Survey of the

antimicrobial activity
of Australian honey
Julie Irish
Dee Carter
Shona Blair
School of Molecular and Microbial Biosciences
University of Sydney

Antimicrobial activity of honey

Honey has been well studied for its antimicrobial

and wound healing properties
Low water activity

Low pH

~80% sugars
Gluconic acid
pH 3.2 4.5

Hydrogen peroxide

Glucose oxidase

Antimicrobial activity of honey

Floral factors

Non-peroxide activity (Leptospermum spp.)

Also known as Unique Manuka Factor (UMF)
Advantage in clinical situations

Leptospermum honey
+ catalase

Leptospermum honey

Brush box honey

+ catalase

Brush box honey

Staphylococcus aureus

Non-peroxide activity is caused by

methylglyoxal (MGO)

High concentrations of MGO in manuka

honey that correlate strongly with nonperoxide activity
MGO is naturally produced in all cells as a byproduct of metabolism
Highly toxic

Modifies DNA, RNA, and proteins

Targets them for degradation by the cells own
machinery
Some bacteria and fungi have well-defined
detoxification systems

Non-peroxide activity is caused by

methylglyoxal (MGO)

How does this fit with the current theory about

medicinal honey?

MGO produced by stressed plants

May explain why some organisms are more sensitive
to honey than others
Combination of MGO and honey makes it non-toxic to
human cells?

Need further studies on source, mode of action,

standardisation in medicinal honeys

Survey of Australian honeys for

antibacterial activity

Important to find the most

highly active honeys for
therapeutic use
345 New Zealand honeys
tested (Allen et al. (1991))
No published data for
Australian honey
Overview of activity of
Australian honey

Testing the antibacterial activity

of honey

Compares the antibacterial activity of a given honey to

that of phenol
Staphylococcus aureus is mixed into agar plate
Holes cut into agar
Various solutions added to wells
25% honey in water
(tests total activity)
OR
25% honey in catalase solution
(tests non-peroxide activity)
OR
Solutions of 2 to 7% phenol
(standard curve)

Testing the antibacterial activity

of honey

Solutions diffuse out of the wells during

incubation
If they prevent the growth of S. aureus
zones of inhibition
Zones are measured,
mean diameter squared
Standard curve generated
from phenol solutions

Mean diameter squared (mm)

Testing the antibacterial activity

of honey
900
800
700
600
500
400
300
200
100
0

y = 137.86x - 108.1
R2 = 0.9889

Phenol concenration (% w/v)

Mean diameter squared (mm)

Testing the antibacterial activity

of honey
900
800
700
600
500
400
300
200
100
0

y = 137.86x - 108.1
R2 = 0.9889

Phenol concenration (% w/v)

Honey compared to phenol (x 4.69 for dilution and density)

400 mm2 zone of inhibition = 17.3% phenol equivalent
The higher the number, the more active the honey
Allows comparison between different honeys

Results

503 honey samples tested

477 Apis mellifera

26 Trigona spp.

Activity (phenol equivalent) varies greatly

<5
5-10
>10
>20

Insignificant therapeutic value

Low activity
Therapeutically beneficial
Highly active

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

Total activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

93

135

40

275 (57.7%)

175 (64%)

VIC

14

21 (4.4%)

6 (29%)

TAS

23

20

46 (9.6%)

21 (46%)

QLD

12

28 (5.9%)

16 (57%)

SA

10

15 (3.2%)

5 (33%)

WA

39

20

31

92 (19.2%)

51 (55%)

Sum

191
(40%)

12
(2.5%)

477
(100%)

274
(57.5%)

189
85
(39.6%) (17.9%)

60% of all samples had antibacterial activity

57.5% of all samples had therapeutically beneficial antibacterial activity

Number of samples

Distribution of total activity

200
180
160
140
120
100
80
60
40
20
0
<5

5-10

10-15

15-20

20-25

25-30

30-35

Total antibacterial activity (% (w/v) phenol equivalent)

Of the honeys that have antibacterial activity, most

are in the therapeutically beneficial range

Average total activity (n=286): 17.8 5 (7.4 34.3)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

Non-peroxide activity
<5

5 10

10 20

>20

Sum

Sum >10

(% of country)

(% of state)

NSW

209

50

10

275 (57.7%)

60 (22%)

VIC

21

21 (4.4%)

0 (0%)

TAS

43

46 (9.6%)

3 (6.5%)

QLD

22

28 (5.9%)

5 (18%)

SA

14

15 (3.2%)

0 (0%)

WA

88

92 (19.2%)

0 (0%)

54
(11.4%)

14
(3.0%)

477
(100%)

68
(14.3%)

Sum

397
12
(83%) (2.6%)

17% of all samples had non-peroxide activity

14.3% of all samples had therapeutically beneficial non-peroxide activity

Distribution of non-peroxide activity

30

350

Number of samples

Number of samples

400
300
250
200
150
100
50
0

25
20
15
10
5
0

<5

5-10

10-15

15-20

20-25

25-30

Antibacterial activity (% (w/v) phenol equivalent)

5-10

10-15

15-20

20-25

Antibacterial activity (% (w/v) phenol equivalent)

Of the honeys that have non-peroxide activity,

most are in the therapeutically beneficial range

25-30

High non-peroxide activity is rare

Non-peroxide activity

Of the 80 honeys with non-peroxide activity,

62 (77.5%) were from Leptospermum, or
contained Leptospermum

Mean non-peroxide activity 17.2 4.1 (9.8 25.9)

Some species/areas more reliable than others

L. polygalifolium (Jelly bush): 29 samples, 28 had nonperoxide activity

L. scoparium (Manuka): 11 samples, none had nonperoxide activity, some inactive
Most active Leptospermum honeys from NSW-QLD border

Non-peroxide activity

18 non-Leptospermum samples had

non-peroxide activity
Mean non-peroxide activity 10.1 1.7 (8.1
15.9)

Spotted gum (3/6 samples)

South west Tasmanian wildflowers (3/5 samples)
Stonefruit orchard (1/2 samples)
Clover (1/5 samples)
Melaleuca (2/28 samples)
Moort (1/1 samples)
Forest red gum (1/2 samples)
Messmate (1/6 samples)
Unspecified mixed flora (5/64 samples)

Floral sources

477 honey samples from 142 different floral

sources, including mixed flora
78% of honeys were from native
Australian flora, another 17%
were likely to contain natives
38% of honeys contained
eucalypts
24% of honeys contained
Leptospermum
(likely to be biased)

Eastern Australia
Inactive

Hydrogen
peroxide
activity

Non-peroxide
activity

Eastern Australia

Highest total activity: 34.3 (Apple box/Red stringybark)

Highest non-peroxide activity: 25.9 (Jelly bush)

High non-peroxide activity (>20% phenol equivalent) in

Leptospermum samples from QLD and northern NSW

12 non-Leptospermum honeys
had non-peroxide activity

Western Australia
Inactive

Hydrogen
peroxide
activity

Non-peroxide
activity

Western Australia

Highest total activity: 31.9 (Stonefruit)

Highest non-peroxide activity: 9.7 (Melaleuca)
31 honeys with total activity >20
(many from flora endemic to WA)
Jarrah: 19 samples

Karri: 3 samples

Activity range 17.7 to 31.4

1 sample was inactive
Activity range 18.5 to 29.6

Marri: 9 samples

Activity range 18 to 29.7

1 sample was inactive

Important message

It is impossible to predict the activity of a

given honey, must test every batch

32 samples, same beekeeper, same floral source,

same time, different hives

31 samples had total activity between 11 and 19

1 sample was inactive

30% of Leptospermum honeys were inactive,

15% had hydrogen peroxide activity only
Activity relates to floral source to an extent, but there
are clearly many other factors involved

Environmental conditions, soil type, soil and plant

microbiology, bee health

Stingless bee honey

26 samples of Trigona spp.

honey from QLD

Mixed flora, various times of year

23 samples had total activity >20

(12.4 32.1)

24 samples had non-peroxide

activity >10 (11.5 23.7)

Conclusions

Australian honeys show a wide range of

antibacterial activities
Many have potential for therapeutic use
Non-peroxide activity exists in honeys from
various floral sources
Leptospermum spp. remain the most
reliable source of high non-peroxide activity
Statistical analysis is ongoing
Identified areas to focus on in
future studies
Role of MGO?

Acknowledgments

The 124 beekeepers who supplied honey

samples for the survey
Rob Manning, Dept of Agriculture, WA
Tim Heard, CSIRO Entomology, QLD
Comvita, New Zealand
RIRDC