Antimicrobial Activity

of Stingless Bee
Honey on
Staphylococcus
aureus
By:
1. Siti Nursyuhada binti Zulkefle
2. Nuryasmin binti Mohd Razak Supervised by:
Yanti binti Yaacob
3. Nurul Amirah binti Mahyuddin
4. Muhammad Hifni Nasif bin Muhammad Taufik
Introduction
 Background of Study

Anti-
inflammatory Anti-bacterial
Antioxidant

Stingless Bee Antidiabetic

Antiviral Honey Biological

Properties (Erejuwa et al., Suction of honey
2010, Kishore et al., 2011 and Viuda-
Martos et al., 2008).

Antihyperlipidemic

Anti-ulcer Anticancer
activities
 Identified in 1880 in
Aberdeen, Scotland,
by surgeon Sir
Alexander Ogston in
Continue… pus from a surgical
abscess in a knee joint

Microscopic:
Cocci in grape-
like clusters
Staphylococcus Found in:
• Nose

Morphology
aureus • Respiratory tract
• The skin
• gram +ve
coccus
• anaerobic

Skin infection :
• Hair follicle infections
macroscopic : including abscess and
Round, usually sycosis (beard infection)
golden-yellow • Ecthyma (crusted ulcers)
colonies, • Cellulitis
 Significance of
Study

To prove that stingless

To cure several skin bee honey has To produce an organic
disease caused by antimicrobial antimicrobial product by
Staphylococcus aureus properties against using stingless bee honey
Staphylococcus aureus
Hypothesis

 Stingless bee honey has properties that can inhibit the growth of
Staphylococcus aureus
Objectives

To determine the effectiveness of stingless

bee honey as an antimicrobe

To compare the effectiveness of honey

from stingless bee at different
concentration on Staphylococcus aureus.

To determine the antimicrobial activity

towards Staphylococcus aureus.
Literature Review

Stingless bee honey has a broad-
spectrum antibacterial activity
(Boorn K.L, et al.2010)
Stingless Bee
Honey as an
Antimicrobe
Two recent publications have
described the antimicrobial
activity of honey derived from the
Australian native stingless bee
Trigona carbonaria. (Irish et al. 2008;
Kimoto-Nira and Amano 2008).
Stingless bee honey have similar
activities to medicinal honey ,
therefore they have roles as a
medicinal agent. (Cortopassi-Laurino et al.
2006)
Stingless bee honey was used in
traditional medicine in Central
and South America, and Africa.
(Cortopassi-Laurino et al. 2006)

Composition of
Honey
Non-peroxide
components such as
antioxidants may
contribute to
controlling the growth
of some foodborne
pathogens (Taormina, et al,
2001)
Flavonoids is derived from
propolis, a resinous material
collected by bees from gum
exudates of trees, and used as
an antibacterial agent in hives
(Marcucci, 1995)
Two important classes
of these inhibines are
the flavonoids and the
phenolic acids
(Wahdan, H. A. L., 1998)
Antimicrobial activity
of honey has been Phenolic components in
attributed to hydrogen nectar has antioxidant activity
(Gil et al., 1995 and Ferreres et al., 1996 )
peroxide, which is
produced by naturally
occurring glucose
oxidase (Taormina ,et al, 2001)
The presence of Flavonoid and Phenolic Acid
(using FTIR)
 Ecthyma-gangrenosum-like lesions Skin and soft tissue infections are usually caused
associated with methicillin-resistant by Staphylococcus aureus (S. aureus) and
Staphylococcus aureus infection(Şen, Hüseyin, Streptococcus pyogenes(Sharma, S., and K. K. Verma. 2001)
et al., 2009)

Staphylococcus
Impetigo aureus Causes Skin
Diseases

Resident gram- positive bacteria include

Epidermal infections caused by S. aureus Staphylococcus, Micrococcus, and Corynebacterium
and S. pyogenes include impetigo and sp. Staphylococcus aureus and Strepto coccus in
ecthyma(Chiller et al,2001) order to be pathogenic, they must be able to adhere
to, grow on, and invade the host(Chiller et al,2001)
Material and Methodology
 All apparatus and chemicals
(nutrient agar, broth and
distilled water) was sterilized
by using an autoclave for
15-20 minutes at 121ºC and
disinfected by using 70%
ethanol before use.

Sterilization

The inoculating loop and the

mouth of culture tube was
sterilized by dry heat every
time it was used.

Preparation of Preparation of
Broth Add 25g broth
powder to 1 liter of
Nutrient Agar
distilled water and Add 28g agar powder in
autoclave 1 liter of distilled water into
glass bottle. Autoclave
and pour into the plate .
 Dilution of Honey
Staphylococcus The honey has been diluted 4 times
aureus Isolation with dilution factors 10-1, 10-2, 10-3
and 10-4 with sterile distilled water

• The original inoculum containing a

mixture of bacteria was streaked into 4
Step 1 quadrants on solid media (agar).

• The individual colony that was identified by

macroscopic appearance s. aureus then was
separated to another agar plate by streaking method
Step 2 and broth.

• The staphylococcus aureus was then tested

under a microscope to confirm its microscopic
Step 3 characteristic.

• Staphylococcus aureus was then sub

cultured to another agar plate and broth to be
Step 4 tested with honey.
Agar plate Broth
(Magnification at x100)
 Bacteria Test
100 µl of each dilution was transferred and absorbed into disc.
Disc
Diffusion Positive control = steroid cream was swapped on the surface of the disc.

Negative control =100 µl of sterilized distilled water onto the disc.

200 µl of bacteria was transferred to the agar plate by spreading before placing the
disc on it.

After overnight incubation, the plates were examined. The diameter (d) of the zone
of inhibition for each disk were measured every day for 5 days and recoded.
Result and Discussion
 Day 1
DAY 1
14

Present sample Zone of inhibition (mm) 12

Day 1

diameters of inhibition zone (mm)

Negative control 6.63
10
(sterilized distilled
water)
Positive control 7.75 8
(eumovate)
1st dilution of 10.50
6
honey ()
nd
2 dilution of 8.00
honey () 4
rd
3 dilution of 7.00
honey () 2
4th dilution of 6.70
honey ()
0
Negative Positive 1st dilution 2nd dilution 3rd dilution 4th dilution
control control
Samples
Day 1
 Day 2

Present sample Zone of inhibition (mm)

DAY 2
14

diameters of inhibition zone (mm)

Day 2 12
Negative control 6.75
(sterilized distilled water) 10

Positive control 7.85 8

(eumovate)
st
1 dilution of honey () 11.50 6

2nd dilution of honey () 10.85 4

2
3rd dilution of honey () 8.00
0
4th dilution of honey () 7.00 Negative Positive 1st dilution 2nd dilution 3rd dilution 4th dilution
control control
Samples
Day 2
 Day 3

DAY 3
Present sample Zone of inhibition (mm) 14

diameters of inhibition zone (mm)

12
Day 3

Negative control 6.75 10

(sterilized distilled water)
8
Positive control 7.85
(eumovate)
6
1st dilution of honey () 12.00
4
2nd dilution of honey () 11.50
3rd dilution of honey () 8.50 2

0
Negative Positive 1st dilution 2nd dilution 3rd dilution 4th dilution
4th dilution of honey () 7.50 control control
Samples
Day 3
 Day 4

Present sample Zone of inhibition (mm) DAY 4

14

diameters of inhibition zone (mm)

12
Day 4
10
Negative control (sterilized 6.63
8
distilled water)
Positive control 7.25 6
(eumovate)
st
1 dilution of honey () 11.50 4

2
2nd dilution of honey () 9.50
0
3rd dilution of honey () 8.00 Negative Positive 1st dilution 2nd dilution 3rd dilution 4th dilution
control control
4th dilution of honey () 6.00 samples
Day 4
 Day 5

Present sample Zone of inhibition (mm) DAY 5

12

diameters of inhibition zone (mm)

10
Day 5

Negative control 0.00 8

(sterilized distilled water)
Positive control 6.25 6
(eumovate)
st
1 dilution of honey () 10.00 4

2nd dilution of honey () 9.50 2

3rd dilution of honey () 8.00

0
Negative Positive 1st dilution 2nd dilution 3rd dilution 4th dilution
4th dilution of honey () 0.00 control control
Samples
Day 5
 Summary
Graph summary
Present sample Zone of inhibition (mm) 14
Day 1 Day 2 Day 3 Day 4 Day 5
Negative control 6.63 6.75 6.75 6.63 0.00 12
(sterilized distilled

diameters of inhibition zone (mm)

water)
10
Positive control 7.75 7.85 7.85 7.25 6.25
(eumovate)
8
1st dilution of honey () 10.50 11.50 12.00 11.50 10.00

2nd dilution of honey () 8.00 10.85 11.50 9.50 9.50 6

3rd dilution of honey () 7.00 8.00 8.50 8.00 8.00 4

Negative control
Positive control
4th dilution of honey () 6.70 7.00 7.50 6.00 0.00 2
1st dilution
2nd dilution
0
3rd dilution
Day 1 Day 2 Day 3 Day 4 Day 5
4th dilution Samples
Conclusion
Stingless bee honey has antimicrobial activities
towards Staphylococcus aureus.

The1st dilution of honey gave the largest inhibition

zone compared to other dilutions, since it contained
higher concentration of honey.

Higher concentration of honey indicates the higher

composition of hydrogen peroxide, flavonoid and
phenolic acid that acts as inhibines (antimicrobial
activity).
Future Recommendation

Using different types (in colour)of stingless

Using high performance liquid
bee honey to determine which type of
chromatography (HPLC) equipped with a UV
honey contains the higher content of
detector method to get more confirmation
flavonoid and hydrogen peroxide which
on the identification of the phenolic acids
gives the more effectiveness in antimicrobial
and flavonoids
activity.
References

 Boorn, K. L., et al. "Antimicrobial activity of honey from the stingless bee Trigona carbonaria determined by
agar diffusion, agar dilution, broth microdilution and time‐kill methodology." Journal of applied
microbiology 108.5 (2010): 1534-1543.
 Wahdan, H. A. L. "Causes of the antimicrobial activity of honey." Infection 26.1 (1998): 26-31.
 Viuda‐Martos, M., Ruiz‐Navajas, Y., Fernández‐López, J., & Pérez‐Álvarez, J. A. (2008). Functional properties of
honey, propolis, and royal jelly. Journal of food science, 73(9).
 Erejuwa, O. O., Sulaiman, S. A., Wahab, M. S. A., Sirajudeen, K. N. S., Salleh, M. S. M., & Gurtu, S. (2010).
Antioxidant protective effect of glibenclamide and metformin in combination with honey in pancreas of
streptozotocin-induced diabetic rats. International journal of molecular sciences, 11(5), 2056-2066.
 Kishore, R. K., Halim, A. S., Syazana, M. N., & Sirajudeen, K. N. S. (2011). Tualang honey has higher phenolic
content and greater radical scavenging activity compared with other honey sources. Nutrition
Research, 31(4), 322-325.
 Cortopassi-Laurino, M., Imperatriz-Fonseca, V. L., Roubik, D. W., Dollin, A., Heard, T., Aguilar, I., ... & Nogueira-
Neto, P. (2006). Global meliponiculture: challenges and opportunities. Apidologie, 37(2), 275.
References

 Cortopassi-Laurino, M., Imperatriz-Fonseca, V. L., Roubik, D. W., Dollin, A., Heard, T., Aguilar, I., ...
& Nogueira-Neto, P. (2006). Global meliponiculture: challenges and
opportunities. Apidologie, 37(2), 275.
 Irish, J., Carter, D. A., Blair, S. E., & Heard, T. A. (2008). Antibacterial activity of honey from the
Australian stingless bee Trigona carbonaria. International journal of antimicrobial agents, 32(1),
89-90.
 Kimoto-Nira, H., & Amano, K. (2008). Antimicrobial activity of honey produced by stingless
honey bees. Journal of apicultural research, 47(4), 325-327.
 Taormina, P. J., Niemira, B. A., & Beuchat, L. R. (2001). Inhibitory activity of honey against
foodborne pathogens as influenced by the presence of hydrogen peroxide and level of
antioxidant power. International journal of food microbiology, 69(3), 217-225.
 Marcucci, M. C. (1995). Propolis: chemical composition, biological properties and therapeutic
activity. Apidologie, 26(2), 83-99.
Q&A

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