Extraction and Antistaphylococcal Study of the Essential Oil of Origanum vulgare L. (Guelma-Algeria)

Abstract

Abstract
Antimicrobial properties of plant essential oils (EO) have been investigated through several observations and clinical studies which
purpose them as potential tools to overcome the microbial drug resistance (MDR) problem. The aim of this research was to study
the antibacterial effect of a traditional plant EO, Oraginum vulgare L., against clinical isolates of methicillin resistant Staphylococcus
aureus (MRSA) through disk diffusion and agar dilution methods.
The EO showed very effective bactericidal activity towards the majority of the tested bacterial strains with inhibition zone
diameters in the range of 9.9-31.9 mm and a minimum inhibitory concentration (MIC) ranging from 0.314 to 0.628 mg/ml.
These results suggest that the essential oil of Origanum vulgare L. may be a useful alternative to antibiotics for the control of the
infections caused by Staphylococcus aureus.
Keywords: Origanum vulgare L., Essential oil, Antibacterial activity, Staphylococcus aureus MRSA.

Full text

International Journal of Innovative Approaches in Agricultural Research
Volume 3, Issue 1 March 2019
ijiaar.penpublishing.net
ISSN: 2602-4772 (Online)
Extraction and Antistaphylococcal Study of the Essential Oil of Origanum vulgare L.
(Guelma-Algeria)
Karima Ounaissia, Nora Mahfouf, Salima Bennadja, Abdelghani Djahoudi & Hichem Nasri
To cite this article
Ounaissia, K., Mahfouf, N., Bennadja, S., Djahoudi, A. & Nasri, H. (2019). Extraction and Antistaphylococcal Study of the Essential Oil of
Origanum vulgare L. (Guelma-Algeria). International Journal of Innovative Approaches in Agricultural Research, 3(1), 123-131. doi:
10.29329/ijiaar.2019.188.12
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Uluslararası Tarım Araştırmalarında Yenilikçi Yaklaşımlar Dergisi
International Journal of Innovative Approaches in Agricultural Research 2019, Vol. 3 (1), 123-131
https://doi.org/10.29329/ijiaar.2019.188.12
Copyright © 2019. This is an open access article under the CC BY-NC-ND
123
Original article
Extraction and Antistaphylococcal Study of the Essential Oil of
Origanum vulgare L. (Guelma-Algeria) 1
Karima Ounaissia a, b,
*
, Nora Mahfouf a, c, Salima Bennadja a, c ,
Abdelghani Djahoudi a, d & Hichem Nasri e
a University of Annaba, Annaba, Algeria
b Laboratory of Medical Botany, Faculty of Medicine
c Laboratory of Biochemistry and Environmental Toxicology
d Laboratory of Microbiology, Faculty of Medicine
e Laboratory on Biodiversity and Ecosystem Pollution, University of El Taref, Algeria
Abstract
Antimicrobial properties of plant essential oils (EO) have been investigated through several observations and clinical studies which
purpose them as potential tools to overcome the microbial drug resistance (MDR) problem. The aim of this research was to stud y
the antibacterial effect of a traditional plant EO, Oraginum vulgare L., against clinical isolates of methicillin resistant Staphylococcus
aureus (MRSA) through disk diffusion and agar dilution methods.
The EO showed very effective bactericidal activity towards the majority of the tested bacterial strains with inhibition zone
diameters in the range of 9.9-31.9 mm and a minimum inhibitory concentration (MIC) ranging from 0.314 to 0.628 mg/ml.
These results suggest that the essential oil of Origanum vulgare L. may be a useful alternative to antibiotics for the control of the
infections caused by Staphylococcus aureus.
Keywords: Origanum vulgare L., Essential oil, Antibacterial activity, Staphylococcus aureus MRSA.
Received: 12 July 2018 * Accepted: 19 January 2019 * DOI: https://doi.org/10.29329/ijiaar.2019.188.12
*
Corresponding author:
Karima Ounaissia, Laboratory of Medical Botany, Faculty of Medicine. University o f Annaba. Algeria.
Email: ounaissia_k@yahoo.fr
1 A part of this study was presented at the International Agricultural, Biological and Life Science Conference, Edirne, Turkey, September 2 -5, 2018.
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INTRODUCTION
The emergence and spread of multidrug-resistant (MDR) bacterial pathogens have substantially
threatened the current antibacterial therapy (Boucher et al., 2009). MDR bacterial infections often lead
to increased mortality, longer stays in hospitals, and higher cost of treatment and care (Boucher et al.,
2009; Giamarellou, 2010).
The resistance to antibiotics by microorganisms has increased because, generally, bacteria have
the genetic ability to transmit and acquire resistance to them.
Bacteria that belong to the Staphylococcus genus constitute one of the most serious
epidemiological problems. S. aureus has the strongest virulence potential among all the staphylococcal
species. It may become a part of the human bacterial flora (S. aureus nasal carriage) but increases the
risk of infection development, both nosocomial and community-acquired (DeLeo et al., 2010; Rafee et
al., 2012; Wang et al., 2010).
Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing nosocomial
infections, but it has emerged as a problematic pathogen in the community setting as well. These strains
called Community-Associated Methicillin-Resistant Staphylococcus aureus (CAMRSA) cause
infections in healthy individuals without predisponent risk factor and outside the hospital setting. MRSA
and CA-MRSA present a significant threat to public health and are difficult to manage (Li et al., 2009).
The therapeutic options for these pathogens are extremely limited and physicians are forced to use
expensive or previously discarded drugs that are associated with significant side effect to the patients
health (Boucher et al., 2009). Therefore, it is necessary to search the other alternatives that can
potentially be effective in the treatment of these problematic bacterial infections.
For a long period of time, plants have been a valuable source of natural products for maintaining
human health. The use of plant compounds for pharmaceutical purposes has gradually increased in the
world according to World Health Organization (Santos et al., 1995). About 80% of individuals from
developed countries use traditional medicine, which has compounds derived from medicinal plants
(Ellof, 1998).
The use of essential oils has been shown to possess potential in the treatment of infections, and is
safe in terms of human and animal health. Essential oils generally regarded as safe show antimicrobial
proprieties and antibacterial resistance that has not been reported after prolonged exposure (Pozzo et al.,
2012).
Among many plants scientifically studied regarding their antimicrobial properties, Origanum
vulgare L. (Oregano), Lamiaceae, has showed prominent results (Skandamis et al., 2002; Souza et al.,
2007). O. vulgare L. essential oil presented interesting results in inhibiting the growth of bacteria and
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synthesis of microbial metabolites, including the pathogen Staphylococcus aureus (Barros et al., 2009;
Baydar et al., 2004; Souza et al., 2006).
Oregano oil and its major phenolic components, carvacrol and thymol, are known for their wide
spectrum of antimicrobial activity, which has been the subject of several investigations in vitro (Dorman
et al., 2000; Lambert et al., 2001) and in vivo (Adam et al., 1998, Manohar et al., 2001).
In this context, the aim of this study was to evaluate the antimicrobial activity in vitro of the
essential oil of oregano from the region of Guelma (Algeria) against eight (8) methicillin-resistant
Staphylococcus aureus.
Material and Methods
Plant material
Leaves of O. vulgare were harvested at flowering stage in mid-June, 2014 from wild grown plants
at Guelma (East of Algeria) situated at latitude: 36° 36ˊ 41̋ , longitude: 7° 30 ˊ48̋ where the climate is
mild and rainy in winter and hot in summer with an annual average temperature of 17.3°C. Dried leaves
of O. vulgare were subjected to hydro distillation using a Clevenger-type apparatus and has been
analyzed by GC/MS, as described in our latest publication (Mahfouf et al., 2017).
Bacterial Strains
Bacterial strains of MRSA used were clinically isolated from specimens of different infectious
diseases obtained from Hakim Okbi hospital in Guelma city. They were provided by laboratory of
microbiology, faculty of medicine, university of Badji Mokhtar, Annaba.
The isolates were identified on the basis of Gram’s staining, mobility, cultural characterization
and biochemical screening routine methods were used. The antibiotic susceptibility was tested by the
antibiogram method.
Diffusion method in agar
Disc diffusion assay
A 24 h culture was diluted with sterile physiological saline solution with reference to the
McFarland standard, to achieve an inoculum of approximately 106 CFU ml. A 5ml portion of this
inoculum was placed on to the surface of pre-dried Mueller–Hinton agar Petri dishes, and allowed to
remain in contact for 1 min. Excess inoculums was removed using a sterile syringe and the Petri dishes,
were allowed to dry for 20 min at room temperature. Sterile 6 mm filter paper discs were placed on the
plates and immediately 20 µl of the essential oils were added. Sterile paper disc was used as control.
After allowing 1 h at room temperature for the essential oil to diffuse across the surface, Petri dishes
were incubated at 37°C for 24 h. The inhibition zone was measured in millimeter (Ahmad et al., 1999).
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Dilution method
The essential oil to be tested was incorporated into a semi-solid agar medium with different
concentrations of essential oil diluted in dimethyl sulfoxide (DMSO). After incubation, the absence of
microbial growth in Petri dishes was determined by the naked eye.
The minimum inhibitory concentration (MIC) was defined as the lowest concentration of essential
oil inhibiting any visible growth to the naked eye after 16 to 20 hours incubation at 37 ° C.
Microorganisms, however, remain viable.
The MIC values were evaluated according to published procedures (National Committee for
Clinical Laboratory Standard, 2000). The MICs were determined only with micro-organisms that
displayed inhibitory zones. Dilutions of the oil within a concentration range of 0.314-10.05 mg/ml.
Results and Discussion
Chemical composition of the essential oil of O. vulgare L.
Fifty five volatile constituents were identified in this EO, representing 98.7% of the total
composition (Table 1).The most abundant component was thymol (32.58%). Other components were
identified as γ-terpinene (18.76%), phenol (17.92%),1,2,3,4-tetramethylfulvene (11.40%),
isodiprene(2.79%), β thujene (1.94%), caryophyllene (1.80%) , β sesquiphellandrene (1.43%) and
linalool (1.22%) (Mahfouf et al., 2017).
Antistaphylococcal activity
Table 1. Antibacterial activity of O.vulgare L. essential oil against the bacterial strains tested based on
MIC and disc diffusion method
* MIC: Minimal Inhibitory Concentration, concentration range: 0.314-0.628 g/ml
** Disc diameter 6 mm average of three consecutive trials
Micro-organisms
**Disc diffusion assay
(inhibtion zone mm)
*MIC (mg/ml)
MRSA 1
25.2
0.628
MRSA 2
31.9
MRSA 3
25.1
MRSA 4
9.9
MRSA 5
19.3
0.314
MRSA 6
26.2
MRSA 7
28.9
MRSA 8
26.1
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Among the 8 strains of MRSA tested, 6 strains were very sensitive to the essential oil tested with
diameters of inhibition zones ranging from 25.1 mm to 31.9 mm, one strain had average sensitivity with
a diameter of inhibition zone of 19.3 mm and another strain with a limited sensitivity with diameter of
inhibition zone of 9.9 mm. The MIC was relatively low, ranging from 0.314 mg/ml to 0.628 mg/ml
(Table 1, Fig.1).
The essential oil from oregano was inhibitory to the growth of all the bacteria under test (Table
1). The obtained results, in accordance with the literature, showed that Oregano essential oil has
antibacterial properties. The activity is due to the high content of phenolics compounds such as thymol,
which account for over 32% of the ingredients of the oregano oil (Bouhdid et al., 2012).
Generally, the major compounds determine antimicrobial activity. Carvacrol, terpinen-4-ol and
thymol are the major compounds in oregano EO (Barros et al., 2009). The antimicrobial activity of
oregano oil is mostly attributed to the action of its principal phenolic components, carvacrol and thymol,
which exhibit significant bactericidal activity when tested separately (Juven et al., 1994; Ultee et al.,
1998; Lambert et al., 2001; Friedman et al., 2002). Due to their hydrophobic nature, carvacrol and
thymol interact with the lipid bilayer of cytoplasmic membranes causing loss of integrity and leakage
of cellular material such as ions, ATP and nucleic acid (Helander et al., 1998; Ultee et al., 1999; Lambert
et al., 2001; Trombetta et al., 2005).
Studies on the antibacterial mechanism of phenolic compounds found in essential oils focused on
their effects on the cellular membrane, changing its structure and permeability (Shetty et al., 2004). Lin
et al. (2004) stated that the damage to cell membrane might explain the observed effects, since phenolics
could cause sublethal injury to cell membranes, causing disruption of proton motive force due loss of
H+-ATPase. This could make bacteria more susceptible to acid environment. Moreover, at low pH, the
hidrophobicity of an essential oil increases, enabling it to more easily dissolve in the lipids of the cell
membrane of target bacteria (Juven et al., 1994).
Although the antibacterial activity of oregano EO is more pronounced against Gram-positive than
Gram-negative bacteria (Marino et al., 2001).
Overall, we can conclude that oregano EO is one of the most promising natural compounds that
can be used to develop safer antibacterial agents and that its effective combination with antibacterials
may be used in the future to treat diseases caused by Staphylococcus aureus.
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Figure 1. Aromatograms: qualitative effects of the essential oil of oregano observed on the strains of
Staphylococcus aureus by diffusion from impregnated discs on agar medium. (A) MRSA 1; (B) MRSA
2; (C) MRSA 3; (D) MRSA 4; (E) MRSA 5; (F) MRSA 7; (G) MRSA 6; (H) MRSA 8.
Conclusion
Plant extracts have great potential as antimicrobial compounds against microorganisms. Thus,
they can be used in the treatment of infectious diseases caused by resistant microbes.
The results ofthis study suggest that O. vulgare essential oil possesses antibacterial properties due
to its richness in thymol. It gave inhibition zone diameters in the range of 9.9-31.9 mm and MIC ranging
from 0.314 to 0.628mg/ ml.
This EO could be used as bacterial growth inhibitory agent in new drugs in therapy of
Staphylococcus diseases. Although this study has investigated in vitro antibacterial activity, extensive
in vivo studies could confirm the potential usefulness of this plant's essential oil in combating
antimicrobial resistance.
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