A Comparative Study of the Antimicrobial Activity of Wild (Tualang)

Honey and Artificial Honey Against Methecillin-resistant

Staphylococcus Aureus, Streptococcus Pneumoniae and Klebsiella

Pneumoniae

May Florence Dela Cruz Bacayo

, Shazwan Fahmi Shafi

, Wong Charng Choon

, Santosh Fattepur

Kiran Chanabasappa Nilugal

, Jiyauddin Khan

, Fadli Asmani

, Eddy Yusuf

School of Pharmacy, Management and Science University, 40100 Shah Alam Malaysia

Keywords : Tualang honey, Artificial honey; Antimicrobial activity.

Abstract : Antimicrobial agents are becoming less effective due to the emergence of antibiotic-resistant microbes, thus

the discovery of alternative antimicrobial agents are urgently needed. Honey is believed to have antimicrobial

activity and are used in traditional medicine as skin healing dressing for wound. To find an alternative

substance as a substitute for the current antimicrobial agent for the three strains of microbes and to make a

comparison between the antimicrobial activity of the wild (Tualang) honey and the artificial honey. The 50 %

(w/v) of honey were diluted using double dilution method and are tested for its antimicrobial activity using

disc diffusion method with Ampicillin as a standard antibiotic for Streptococcus pneumoniae, Gentamycin for

Klebsiella pneumonia and Vancomycin for Methicillin-resistant Staphylococcus aurues as positive control.

The MIC and MBC were determined. Based on the results obtained from the disc diffussion assay, it shows

that wild (Tualang) honey has antimicrobial activity at a concentration of 10 % (w/v) for MRSA and S.

pneumoniae while at 25 % (w/v) for K. pneumoniae. Meanwhile it need 100% (w/v) concentration of artificial

honey to shows its antimicrobial activity. Wild (Tualang) honey was recorded as the most potent honey

against S. pneumoniae, in which a dilution of 10% (w/v) was required to inhibit the growth and kill S.

pneumoniae colony at 20% (w/v). The concentration of 20% (w/v) was required to inhibit MRSA and 25%

(w/v) to kill MRSA. Meanwhile, the highest concentration were required to inhibit K.pneumonia at 50% (w/v)

and no bactericidal effect were recorded. The comparative study between Tualang honey and Artificial honey

gives a promising result that the Tualang honey has a highest antimicrobial activity against MRSA, Klebsiella

pneumoniae, Sterptococcus pneumoniae compared to the Artificial honey.

1 INTRODUCTION

Honey is a natural syrup which have a complex

function of physicochemical properties (color, flavor

and texture), mainly determined by their botanic and

geographic origins. Honey is a concentrated aqueous

solution of glucose (31%) and fructose (39%). It

contains free amino acids at a level of 1%, pollen

being one of their sources. Proline, which might

originate from bees, is the prevalent amino acid and

makes up 50–85% of the amino acid fraction (White,

1975). Organic acids are present in honey at low

concentrations (<0.5%) and it is related to the color,

flavor and physico-chemical properties of the honey,

such as pH, acidity, and electrical conductivity.

Organic acids chelate metals can synergistically

enhance the antioxidant action of phenolic

compounds (Gheldof, Wang & Engeseth, 2002).

Moreover, acetic acid and ethanol can be used as

fermentation indicators and formic acid as an

indicator for the treatment of Varroa infestation

(Calderone, 2000).

It was used as a wound treatment that are

non-responsive to conventional therapies, such as

diabetic ulcers, and wounds infected with

antibiotic-resistant bacteria. There were countless

studies that recount the antimicrobial activity of

honey against microbes. (Dunford C, Cooper R,

Molan P, et al, 200)

With the emergence of antibiotic-resistant

microbial strains, such as Methicillin-resistant S.

aureus that can cause difficult-to-treat wound

Bacayo, M., Shaﬁ, S., Choon, W., Fattepur, S., Nilugal, K., Khan, J., Asmani, F. and Yusuf, E.

A Comparative Study of the Antimicrobial Activity of Wild (Tualang) Honey and Artiﬁcial Honey Against Methecillin- resistant Staphylococcus Aureus, Streptococcus Pneumoniae and Klebsiella

Pneumoniae.

DOI: 10.5220/0009842900002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

infections, honey has again caught the attention of

medical researchers. Scientists had first reported the

ability of honey to treat infections caused by

microbes in the late 1800s, but with the advent of

antibiotics in the early 1900s, the scientific interest in

honey decreased. (Fry DE, Barie PS, 2001)

The aim and objective of this study is to

determine the antimicrobial activity of honey against

MRSA, Klebsiella pneumoniae, Streptococcus

pneumoniae which can be an alternative to the

existing antimicrobial agent. It is so that the case of

antimicrobial resistance will reduce significantly.

This study is also to compare antimicrobial activity

of the wild (Tualang) honey and artificial honey.

Thus comparing also the antimicrobial activity of

wild (Tualang) honey, artificial honey and the

standard antibiotics.

2 METHODOLOGY

2.1 Preparation of Media

38g of MHA is dissolved into 1 liter of saline water

and the solution is stirred homogenously. The agar

solution is then autoclaved for 2 hours and poured

into the petridish until the height is not more than

5mm keeping the petridish dry. The pH of the

Mueller-Hintom medium should be maintained at 7.2

to 7.4 at room temperature and keep in the incubator

set to 37°C prior to use . (Mm & Fatema, 2009)

2.2 Minimum Inhibitory

Concentration (MIC)

The 50% w/v was prepared by diluting 10 g of honey

in 20 ml of Mueller Hinton broth and the two-fold

dilution of the honey solution in Mueller Hinton

broth were prepared. Ten sterile tubes were labeled

each and placed in a test tube rack. Tube 1 with 1 ml

of honey solution was labeled as the honey control

and tube 8 was labeled as growth control. 1 ml of

Mueller Hinton broth was added to test tube 1 to 4.

Using a sterile micropipette, take 1 ml from tube 1

and transfer to tube 2 and thoroughly mixed. 1 ml

again was transferred from tube 2 to tube 3. This

process was repeated until tube 4 except for growth

control tube. Using fresh micropipette for each

dilution. The last tube received no honey solution and

was served as growth control then add 20% (4g in 20

ml), 15% (7.5g in 50 ml), 10% (1g in 10 ml), and 5%

(2.5 in 50 ml) in test tube 5 to 8 respectively. Each

tube was inoculated including the growth control

except honey control with 1 ml of the culture of

respective organism. The tubes were incubated at

37°c for 24 hours. The tubes were examined for

growth and were determined the MIC of the tested

antibiotics, which is bacteriostatic for the test

organism. The tubes were examined for visible

growth (cloudy) and was recorded growth as (+) and

no growth as (-).

2.3 Minimum Bactrialcidal

Concentration (MBC)

The MBC test was used to determined the lowest

concentration needed to kill the bacteria. MBC was

perform after MIC test. Each honey dilution with no

bacteria growth from the mic test was determined.

For each honey solution that has no growth was

incubated onto Mueller Hinton agar plate. It was

spread evenly and incubated at 37°c for 24 hours.

MBC were determined by minimum concentration

that allowed less than 1% of bacterial growth.(

Fakruddin,M., 2013)

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Figure 3: (a) Minimum inhibitory concentration (MIC) and (b) Minimum Bactericidal Concentration (MBC) (Yilmaz, 2012)

2.4 Disc Diffusion Assay for

Determination of Zone of

Inhibition

The antimicrobial activity of the diluted honey

concentration is tested by disc diffusion susceptibility

method . The Mueller-Hinton Agar (MHA) plate is

used as the agar plate for the bacterial culture because

it gives a good result in batch to batch reproducibility,

it is low in of sulfonamide, trimethoprim and

tetracycline inhibitors. (Zainol, Mohd Yusoff, &

Mohd Yusof, 2013) Tualang and artificial honey

were prepared by double dilution method from 100%

concentration until 6. 25% of the concentration is

diluted with distilled water. A sterile swab is used to

spread the suspension of the pure culture on one side

face of the agar plate. The honey solution and

standard antibiotic is applied to the opposite side face

of the agar plate and distilled water was employed as

negative control. The agar plate was incubated for 24

hours at a temperature of 37°C. The size of the zone

of inhibition were measured using a metric ruler. The

larger zone of inhibition represents the antimicrobial

activity.

3 DATA ANALYSIS

The data obtained from different formulations will be

analyzed by Mean, Standard deviation and one-way

analysis of variance (ANOVA) procedure using

the Statistical Package for the Social Science (SPSS)

program (IBM SPSS Statistics 22.0).

When there is a statistically significant difference,

a post-hoc Tukey test will be then conducted to detect

the differences among the pairs. A statistically

significant difference is considered at p < 0.05.

4 RESULTS

Several evaluation tests were conducted in order to

determined the effectiveness of Tualang honey and

artificial honey against sample bacteria which is disc

diffussion test, Minimum Inhibition Test and

Minimum Bactericidal Test.

From the result obtain, it shows that Tualang

Honey has antimicrobial activity against MRSA and

S. pneumoniae at lowest concentration of 10 %(w/v),

while against K. pneumoniae at concentration 25

%(w/v). It means that Tualang honey has quite potent

antimicrobial activity against MRSA and S.

pneumoniae as it only need 10 %(w/v) concentration.

Meanwhile, K. pneumoniae has higher resistance

compare to MRSA and S. pneumoniae as it need 25

%(w/v) of Tualang honey to shows its antimicrobial

activity.

A Comparative Study of the Antimicrobial Activity of Wild (Tualang) Honey and Artiﬁcial Honey Against Methecillin- resistant

Staphylococcus Aureus, Streptococcus Pneumoniae and Klebsiella Pneumoniae

Figure 2: Zone of Inhibition for MRSA against Tualang

Honey, Artificial Honey and Positive Control.

Figure 3: Zone of Inhibition for Streptococcus pneumoniae

against Tualang Honey, Artificial Honey and Positive

Control.

Figure 4: Zone of Inhibition for Klebsiella pneumoniae

against Tualang Honey, Artificial Honey and Positive

Control.

Table 1: MIC and MBC values of Tualang honey and

artificial honey.

Honey

Sample

MIC & MBC %(w/v)

Methicillin

Resistence

Streptococcus

Aerus (MRSA)

Streptococcus

pneumoniae

Klebsiella

pneumoniae

MIC

MBC

MIC

MBC

MIC

MBC

Tualan

Honey

>50

Artifici

Honey

>50

5 DISCUSSION

This study was about investigating the antimicrobial

activity of Tualang and artificial honey against

MRSA, S.pneumoniae and K. pneumoniae as there are

limited studies available comparing different type of

honey. Based on the results obtained from the disc

diffussion test, it shows that Tualang honey has

antimicrobial activity at concentration of 10 % (w/v)

for MRSA and S. pneumoniae while at 25% (w/v) for

K. pneumoniae. Meanwhile it need 100% (w/v)

concentration of artificial honey to shows its

antimicrobial activity. The results shows that Tualang

honey has high antimicrobial activity against the

selected bacteria as its only need as low as 10%(w/v)

to 25%(w/v) concentration to inhibit the growth of

MRSA, S.pneumoniae and K pneumoniae. These

result were due to the two important enzymes known

to contribute to the major biological activities of

honey which are bee-origin glucose oxidase and

floral-origin catalase. These enzymes will determine

the antimicrobial activity of Tualang honey. (Zainol,

Mohd Yusoff, & Mohd Yusof, 2013).

For MIC and MBC values, Tualang honey was

recorded as the most potent honey against S.

pneumoniae, in which a dilution of 10% (w/v) was

require to inhibit and kill them at 20% (w/v). The

concentration of 20% (w/v) are require to inhibit

MRSA and 25% (w/v) to kill them. Meanwhile, the

highest concentration are require to inhibit K.

pneumoniae at 50% (w/v) and no bactericidal effect

were recorded. Overall, the bactericidal activities of

Tualang honey were recorded to be one reading

higher than their inhibitory effect. Results of artificial

honey showed that only S. pneumoniae show were

inhibited at concentration 50% (w/v) and no

bactericidal value. It also shows no MIC and MBC

value against other bacteria.The statistical analysis

showed that the antimicrobial activity of the Tualang

honey and artificial honey were significant when

compared with positive control with the P value of

<0.05 against MRSA, S.pneumoniae and

K.pneumoniae.

11,9

9,73

6,33

1,67

1,3

0 0 0 0 0

Tualang Honey Artificial Honey

10,7

11,3

5,7

3,3

0 0

6,3

2,3

0 0 0 0

Tualang Honey Artificial Honey

1…

7,7

4,7

0 0 0

3,7

0 0 0 0 0

Tualang Honey Artificial Honey

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

6 CONCLUSION

The in-vitro comparative study of antimicrobial

activity between Tualang honey and artificial honey

against the selected gram-negative bacteria concludes

that the Tualang honey has highest antimicrobial

activity against MRSA, Streptococcus pneumoniae

and Klebsiella pneumoniae compared to artificial

honey.

ACKNOWLEDGEMENT

The author is grateful to the research committee and

lecturers of Management and Science University,

Malaysia for providing all the needed support and

guidance to complete this project.

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A Comparative Study of the Antimicrobial Activity of Wild (Tualang) Honey and Artiﬁcial Honey Against Methecillin- resistant

Staphylococcus Aureus, Streptococcus Pneumoniae and Klebsiella Pneumoniae