Antibacterial Activity of Several Indonesian Endemic Plants Against

Staphylococcus Epidermidis, Staphylococcus Aureus and Methicillin-

Resistant Staphylococcus aureus

Ariyani Kiranasari*

, Angela Bonita

, Elizabeth Melina

, Kevin Winston

, Naivedh Baht

, Nathania

Sutandi

, Beti Ernawati Dewi

, Ika Ningsih

, Fithriyah Sjatha

Department of Microbiology, Fculty of Medcicine, Universitas Indonesia,Jl. Pegangsaan Timur no. 16 Jakarta 10320,

Undergraduate Program, Faculty of Medicine, Universitas Indonesia,,

Undergraduate Program, International

Class, Faculty of Medicine, Universitas Indonesia

Keywords: Antibacteria,Plant Extract, MRSA, Staphylococcus Epidermidis.

Abstract: In this current era, infectious diseases worldwide is increasing and due to misuse of antibiotics for treating

infections eventually leads to the emergence of antibiotic resistant microbes. As a country with abundance

of natural resources, Indonesia must be the forefront on research in finding new antibacterial candidates

resourced from endemic medicinal plants. The objective of this research is to assesed the activities of

several Indonesian endemic plants extract to inhibit several bacteria in-vitro by microdilutin method to

obtain Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) and agar

diffusion method to obtain inhibition zone. In this study, extract of Syzigium aromaticum, Piper betle and

Aleurites moluccana were show anti bacterial activity against MRSA (Methicillin-resistant Staphylococcus

aureus), Staphylococcus aureus and Staphylococcus epidermidis. On the other hand, extract of Curucuma

longa and Samanea saman didnt show any anti bacterial properties. This study show the potency of several

endemic plants extract to inhibit Staphylococcal bacteria.

1 INTRODUCTION

Antimicrobial agents in today medicine play a very

important role in treating infectious disease that was

once fatal and incurable (Katzung B et al, 2012) In

this modern age, there is a rising concern of

antimicrobial resistance due to extensive and

unregulated antimicrobial used in clinical setting by

medical professionals (Fraise A P, 2002; Nikaido H,

2014). Consequently, more and more antimicrobial

resistance is being reported and in addition, it is

discovered that the highest rate of antimicrobial

resistance is located in developing world (Nikaido

H. 2014). Nevertheless, the demand of new novel

effective anti-microbial to combat pathogenic

microorganisms in clinical setting has increased

(WHO, 2002).

Staphylococcus epidermidis is a gram-

positive bacteria belonging to the coagulase-negative

staphylococci group. This bacteria is a normal flora

of human skin but also the most common cause of

infection in the use of medical devices (WHO,

2002). Increased resistance of S. epidermidis to

various antibiotics led to the treatment of

nosocomial infections more difficult (Yuwono H,

2010). Based on a study conducted by Najar-

Peerayeh et al, 2014, 92.2% of the 64 isolates of

S.epidermidis have mecA genes that play a role in

penicillin-binding expression protein (PBP2a) that

decreases the affinity of the beta-lactam antibiotic.

In addition, the ability of S. epidermidis to form

biofilms makes this bacteria able to avoid the

immune system and antibacterial drugs (Solati SM

et al, 2015; Abidi et al, 2015).

Staphylococcus aureus is a bacteria that

can cause various diseases because of the toxin it

produces or direct invasion that damages the tissue

(Murray PR et al, 2013). In early 1940, the infection

caused by S. aureus was successfully treated with

penicillin, a beta-lactam antimicrobial group, which

rapidly replaced by the new resistant strain encodes

a betalactamase enzyme. This new strain is actually

can be resolved through the administration of new

antimicrobial methicillin (Yuwono H, 2010) but in

the early of 1980s, methicillin resistant S. aureus

(MRSA) strains spread rapidly and alter the

Kiranasari, A., Bonita, A., Melina, E., Winston, K., Baht, N., Sutandi, N., Dewi, B., Ningsih, I. and Sjatha, F.

Antibacterial Activity of Several Indonesian Endemic Plants against Staphylococcus epidermidis, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus.

DOI: 10.5220/0009844100002406

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

ISBN: 978-989-758-347-6

therapies available for S. aureus infection. In 2003, a

new strain of MRSA caused outbreak of cutaneous

infection and pneumonia.

Compared to other resistant bacteria, MRSA

infection is epidemiologically significant. Studies

conducted by CDC more than half Staphylococci

bacteria ehich caused Hospital Acquired Infections

are resistant to oxacillin (De Angelis G et al, 2010)

and its infection nowadays is an endemic in US

hospitals and communities (Klein E et al, 2007;

Crum NFet al, 2006). Furthermore, MRSA in Cipto

Mangukusumo Hospital, Indonesia also show an

increase from 28.5% in 2009 to 32% in 2010 (Liana

P., 2014).

Several research to find a novel anti

Staphyococcal bacteria from plants is also

progressing. Pradhan D et al, 2013 and Dwivedi Vet

al, 2014 reported Piper betle leaves and leaves

extract have antimicrobial, anti-inflammatory,

antioxidant and antiseptic properties. Specifically, P.

betle shown to have antibacterial activity against S.

aureus, Streptococcus pyogenes, E. coli,

Pseudomonas aeruginosa, Enterococcus fecalis,

Klebsiella pneumoniae, and others. The content of

sterols in betel leaf extract interact with bacterial cell

wall, disturbing its permeability.

Other plant that also potential to be an

antibacterial are bark of candlenut (Aleurites

moluccana) which is used traditionally for the

treatment of diarrhea and thypoid fever

(Alimboyoguen AB, et al 2014). Research shows

3acetyl aleuritolic acid from bark extract has an

antimicrobial activity. Moluccanin from A.

moluccana also has antibacterial including S. aureus

and antiviral activity (Othman AS and Rasyidah

MR, 2010).

Albizia saman (Jacq.) Merr. formerly known as

Samanea saman is having several phytochemical

components which are flavonoids, alkaloids, tannins,

carbohydrates, glycosides, saponins, steroids, and

reducing sugar are widely used as the remedy for

colds, diarrhea, headache, and stomach ache.

According to Perry in 1980, the alcoholic extract of

S. saman is also proven to inhibit the growth of

Mycobacterium tuberculosis (Kirithika T.2013).

The clove plant (Syzygium aromaticum) contain

chemical compounds that provide its aromatic and

antibacterial nature. The active compound being

studied is eugenol, one of many phenolic

compounds. Eugenol has been widely used in dental

care settings, and has been proven as an effective

anesthetic and antiseptic (Cortés-Rojas Det al, 2016;

Neveu Vet al, 2010).

With its promising properties as antibacterial and

its abundancy worldwide, we assessed several

concentration of P. betle leaves extract, A.

moluccana stem bark extract, S. saman extract, C.

longa extract and S. aromaticum flower bud extract

against S. epidermidis, S. aureus and MRSA. As our

result show that several extracts have a good

potency as anti-staphylococcal infection.

2 MATERIAL AND METHODS

2.1 Bacteria, Medium and Extract

S. epidermidis, S. aureus and MRSA bacteria were

grown in nutrient agar. All bacteria were from

Microbiology Department culture collection, Faculty

of Medicine Universitas Indonesia, which identified

using commercial Vitex identification kit and tested

for its resistancy according to CLSI. Broth Brain

Heart Infusion (BHI) medium and Muller Hinton

Agar (MHA) for antibacterial testing and Plate

Count Agar (PCA) were provided by Department of

Microbiology, Faculty of Medicine, Universitas

Indonesia. Extracts of P. betle leaves, A. moluccana

stem bark, S. saman, C.longa and S. aromaticum

flower bud in ethanol were prepared by Medical

Pharmacy Department, Faculty of Medicine,

Universitas Indonesia. Antibiotic ciprofloxacin or

clindamycin was used for positive control.

2.2 Antibacterial Assay

2.2.1 Agar diffusion method

An overnight bacteria culture was diluted into 0.9%

NaCl to reach McFarland value of 0.5. Bacterial

suspension was then applied into MHA followed by

creating 7 diffusion wells in the media using blue

tips. Each extract at several concentrations was

applied in to the well which are : P.betle extract at

62,5; 125; 250; 500 and 1000 mg/ml was tested

against S. epidermidis and A.moluccana extract at

50; 100; 200; 400 and 800 µg/ml against MRSA.

With addition for aquadest and antibiotics at 20

µg/ml as negative and positive control respectively.

Plate was then incubated for 16 – 18 hours at 37

Observed inhibitory zone was measured using

calipers.

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

2.2.2 Dilution method

An overnight bacteria culture were diluted into BHI

media followed by addition of final concentration of

S. saman and C. longa extract at 12.5%, 6,25%,

3,125%, 1.563%, 0.782% and 0.391% meanwhile

for S. aromaticum extract at 0.0488%, 0.0977%,

0.19530%, 0.3906%, 0.7813%, 1.563%, and

3.125% all against MRSA and non-MRSA.

Aquadest and antibiotic at 20 µg/ml were used as

negative and positive control respectively. Culture

were then incubated for 18 – 24 hours at 37

MIC value defiened as the smallest extract

concentration that inhibits bacteria growth in BHI

media. Two cultures at higher and lower

concentration of MIC value were smeared at PCA

followed by incubation for 18 – 24 hours at 37

C to

obtain number of bacterial colony from the tested

concentration. Lowest extract concentration giving

smaller amount of 30 CFU/ml (colony forming unit)

bacteria defined as MBC value.

3 RESULT AND DISCUSSION

Several plants extract tested in this study are briefly

conclude in table 1. Extract of C. longa, A.

moluccana and S. saman didn’t show any

antibacterial activity against S. aureus and MRSA

by dilution method. Our result is contrary with

several studies which show anti bacterial inhibition

of S. saman and C. longa against several bacteria

including S. aureus and MRSA due to lower extract

concentration that we use in this study (Rita et al,

2013, Thippeswamy et al, 2011, Prasad et al, 2008,

Bengmark et al, 2009 and Moghadamtousi et al,

2014, Othman et al, 2009). Obasi et al, 2011, found

that tannin is one of phytochemical compund found

in S. saman that can inhibit the growth of

microorganism by precipitating the microbial protein

needed for their growth, resulting in protein

deprivation of the microorganism. Experiments

performed by Ibrahim A, 2011 showed A.

moluccana bark extract produced an antimicrobial

effect against Salmonella thyphii and Vibrio

cholerae but not tested against MRSA or other

positive gram cocci bacteria.

Our research show the potency of P. betle leaf

extract formed the inhibitory zone of S. epidermidis

on MHA for all tested concentrations as listed in

table 2. The results were greater than the positive

control of ciprofloxacin which resulted in an average

inhibitory zone diameter of 31.70 ± 0.94. Based on

the Pearson correlation test, it was found that the

concentration of P. betle leaf extract correlated with

moderate (r = 0.642) to the large increase in

inhibition zone diameter of S. epidermidis bacteria

(Tumbelaka AR et al, 2011). Chakraborty, et al,

2011 tested metalloic leaf extract of P. betle against

S. aureus and proved an increase in antibacterial

activity assessed by measurement of inhibitory zone

diameters along with increased concentration of

extract (5 mg/ml, 10 mg/ml, 25 mg/ml, 50 mg/ml,

100 mg/ml).

Our result for S. aromaticum flower bud extract

show anti S. aureus and MRSA with similar MIC

and MBC value at 0.7813% and 0.3906%

respectively. Interestingly, our result show inhibition

and bactericidal value of S. aromaticum extract

against MRSA is lower than S. aureus which may

need further research. We hypothesize the effect of

mecA and change in the cell wall structure of MRSA

increased sensitivity towards eugenol, confirmed

phenolic compund of S. aromaticum (Cortés-Rojas

Det al, 2016; Neveu Vet al, 2010).

A research on the effect of Indian spices on food

borne pathogens showed that an aqueous extract of

S. aromaticum has inhibitory activity against S

aureus at 1% concentration, and complete

bactericidal activity at 3% concentration (Sofia P et

al, 2007). Another research compared aqueous clove

extracts from Sri Lanka and Zanzibar, and its results

suggested that the end-point of antimicrobial activity

against S aureus is at 6.25% (Nzeako BCet al,

2006). These results suggest that multiple external

factors influence the content and potency of the

herb. These factors also influence the inhibitory and

bactericidal effects. In this research, S aureus is

inhibited at 0.7813%, which is much lower to other

results.

4 CONCLUSION

Our study shows that extract from Piper betle and

Syzygium aromaticum were found to have a good

anti Staphylococcal activity which can be further

analyed for purification and bacterial inhibition

mechanism.

Antibacterial Activity of Several Indonesian Endemic Plants against Staphylococcus epidermidis, Staphylococcus aureus and

Methicillin-resistant Staphylococcus aureus

Table 1. Plant extract, antibacterial method and result

Plant extract

Antibacterial method

Bacterial tested

Inhibition

Curcuma longa

Dilution

S. aureus, MRSA

Aleurites moluccana

Diffusion

MRSA

Samanea saman

Dilution

S. aureus, MRSA

Piper betle

Diffusion

S. epidermidis

Syzigium aromaticum

Dilution

S. aureus, MRSA

Table 2. Inhibition zone of ciprofloxacin and P.betle leaf extract against S. epidermidis

Treatment

Concentration (mg/ml)

Average inhibition zone (nm) ± SD

Aquadest

Ciprofloxacin

31,70 ± 0,94

P. betle extract

1000

32,38 ± 2,63

P. betle extract

500

30,05 ± 1,38

P. betle extract

250

27,85 ± 1,91

P. betle extract

125

23,80 ± 2,1

P. betle extract

62,5

23,78 ± 0,47

ACKNOWLEDGEMENTS

Publication of this was study supported by Hibah

PITTA UI 2018/2019.

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Antibacterial Activity of Several Indonesian Endemic Plants against Staphylococcus epidermidis, Staphylococcus aureus and

Methicillin-resistant Staphylococcus aureus