carter D, Shokohi T, Blair S (2006) Honey has an antifungal effect
against Candida species. Med Mycol 44: 289–291.
39. Kwakman PHS, te Velde AA, de Boer L, Speijer D, Vandenbroucke-
Grauls CMJE, et al. (2010) How honey kills bacteria. FASEB 24: 2576–2582.
40. Dag A, Fetscher AE, Afik O, Yeselson Y, Schaffer A, et al. (2003) Honey bee
(Apis mellifera) strains differ in avocado (Persea americana) nectar foraging
preference. Apidologie 34: 299–309.
41. Somerville DC (2008) A study of New Zealand beekeeping: lessons for Australia.
Rural Industries Research and Development Corporation Publication No 08/
060.
42. Castro-Vazquez L, Diaz-Maroto MC, Gonzalez-Vinas MA, de la Fuente E,
Perez-Coello MS (2008) Influence of storage conditions on chemical composition
and sensory properties of citrus honey. J Agric Food Chem 56: 1999–2006.
43. Gallardo-Chacon JJ, Casellies M, Izquierdo-Pulido M, Rius N (2008) Inhibitory
activity of monofloral and multifloral honeys against bacterial pathogens.
J Apicul Res 47: 131–136.
44. Molan PC (1992) The antibacterial activity of honey. 1. The nature of the
antibacterial activity. Bee World 73: 5–28.
45. White JW, Subers MH (1964) Studies on honey Inhibine. 4. Destruction of the
peroxide accumulation system by light. J Food Sci 29: 819–828.
46. Flores MJ, Ehrlich SD, Michel B (2002) Primosome assembly requirement for
replication restart in the Escherichia coli holDG10 replication mutant. Molecular
Microbiology 44: 783–792.
Antibacterial Honey from Australian Plants
PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e18229
