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

, M. Rasny

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.

METHOD: 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. RESULT: 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. CONCLUSION: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 (Khan et al., 2015). 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

(Rosenthal et al., 2015). 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

(Khan et al., 2015).

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 (Tisserand & Young, 2014). 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

(Hamid et al., 2014). Apart from that, alcohols,

esters, aldehydes and ketones can also be discovered

on essential oil (Worwood, 1991).

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

(Moghadda et al., 2011). The plant is used as

traditional medicine for gastrointestinal problems

like diarrhea, dyspepsia, intestinal spasm and

gastritis, also as antitussives, antihelminths and

analgesics (Shirazi et al., 2014).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 (Moghadda et al., 2011). It displayed

significant antibacterial activity against

Staphylococcous aureus, Salmonella enterititis and

202

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: 10.5220/0008360002020208

In Proceedings of BROMO Conference (BROMO 2018), pages 202-208

ISBN: 978-989-758-347-6

Escherichia coli while being antiseptic against

Proteus vulgaris, Bacillus subtilis and Salmonella

parathypha (Shirazi et al., 2014).

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 (Theophel et

al., 2014). 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 (Zhang et al., 2015). 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. (Diao et al., 2014). This experiment was

carried out in three replicates.

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

203

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).

Table 1 The mean diameter zone of inhibition of Ocimum basilicum essential oil against Escherichia coli and

Staphylococcus aureus

Extract Mean zone of inhibition

(

)

E. coli S. aureus

O. basilicum EO

(

0.092

/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.

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

204

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

0,1

0,2

0,3

0,4

0,5

0,6

0,7

12468101224

Absorbance

ESCHERICHIA COLI GROWTH KINETIC

EO 1.470 g/ml

EO 0.735 g/ml

EO 0.368 g/ml

EO 0.184 g/ml

EO 0.092 g/ml

Gentamicin

Negative control

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

12468101224

Absorbance

EO 1.470 g/ml

EO 0.735 g/ml

EO 0.368 g/ml

EO 0.184 g/ml

EO 0.092 g/ml

Gentamicin

Negative control

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

205

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 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 (Shirazi et al., 2014). 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 (Moghadda et al., 2011; Shirazi et al.,

2014).

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 (Aiemsaard et al.,

2014; Luís et al., 2016). The essential oil of

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

206

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 (Diao et al.,

2014).

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 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 (Zhang et al., 2015).

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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