Mechanism of Antimicrobial Action of Ocimum Basilicum Essential

Oil Against Nosocomial Bacteria

Fadli Asmani

, Kiran Chanabasappa Nilugal

, Sherilyn Fenn Karel

Santosh Fattepur

, May

Florence Dela Cruz Bacayo

, Wong Charng Choon

, Rasny M

and Eddy Yusuf

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

International Center for Halal Studies, Management and Science University, 40100 Shah Alam, Selangor, Malaysia

Keywords: Essential oil, Ocimum basilicum, nosocomial infection, Antimicrobial mechanism

Abstract: Nosocomial infection is a major concern in the healthcare sector. Ocimum basilicum essential oil is one of

the main essential oil being investigated for its antimicrobial activities. This study was conducted to

determine the effect of Ocimum basilicum essential oil on bacterial cell growth and cell membrane integrity.

The O. basilicum essential oil (EO) was purchased from Natur Aromatherapy and Wellness and different

concentrations of the essential oil (0.092 – 1.470 g/ml) was prepared using 10% DMSO. Disc diffusion test

and MIC using broth dilution for Escherichia coli and Staphylococcus aureus, with Gentamicin as positive

control were conducted. 24-hour growth kinetic analysis of the bacteriaand their cell membrane integrity

were investigated by measuring the absorbance value. The zone of inhibition by the essential oil was greater

in S.aureus compared to E.coli and the MIC was 0.368g/ml and 0.735g/ml respectively. EO concentrations

at 1.47 g/ml and 0.735 g/ml are the only one showed effective inhibition on the growth of the bacteria. The

EO was more effective in inhibiting the growth of S.aureusthan E.coli. The absorbance of the bacterial cell

constituents increased from the negative control, 2 x MIC, MIC and Gentamicin in both bacteria. Ocimum

basilicum essential oil exert its antimicrobial action by affecting bacterial growth and acting on cell

membrane integrity of the bacteria.

1. INTRODUCTION

Nosocomial infections are infections which are

acquired by patients under medical care during

their hospital stay and also by the visitors and

healthcare workers. It is also known as hospital

acquired infection [1]. The International

Nosocomial Infection Control Consortium

(INICC) has reported the rates of device-associated

hospital acquired infections (DA-HAIs) in various

countries including Malaysia, from 2007 to 2012 is

high [2]. The agents most commonly associated

with nosocomial infections are Staphylococcus

aureus, Escherichia coli, Pseudomonas

aeruginosa, Klebsiella pneumonia, Streptococcus

spp., Acinetobacter spp., Bacillus cereus, Proteus

mirablis and Serratia marcescens [1].

Essential oils or also known as volatile oils are

the mixture of volatile, organic compounds that

comes from one plant source and it is the essence

that is responsible for the flavor and fragrance of

the plant. They can be obtained from different

parts of plants [3]. They are secondary metabolites

of plants which are produced in response to stress.

There are three broad categories of plant secondary

metabolite that encompass terpenes and terpenoids

(oxygenated compound), alkaloids and the

phenolic compounds [4]. Apart from that, alcohols,

esters, aldehydes and ketones can also be

discovered on essential oil [5].

Ocimum basilicum is an aromatic herb with a

strong odour and sharp taste that belongs to the

Lamiaceae family which has white, rose or violet

labiates flowers with bilabiate calyx and four lobed

corolla [6]. The plant is used as traditional

medicine for gastrointestinal problems like

diarrhea, dyspepsia, intestinal spasm and gastritis,

also as antitussives, antihelminths and analgesics

[7].The main constituents of O.basilicum essential

oil are chavicol methyl ether or astragole, linalool

and eugenol where the antibacterial activity is said

to be due to linalool [6]. It displayed significant

antibacterial activity against Staphylococcous

aureus, Salmonella enterititis and Escherichia coli

Asmani, F., Nilugal, K., Karel, S., Fattepur, S., Bacayo, M., Choon, W., Rasny, M. and Yusuf, E.

Mechanism of Antimicrobial Action of Ocimum Basilicum Essential Oil Against Nosocomial Bacteria.

DOI: 10.5220/0009844600002406

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

ISBN: 978-989-758-347-6

while being antiseptic against Proteus vulgaris,

Bacillus subtilis and Salmonella parathypha [7].

2. MATERIAL & METHODS

2.1 Ocimum Basilicum Essential Oil

Acquisition

The Ocimum basilicumessential oil was purchased

at Natur Aromatherapy and Wellness.

2.2 Antimicrobial Susceptibility Test

by Disk Diffusion Method

An inoculum suspension was prepared by

inoculating some of the colony from the bacteria

subculture into a sterile saline solution. This

inoculum suspension was used within 15 minutes

to avoid further bacteria growth. The bacteria

inoculum was inoculated on Mueller Hinton agar

plate. On each plate, the Ocimum basilicum

essential oil disk (0.092 g/ml) will be placed. The

positive control is gentamicin disc (10 μg). The

agar plate was incubated at 37 ⁰C for 24 hours.

After that, the zone of inhibition of each agar was

measured and sensitivity of the bacteria to the

essential oil was identified. This experiment was

conducted in three replicates (Luis et al., 2016).

2.3 Minimum Inhibitory Concentration

Using Broth Dilution Method

1 ml of peptone water was placed inside

microcentrifuge tube, then 100 μl of bacterial

inoculums was placed inside each test tube.100 μl

of Ocimum basilicum essential oils which has been

diluted by two fold dilution, was placed in to each

respective microcentrifuge tube. The mixtures are

mixed well then incubated for 24 hours at 37 ⁰C.

The control consists of 100 μl gentamicin solution.

The lowest concentration of the essential oil that

causes complete inhibition of the bacterial growth

will be taken as the MIC. This experiment was

conducted in three replicates (Luis et al., 2016).

2.4 Growth kinetic analysis of

Staphylococcus aureus and

Escherichia Coli Bacteria

0.3 ml bacterial inoculum suspension was placed

into a test tube containing 8 ml of peptone water

and they were mixed well by slightly vortexing

them. The mixture was then placed into the test

tube which contained 0.092 g/ml Ocimum

basilicum essential oil. The process was repeated

for each concentrations of serially diluted O.

basilicum essential oil. The positive and negative

controls in the experiment were prepared by using

0.3 ml gentamicin solution and 0.3 ml peptone

water respectively. The test tubes were incubated

at 37 ⁰C and analyzed over period of 24 hours [8].

A sample from each test tubes were taken at

different time intervals and analyzed at 660 nm

using the visible spectrophotometer. The

absorbance value was recorded and the figure 1 of

Absorbance against Time was plotted. The

experiment was carried out in three replicates.

2.5 Staphylococcus aureus and

Escherichia Coli Bacterial Cell

Membrane Integrity

100 ml of bacterial inoculums suspension was

prepared and centrifuged for 12 minutes at 3000g.

The solution was drained from the centrifuge tube

and bacterial cells which was suspended at bottom

of tube was retained. The bacterial cells were

washed for three times and resuspended using 0.1

M phosphate buffer solution (PBS, pH 7.4). The

cell suspension (100 mL) was incubated at 37 ⁰C

under agitation for 4 hours in the presence of two

different concentrations of Ocimum basilicum

essential oil (MIC, and 2 x MIC). Then, 25 ml

sample from each centrifuge tubes were collected

and then centrifuged at 6000 g for 5 minutes [9].

The supernatant (3 mL) from each samples were

taken and their absorbance value wasmeasured

using UV spectrophotometer at 260 nm

wavelength. Same procedure was conducted for

positive and negative control. [10]. This

experiment was carried out in three replicates.

3. Results

3.1 Antimicrobial Susceptibility Test

by Disk Diffusion Method

Disk diffusion method was conducted by Kirby-

Bauer method. The essential oil was compared to

gentamicin in terms of the zone of inhibition and it

showed smaller diameter of inhibition (Table 1).

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

Table 1 The mean diameter zone of inhibition of

Ocimum basilicum essential oil against Escherichia coli

and Staphylococcus aureus.

Extract

Mean zone of inhibition

(cm)

E. coli

S. aureus

O. basilicum EO

(0.092 g/ml)

0.700

(±0.173)

0.733

(±0.252)

Gentamicin (10

µg)

3.000

(±0.200)

3.467

(±0.153)

Values represent three independent replicates ±SD with

significant differences between the groups (p<0.05).

3.2 Minimum inhibitory concentration

using broth dilution method

Broth dilution method were used in minimum

inhibitory concentration test where peptone water

was mixed with the essential oil and then was

serially diluted by two folds. Table 3.2 showed that

the concentration of essential oil needed to inhibit

the Escherichia coli (E.coli) was higher.

Table 2. The minimum inhibitory concentration of

Ocimum basilicum essential oil against Escherichia coli

and Staphylococcus aureus.

Minimum Inhibition

Concentration (MIC) (g/ml)

E.coli

0.735

S. aureus

0.368

3.3 Growth kinetic analysis of

Staphylococcus aureus and

Escherichia coli bacteria

Both of the bacteria’s growth kinetic were

analyzed for a period of 24 hours upon 4 hours

treatment with Ocimum basilicum essential oil at

different concentrations. From figure 1 and 2, the

negative control which consists of bacteria in

peptone water only displayed the growth curve and

act as the main comparison to the bacteria growth

when treated with the essential oil. E.coli showed

less susceptibility to the essential oil in comparison

with S. aureus.

Figure 1 Growth kinetic of Escherichia coli after

treatment with Ocimum basilicum essential oil.

Figure 2 Growth kinetic of Staphylococcus aureus after

treatment with Ocimum basilicum essential oil.

4. Discussions

Ocimum basilicum (O. basilicum) essential oil is

known for its antibacterial activity and this

properties can be used in reducing the nosocomial

infections in healthcare setting. The result from the

study showed that O.basilicum essential oil has

statistically significant effect on the growth and

cell membrane integrity of bacteria. O. basilicum

essential oil was tested for antimicrobial

susceptibility and the outcome was reflected by the

presence of zone of inhibition (ZOI) in the disk

diffusion test. It was observed that the zone of

inhibition was larger in Staphylococcus aureus (S.

aureus) compared to Escherichia coli (E. coli).

However, the difference in diameter zone of

inhibition between the two bacteria was small

where ZOI of E.coli was 0.700 cm and S. aureus

was 0.733 cm. Both of the bacteria were

0,2

0,4

0,6

0,8

1 2 4 6 8 101224

Absorbance

ESCHER ICHIA COLI

GROWTH KINETIC

EO 1.470

g/ml

EO 0.735

g/ml

EO 0.368

g/ml

0,2

0,4

0,6

0,8

1 2 4 6 8 101224

Absorbance

EO 1.470

g/ml

EO 0.735

g/ml

EO 0.368

g/ml

EO 0.184

g/ml

Mechanism of Antimicrobial Action of Ocimum Basilicum Essential Oil Against Nosocomial Bacteria

susceptible to Gentamicin antibiotic (positive

control). Gentamicin also showed larger inhibitory

zone in E. coli than S. aureus with 3.000 cm and

3.467 cm respectively.

The minimum inhibitory concentration was

determined for both bacteria by broth dilution

method. In this method, two fold dilution of O.

basilicum essential oil was done to prepare series

of concentration of 1.470 g/ml, 0.735 g/ml, 0.368

g/ml, 0.184 g/ml and 0.092 g/ml. After an

overnight incubation, results showed that the MIC

for E.coli and S. aureus were 0.735 g/ml and 0.368

g/ml respectively. The lowest concentration of O.

basilicum essential oil that was able to inhibit the

growth of E. coli was higher than S. aureus.

From the antimicrobial susceptibility study that

was conducted,O.basilicum essential oil showed

more antimicrobial activity against gram

positivebacteria than gram negative bacteria. In the

study done by [7]. O.basilicum essential oil also

showed stronger effect on gram positive bacteria

Staphylococcus aureus and Bacillus subtilis

compared to the gram negatives Escherichia coli

and Salmonella typhi. The presence of outer

membrane which consists of lipopolysaccharides

enables the gram negative bacteria to protect itself

by restricting the penetration of hydrophobic

compounds like the essential oil. Thus, the

essential oil may not be able to properly attack the

phospholipid layers of bacteria cell to compromise

its permeability and integrity. O.basilicum

essential oil exhibited high antibacterial activity

which is associated with its major constituents of

astragole and linalool [6] [7].

Based on the figure 1 and 2, Ocimum basilicum

(O. basilicum) essential oil effectively inhibits the

growth of Staphylococcus aureus (S. aureus) more

than that of Escherichia coli (E. coli). In the

growth kinetic analysis of E. coli, O. basilicum

essential oil was able to effectively inhibit the

growth of bacteria with the concentration of 1.470

g/ml and 0.735 g/ml which was represented by the

steady decline of the line graph. Starting at

concentration of 0.368 g/ml and lower, the line

graph started to increase steadily. At concentration

0. 368 g/ml of the essential oil, inhibitory action on

the bacteria growth occurred up to time 6 hours

only, then the bacteria growth curve began to

increase. At concentration of 0.184 g/ml and 0.092

g/ml of the essential oil, it was observed that the

inhibitory action is not effective as the bacteria

continued to grow steadily at lower rate compared

to that of the negative control (containing only

peptone water and E. coli bacteria). In the positive

control test, it was showed that it is able to

significantly decrease the growth of E. coliand the

line graph decreased and remained at a low growth

rate throughout the 24 hours study. In S. aureus

growth kinetic analysis, the growth of bacteria was

inhibited at concentration of 1.470 g/ml, 0.735

g/ml and 0.368 g/ml. It was observed that at

concentration of 0.184 g/ml and 0.092 g/ml, the

growth of S. aureus continued and compared to the

other concentrations of the essential oil the

difference is large. However, this growth was at

lower rate if compared to the negative control.

Gentamicin was also effective in inhibiting S.

aureus growth.

From the growth kinetic analysis study, it was

found that O.basilicum essential oil effectively

inhibit bacteria growth at higher concentrations.

Meanwhile, gentamicin was able to inhibit the

bacteria growth more than that of O.basilicum

essential oil. In the plotted line graph based on the

study, the normal bacteria growth phase which

consists of the lag phase, log (exponential phase),

stationary phase and death phase were not clearly

defined as the study was done only for a 24 hour

period. Nevertheless, from the study we managed

to confirm that O.basilicum essential oil was able

to inhibit bacteria growth. O.basilicum essential oil

acts by different pathways or mechanism in which

it exerts its antibacterial activity such as through

compromising bacteria membrane permeability

and integrity, inhibiting ATP production,

coagulation of bacteria cell constituents and

inhibiting bacteria quorum sensing (QS) system

[11] [12]. The essential oil of O.basilicum plant

may inhibits the bacteria growth through one or

more of these mechanisms.

There are various mechanism of antimicrobial

action for essential oil that have been proposed

however each essential oil have different

mechanisms and it is crucial to investigate the

exact mechanism to develop a more specific

antimicrobial agent. Ocimum basilicum (O.

basilicum) essential oil has the characteristic of

being lipophilic and it is able to penetrate the cell

membrane of bacteria and accumulate there which

then disrupts its integrity causing release of cell

constituents such as proteins, nucleic acids and

reducing sugars [10].

This is confirmed by the result of the study

where there is increased in the absorbance value of

the sample when the concentration of O. basilicum

essential oil was increased from MIC to 2x

MIC.The absorbance value increased from 3.811

to 4.053 when concentration of essential oil was

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

increased from MIC to 2x MIC in E. coli, which is

also displayed in S. aureus(from 4.077 to 4.494).

It was observed that the effect of O. basilicum

essential oil in compromising the cell integrity was

more prominent in S. aureus than E.

coli.Gentamicin was also able to disrupt bacteria

membrane integrity and from this study, it gave a

higher absorbance value than O. basilicum

essential oil. From the study, it was confirmed that

O. basilicum essential oil was able to compromise

bacteria cell membrane integrity. Cell constituents

such as proteins and nucleic acid are important in

maintaining cell structural integrity and cell

genetic identification respectively. When the cell

losses such important constituents, the cell normal

functioning in DNA transcription and translation is

compromised and this eventually leads to cell

death [9].

In the current study, effects of Ocimum

basilicum (O. basilicum) essential oil was studied

in Escherichia coli and Staphylococcus aureus. It

will be more beneficial if other gram negative and

gram positive bacteria and fungal which are

commonly associated with nosocomial infection

being studied. A study on which method of

extraction for O. basilicum essential oil may be

carried out which can help researchers to

determine which method produced higher yield.

Moreover, other tests such as cell membrane

permeability and scanning electron microscope

(SEM) can be conducted to further investigate how

O. basilicum essential oil alter bacterial cell

membrane permeability which allows leakage of

electrolytes and also how it causes changes in the

bacteria morphology.

5. CONCLUSION

Based on the results, it was concluded that

Ocimum basilicum essential oil exerts its

antimicrobial action against bacteria by inhibiting

their growth and disrupting the cell membrane

integrity. The statistical analysis using SPSS

version 21 indicated that there was significant

difference in the growth and cell membrane

integrity of Eschericia coli and Staphylococcus

aureus upon treatment with Ocimum basilicum

essential oil. The essential oil at higher

concentration was effective. The essential oil

ability to disrupt the bacteria cell membrane

integrity causes leakage of cell constituents and

ultimately leading to cell death. Thus, O. basilicum

essential oil can be used as a natural alternative

agent for the treatment of nosocomial infection.

ACKNOWLEDGEMENT

This research was supported Management &

Science University (MSU) for permitting me to

conduct my research in campus with freedom and

also appreciation to School of Pharmacy in

approving my research project.

CONFLICT OF INTERESTS

Author declare there is no conflict of interest.

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