A Comparative Study of the Antibacterial Activity of Clove and Rosemary Essential
Oils on Multidrug Resistant Bacteria
Baydaa Hameed Abdullah*, Suhad Faisal Hatem, Widad Jumaa
Department of clinical and laboratory sciences, Pharmacy college, Al Mustansiriyah University. Baghdad, Iraq
Article Information Received 20 November 2014 Received in revised form 26 Feb 2015 Accepted 27 Feb 2015
Abstract
The essential oils of Syzygium aromaticum (clove bud) and Rosmarinus officinalis L. (rosemary) were obtained by hydro-distillation.The antimicrobial activity of clove bud oil and rosemary oil
was investigated by agar well diffusion method against four multidrug resistant strains namely
Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus feacalis as well as two standard strains, Staphylococcus aureusATCC29213 and Pseudomonas aeruginosaATCC27853. Both essential oils exhibited inhibitory effects towards all the test organisms, clove essential oil had antibacterial activity little higher than of rosemary oil, MICs
ranged from 0.312% (v/v) to 1.25% (v/v) for all tested bacteria while MICs for rosemary oil ranged
from 0.312% (v/v) to 5 % (v/v). Based on this finding, it may be suggested that these essential
oils may be used as natural antibacterial agents to treat infections caused by multidrug resistant
bacteria.
Keywords: Antibacterial, Essential oil clove, Rosemary, MIC *
Corresponding Author: E-mail: [email protected] Tel.: 009647902244154
1 Introduction
Antibiotics provide the main basis for the therapy of microbial
(bacterial and fungal) infections. There was a medical belief since
antibiotics discovery and their uses chemotherapeutic agents that
this would lead to the infectious diseases eradication. However , the
major factor for the dissemination and emergence of muli- drug
resistant bacterial strains was the overuse of antibiotics1.The
worldwide emergence of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus feacalis and Acinetobacter baumanni has become a majortherapeutic problem. Multi-drug resistant strains of
Pseudomonas aeruginosa, Staphylococcus aureus and
Enterococcus feacalis are widely distributed in hospitals and are
increasingly being isolated from community acquired
infections2.Thus, as a result of the rapid global spread of emergence
of resistant bacteria and the need to find new antibacterial agents.
However even new generations of antibiotics have low effectiveness.
For this reason, Natural products provide unlimited opportunities for
new drug. The search for new antibacterial compounds an
alternative way for solving this problem so herbal products are
increasingly turning the researchers attention to develop better drugs
against Multidrug Resistant Bacteria(MDR)3,4,32.
Cloves (Syzygium aromaticum) are belonging to the family
Myrtaceae which are dried unopened floral buds of an evergreen
tree5. Clove is used as flavoring agent and as spice .It has been
known as aromatic, stimulant and carminative, used for dyspepsia
and gastric irritations. Antimicrobial and antioxidant properties have
been known to clove buds and their essential oils 6.
Clove oil is widely used as a perfume and food flavouring7, as a
medicine for the treatment of asthma, rheumatoid arthritis, acne,
warts, scars and various allergic disorders8, as an analgesic,
antispasmodic, and as a general antiseptic in medical dental
practices9.Antimicrobial properties of clove essential oil was tested
and showed inhibitory activity to Listeria monocytogenes,
Campylobacter jejuni, Salmonella enteritidis, Bacillus cereus, Escherichia coli and Staphylococcus aureus10. Importantly, clove oil might be considered as anti-carcinogenic agent due to its antioxidant
properties11.
Rosemary (Rosmarinus officinalis L.) is a spice and medicinal herb widely used around the world. Of the natural antioxidants, rosemary
has been widely accepted as one of the spices with the highest
UK Journal of Pharmaceutical and Biosciences
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UK J Pharm & Biosci, 2015: 3(1); 19 antioxidant activity11. Rosemary essential oil is also used as an
antibacterial, antifungal13,14,15 and anticancer agent16.
The aim of the present study was to investigate the antibacterial
effects of the oil extracts of Syzygium aromaticum and Rosmarinus officinalis against multi-drug resistant strains isolated from clinical infection.
2 Materials and Methods
2.1 Collection and identification of plant materials
The leaves of rosemary plant and the flower buds of clove used in
this study were purchased from the local market of Baghdad,
Iraq.The plants were identified and authenticated at the Department
of Pharmacogonosy, Faculty of Pharmacy, and University of
AlMustansiriah.
2.2 Extraction of volatile oil by steam distillation
Steam distillation method was used in order to obtain the oil extracts
of the dried flower buds of cloves and the dried rosemary by using
the Clavenger apparatus as mentioned by Harbone17.Briefly,120g of
grinded dried two plants were placed in a round bottom flask and the
added water full approximately three-quarter of the flask. The
distillation apparatus was then connected to the flask and by heating
the water boiled, steam carrying volatile condensed by condenser
and over anhydrous sodium sulphate the oil was drained and dried.
Determination the density of the oil was carried according to the
weight: volume ratio (w/v).
2.3 Bacterial test isolates
Four mulidrug resistant bacteria clinical isolates and two standard
strains were selected for this study. The mulidrug resistant bacteria
comprised Acinetobacter baumanni, Pseudomonas aeruginosa,
Staphylococcus aureus and Enterococcus feacalis were isolated from different clinical samples(stool, wound, ear, throat and sputum)
and the standard strains were Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosaATCC27853.The bacteria were cultured on nutrient agar medium. The agar plates were incubated at
37°C for 24 h.
2.4 Antibacterial assay
Determination the antibacterial activities of each plants oils was
carried out by agar well diffusion method18 . Briefly, one hundred
microlitres (100µL) of inoculums (106CFU/mL; 0.5 Mac-Farland) of
each test bacterium was spread on to a sterile Muller-Hinton Agar
plate (Hi Media). The plates were allowed to dry and a sterile cork
borer of diameter 6.0mm was used to bore wells in the agar plates.
Subsequently, a 50μL volume of each dilution of the oil with DMSO
(10% to 0.312% v/v) introduced in triplicate wells into Muller-Hinton
Agar plate. Sterile DMSO served as the negative control .The plates
were allowed to stand for at least 1h for diffusion to take place and
then incubated at (37 ºC for 24h).The antimicrobial activity, indicated
by an inhibition zone surrounding the well containing the extract, was
recorded if the zone of inhibition was greater than 6mm19.
2.5 Determination of minimum inhibitory concentration (MIC)
The MIC was defined as the lowest concentration that completely
inhibited the growth for 24 h20. The MIC for the oils of each plant was
determined by the agar well diffusion method. A two-fold serial
dilution of the clove oil and rosemary oil was prepared in sterile
DMSO to achieve a decreasing concentration range of 10% to
0.039% (v/v). A 50 µL volume of each dilution was added aseptically
into the wells in Mueller Hinton agar plates that had been inoculated
with standardized inoculums (106 CFU/mL) of the test bacteria. The
agar plates were incubated at 37°C for 24 h. All experiments were
performed in triplicate. The lowest concentration of clove and
rosemary oils showing a clear zone of inhibition was considered as
the MIC.
3 Results
The essential oils obtained were yellow in color with a density of 1.04
mg/ml for Syzygium aromaticum flower budsand 1.07 mg/ml and
Rosmarinus officinalis L.
In the present investigation, we have tested the essential oil extracts
of the two plants for their antimicrobial activity against four multidrug
resistant strains. Two ATCC strains, one of Gram-positive bacterium
(Staphylococcus aureus ATCC 29213) and one of Gram-negative
bacterium (Pseudomonas aeruginosa ATCC 27853) were also used
as control sensitive strains.
The two plants essential oils showed antibacterial activity against all
microorganisms tested, as exhibited by an agar diffusion assay.
Acinetobacter baumanni was found to be the most sensitive to rosemary oil at the higher concentration (10% v/v) with an inhibition
zone diameter of 35 mm, followed by Pseuomonas aeruginosa (34 mm). Staphylococcus aureus and Enterococcus faecalis were found to be less sensitive to the test oil with an inhibition zone diameter of
30mm and 32 mm, respectively. However the lower concentrations
of rosemary oil under (5% v/v) were found to be not effective in all
tested multidrug resistant bacteria except Acinetobacter baumanni
as shown in table 1.
The growth of the tested bacteria was also inhibited by clove oil ,the
widest inhibition zone diameter was produced against Acinetobacter baumanni (28mm) at the higher concentration (10% v/v) followed by
Enterococcus faecalis and Pseuomonas aeruginosa produced an inhibition zone diameter of (25mm) and (17mm) respectively (table
UK J Pharm & Biosci, 2015: 3(1); 20 sensitive to the concentration less than (1.25% v/v) and
Staphylococcus aureus was not sensitive to the concentration less than (0.625% v/v).
As it can be seen, both essential oils exhibited inhibitory effects
towards all the test organisms. Clove essential oil exhibited a little
higher antimicrobial activity than that of rosemary essential oil, which
was similar to the results of sensitivity test (Tables 1 and 2). MICs for
clove essential oil ranged from 0.312% (v/v) to 1.25% (v/v) for all test
microorganisms, while MICs for rosemary oil ranged from 0.312%
(v/v) to 5 % (v/v).
Table1: Antibacterial activities of rosemary essential oil
Bacterial isolates
Diameter of growth of inhibition zones ( mm ) Concentration of oil (v/v)
10% 5% 2.5% 1.25% 0.625% 0.312% DMSO
Acinetobacter baumanni 35±1.4* 20±1.8 18±1.6 18±1.7 16±2.1 14±2.5 0
Pseuomonas aeruginosa 34±2.5 18±2.4 - - - - 0
Staphylococcus aureus 30±1.8 20±3.2 - - - - 0
Enterococcus faecalis 32±2.3 21±3.4 - - - - 0
Pseuomonas aeruginosa ATCC
27853 30±2.6 16±1.5 7±3.4 7±1.6 - - 0
Staphylococcus aureus ATCC 29213
25±3.4 22±2.2 - - - - 0
(-) = no activity, *Values, including diameter of the well (6 mm), are means of three replicates ± SD
Table2: Antibacterial activities of clove essential oil
Bacterial isolates
Diameter of growth of inhibition zones ( mm ) Concentration of oil(v/v)
10% 5% 2.5% 1.25% 0.625% 0.312% DMSO
Acinetobacter baumanni 28±2.3 * 24±1.5 21±2.3 15±1.4 14±1 14±0.7 0
Pseuomonas aeruginosa 17±3.3 15±1.7 12±1.2 10±2.2 - - 0
Staphylococcus aureus 20±2.8 18±1.6 15±2.3 13±1.3 10±2.3 - 0
Enterococcus faecalis 25±2.6 22±2.4 19±3.4 15±1.6 13±1.5 7±1.6 0
Pseuomonas aeruginosa ATCC 27853 25±1.4 22±3.5 20±1.3 20±1.8 16±2.4 16±1.4 0
Staphylococcus aureus ATCC 29213 22±1.6 20±2.2 15±2.3 13±2.2 - - 0
(-) = no activity, *Values, including diameter of the well (6 mm), are means of three replicates ± SD
4 Discussion
The increasing occurrence, particularly in hospitals, of multidrug
resistant bacteria has made therapy more difficult21. The spread
control strategies have been proposed and also searched for
treatment MDR bacteria, the alternative treatment of these bacteria
is by using natural compounds investigations22.
The antibacterial activitywere seen against MDR bacteria. However,
the clove oil was more active against Acinetobacter baumanni and
Enterococcus faecalis isolates at concentrations of 0.312% and above while rosemary oil was active against all bacteria tested at
concentrations of 5% and above except Acinetobacter baumanni
which was sensitive at concentrations of 0.312%.
Antibacterial properties of clove and rosemary have already been
reported23,24 but in the present study we tested the antibacterial
activity of clove and rosemary essential oils on multidrug resistance
bacteria. The antibacterial activity of the essential oils of against
clove and rosemary MDR bacteria has not been reported before.
MICs of clove and rosemary essential oils in this study were similar
to the known literature6,9,27,28 ,with a little difference, which could be
UK J Pharm & Biosci, 2015: 3(1); 21 rosemary, different extracting methods of essential oils, andmay be
due to the use of MDR bacteria in the present study.
Different essential oils have different antimicrobial activity because of
the components in them29. The antimicrobial activity of clove
essential oils could be associated with Eugenol (2 methoxy-4
allyl-phenol)25, the main component of clove oil, which is already known
to exhibit antibacterial and antifungal activity30.Clove antimicrobial
activity also due to high tannin content (10-19%)26.The main compounds present in rosemary essential oil were 1,8-cineole,
α-pinene, camphor, etc., which have been evaluated for their
antimicrobial effects31,32,33.
Based on these data, further chemical and pharmacological
investigations are required for clove and rosemary essential oils.
These in vitro results of the present study provide evidence that the two essential oils represent a potentially rich source for medicine and
food preservatives against microorganisms.
5 Conclusion
On comparison of the antibacterial activities of cloveoil and rosemary
oil tested against the MDR bacterial strains, it was finally concluded
that clove oil and rosemary oil emerged as the potent agent
exhibiting high antibacterial activity in the treatment of the infections
caused by multidrug resistant bacteria and the clove oil is more
potent than rosemary oil.
6 Competing interests
Plant extaction, antimicrobial evaluation, multidrug resistance
7 Author’s contributions
BHA carried out literature review, draft the manuscript, data
collection and tabular form. BHA and SHF carried out the bacterial
isolation and antibacterial evaluation. WJ participated in the plants
extraction and data collection. All authors read and approved the
final manuscript.
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