This study investigated the anti-pseudomonal effect of Argan oil on Pseudomonas aeruginosa isolates recovered from burn patients. The isolates showed high resistance to several antibiotics. Argan oil alone and hydrogen peroxide alone had no effect, but their combination did show activity against P. aeruginosa. Mixing Argan oil and hydrogen peroxide in ratios of 1:1, 2:1, and 1:2 resulted in inhibition zones of 23.8 mm, 24.6 mm, and 23.1 mm respectively, demonstrating the anti-pseudomonal potential of the combined compound. The study suggests Argan oil could be developed as an alternative treatment when combined with hydrogen peroxide.

1. Ph ton 270
The Journal of Infectious Diseases. Photon 113 (2014) 270-276
https://sites.google.com/site/photonfoundationorganization/home/the-journal-of-infectious-diseases
Original Research Article. ISJN: 4379-2173: Impact Index: 6.34
The Journal of Infectious Diseases Ph ton
Anti-Pseudomonal Effect of Argan Oil on Pseudomonas aeruginosa
Recovered from Burn Patients in Hilla City, Iraq
Dr. Habeeb Sahib Nahera
*, Hussein Oleiwi Al-Dahmoshib
, Noor Salman Al-Khafajib
, Anwar
Kadhim AL-Saffarb
, Hussein Kadhim AL-Saffarc
a
Dept. Microbiology, College of Medicine, Babylon University, Hillah, Iraq
b
Dept. Microbiology, College of Sciences, Babylon University, Hillah, Iraq
c
Student of Iraqi Board for Medical Specialization, Ministry of Health, Iraq
Article history:
Received: 11 April, 2014
Accepted: 15 April, 2014
Available online: 05 July, 2014
Keywords:
Argan oil, Hydrogen Peroxide, antipseudomonas
compound
Corresponding Author:
Naher H.S.*
Professor
Email: habeebnaher@yahoo.com
Phone: 009647801237560
Al-Dahmoshi H.O.
Lecturer
Email: dr.dahmoshi@yahoo.com
Al-Khafaji N.S.
Assistant Lecturer
Email: noor_micro@yahoo.com
AL-Saffar A. K.
Assistant Professor
Email: anwaralsafar78@gmail.com
AL-Saffar H. K.
Email: husseinkadhim06@gmail.com
Abstract
The goal: This study aimed in part to develop a
novel compound to treat the pseudomonal
infections. Materials: Argan Oil (obtained from
Morocco) and Hydrogen Peroxide (H2O2). Bacterial
isolates: An ideal isolate of Pseudomonas
aeruginosa obtained from Central Laboratory in
Hilla City, Iraq was used as a test organism in this
study. Experimental work: Bacterial isolates,
obtained from the Central Laboratory in Hilla City,
Iraq were preliminary diagnosed in this Lab. as
Pseudomonas aeruginosa, being isolated from
sever skin and burns infections and were diagnosed
as multi-drugs resistant bacteria to antibiotics.
Those isolates were reidentified in our Lab. for
confirmation. The test bacterial isolates were
treated with; H2O2 (1.5%) alone, Argan oil alone
and with physiological saline as a control (by using
the wells in agar method). In other experiment those
isolates were treated with three compounds being
prepared by mixing Argan oil and H2O2 in a rate of
1:1, 2:1 and 1:2 respectively. The treated isolates
were incubated at 37° C for 24 hr. No effect on
bacterial activities and vitality were observed in this
experiment. Results: No effect regarding the growth
and viability of bacterial isolates was observed
when those isolates were treated with Argan alone
and H1O2 alone as well while considerable killing
effect was recorded when Pseudomonas
aeruginosa treated with those agents when
combined with each other, since the diameter of
inhibition zones recorded as high as 23.8 mm., 24.6
mm. and 23.1 mm. for the mixtures of Argan: H2O2
in 1:1, 2:1 and 1:2 respectively. Conclusion:
Combination of Argan, a natural plant product with
Hydrogen Peroxide in defined concentrations
resulted in a combined compound be potentially
active against Pseudomonas aeruginosa. In
comparison with some of traditional antibiotics, e.g.
Amikacin, Tobramycin, Ceftazidime, Aztreonam,
Norfloxacin and Gentamycin. Future prospect:
Experimentation the antimicrobial effects of
plants/herbs products as an alternative for the
current traditional antibiotics. Toxicity of these
products can be investigated as well. Employing of
plants can be of great value as plants/herbs are
available worldwide, cheap and safe for human use.
Citation:
Naher H.S., Al-Dahmoshi H.O., Al-Khafaji N.S., AL-Saffar
A.K., AL-Saffar H.K., 2014. Anti-Pseudomonal Effect of
Argan Oil on Pseudomonas aeruginosa Recovered from
Burn Patients in Hilla City, Iraq. The Journal of Infectious
Diseases. Photon 113, 270-276.
All Rights Reserved with Photon.
Photon Ignitor: ISJN43792173D687605072014

2. Ph ton 271
1. Introduction
1.1. Thermal injuries of skin
Burns are one of the most common and
devastating forms of trauma. Patients with
serious thermal injury require immediate
specialized care in order to minimize morbidity
and mortality (National Center for Injury
Prevention and Control, 2002).
Thermal destruction of the skin barrier and
concomitant depression of local and systemic
host cellular and humoral immune responses
are pivotal factors contributing to infectious
complications in patients with severe burns.
Although burn wound surfaces are sterile
immediately following thermal injury, these
wounds eventually become colonized with
microorganisms (Erol et al., 2004). Bacteria
rapidly colonize open skin wounds after burn
injury. Microorganisms colonizing the burn
wound originate from the patient’s
endogenous skin and gastrointestinal and
respiratory flora. Microorganisms may also be
transferred to a patient’s skin surface via
contact with contaminated external
environmental surfaces, water, fomites, air,
and the soiled hands of health care workers.
1.2. Burns infection
Endogenous gram-negative bacteria from the
patient’s gastrointestinal flora can rapidly
colonize the burn wound surface in the first
few days after injury (Ramzy, 2000). While
Staphylococcus aureus remains a common
cause of early burn wound infection,
Pseudomonas aeruginosa from the patient’s
endogenous gastrointestinal flora and/or an
environmental source is the most common
cause of burn wound infections (Altoparlak,
2004). Despite new techniques and solution
for sterilization and disinfection but P.
aeruginosa still the common nosocomial
infections (Al-Dahmoshi, 2013).
1.3. Control
Silver sulfadiazine has excellent broad-
spectrum antibacterial coverage against
Pseudomonas aeruginosa and other gram-
negative enteric bacteria, although some
bacterial resistance has recently been
reported towards this drug. Also Mafenide
acetate (Sulfamylon) cream has a broad
spectrum of activity against gram-negative
bacteria, particularly P. aeruginosa (Heggers,
et al., 2002).
1.4. Argan oil
Argan oil is plant oil produced from the kernels
of the Argan tree (Argania spinosa L.) that is
endemic to Morocco. It is used for nutritive and
cosmetic properties. The tree is extremely well
adapted to drought and other environmentally
harsh conditions of Southwestern Morocco.
The genus Argania is now endangered and
under protection of UNESCO (Biosphere
Reserve Information, 2007). Argan oil remains
one of the rarest oils in the world due to the
small and very specific growing areas. The
fruits of the Argan tree are nut-sizes and may
be round, oval or conical in shape. The fruits
are covered by a thick peel which covers the
fleshy pulp. The pulp surrounds a hard-shelled
nut which represents approximately 25% of
the weight of the fresh fruit. Argan oil is
extracted from the kernels, with yields varying
from 30% to 55% depending on the extraction
method used (Khallouki et al., 2003). Argan oil
consists of 42.8% oleic acid, 36.8% linoleic
acid, 12% palmitic acid, 6% stearic acid and
0.5% Linolenic acid (Charrouf and Guillaume,
2008). Argan oil has a relative density at 20° C
(68° F) ranging from 0.906 to 0.919
(Monfalouti et. al., 2010). It contains
tocopherols (vitamin-E), phenols, carotenes,
squalene, fatty acids, (80% unsaturated fatty
acids), caffeic acid, oleuropein, vanillic acid,
and it is more resistant to oxidation than olive
oil (Charrouf and Guillaume, 2007).
Virgin Argan oil of edible or beauty grade is
composed of 99-percent acylglycerides
(primarily triglycerides). Unsaponifiable matter,
which represents the remaining one percent, is
composed of carotenes, tocopherols,
triterpene alcohols, sterols, and xanthophylls
(Charrouf and Guillaume, 1999). Fatty acids
that compose acylglycerides are principally
oleic and linoleic acid, 43-49 percent and 29-
36 percent, respectively. Palmitic acid and
stearic acid are saturated fatty acids found at a
concentrations of 11-15 percent and 4-7
percent, respectively (Rahmani, 2005). Some
Argan oil with pharmacological properties are
likely to result from its high unsaturated fatty
acid content. Oleic acid, a monounsaturated
fatty acid, has numerous therapeutic effects
(Lopez-Huertas, 2010) that contribute to the
important properties of Argan oil. Because a
linoleic acid deficiency can induce poor wound
healing (Galli and Calder, 2009). The high
linoleic acid content of Argan oil may
contribute to its traditional indication as a cure
for skin inflammation. Many of Argan oils,
specific health benefits are attributed to its
composition of unsaponifiable matter and high
tocopherol content (Charrouf and Guillaume,
2008). The tocopherol content of Argan oil is

3. Ph ton 272
620 mg/kg, compared to 320 mg/kg in olive oil.
Tocopherols are molecules with strong
antioxidant and free radical scavenging
properties. Gamma-Tocopherol, the most
efficient free radical scavenger of all
tocopherols composes 69% of Argan oil total
tocopherol content (Jiang et. al., 2001).
Because tocopherols and sterols can act
synergistically, the specific combination of
molecules found in the unsaponifiable matter
is theorized to contribute to the therapeutic
aspects of Argan oil (Monfalouti et al., 2010).
Many studies on Argan oil confirmed its
activity as anti-sebum (Dobrev, 2007),
antiproliferative Aactivity (Trichopoulou et al.,
2000; Owen et al., 2000; Khallouki et al.,
2003), antioxidant and hypocholesterolemic
effect (Drissi et al., 2004) and antidiabetic
activity (Samane et al., 2009). According to
our Knowledge, the antibacterial (anti-
pseudomonal) activity of Argan oil was not
investigated until yet and therefore the current
study was suggested and designed to
determine the effect of Argan oil on the Multi-
drugs resistant P. aeruginosa.
2. Materials and Methods
2.1. Bacterial isolates
Thirty five identified pseudomonas aeruginosa
isolates, recovered from burned patients, were
received from the Central Lab. of Hillah
Teaching Hospital, Iraq during a period from
October 2013 to February 2014. All isolates
were confirmed using VITEK 2 compact
system VITEK 2 GN card.
2.2. Susceptibility Test
Antibiotics susceptibility test performed for
Ampicillin, Carbenicillin, Co-timoxazole,
Tetracyclin and Cefuroxime using discs
method according to guidelines of (CLSI,
2012). Also the anti-pseudomonal effect of
Argan oil was tested using well diffusion
techniques. The P. aeruginosa suspension
was standardized with 0.5 McFarland
Nephelometer Standards and streaked on
Muller Hinton agar plate and the well were
made using cork borer. Fifty microliter of
Argan oil alone, 1.5% H2O2 alone and an
aqueous mixtures of Argan oil: 1.5% H2O2
(1:1, 2:1 and 1:2) were dropped in the well and
incubated at 37˚ C for overnight then the
diameter of inhibition zone (mm) was
recorded.
3. Results and Discussion
3.1 Susceptibility to antibiotics
The data reached by this study revealed that
P. aeruginosa isolates exhibited high level of
resistance against each of Ampicillin,
Carbenicillin, Co-trimoxazole, Tetracyclin and
Cefuroxime since the sensitivity values were
(97.14%, 97.14%, 94.28%, 85.71% and
82.85%) respectively. On the other hand, low
level of resistance displayed for Amikacin
(0.00%), Tobramycin (5.71%), Ceftazidime
(5.71%), Aztreonam (11.42%), Norfloxacin
(17.14%) and Gentamycin (34.28%) figure 2.1.
These results were in accordance with Al-
Dahmoshi, 2013 who found that the overall
MAR index of the burn P. aeruginosa isolates
was (0.4). Our results were in agreements with
those presented by other studies in which the
resistance to co-trimoxazole, 94.7% - 90.9%,
Tetracyclin 81.8%, and Carbenicillin 98.4%
(Moazami-Goudarzi and Eftekha, 2013, Garba
et al., 2012). The burn and wound infection is
considered as one of the major health
problems worldwide, and one of the most
frequent and severe complications in patients
who have sustained burn (Zogani et al., 2002).
P. aeruginosa is a common cause of wound
infections, especially of thermal burns, this is
because burns have large exposed areas of
dead tissues free of any defenses and
therefore, are ideal sites for infection by
bacteria from environment or normal
microbiota. The antibiotics susceptibility profile
of P. aeruginosa revesled high resistance for
more than four antibiotics of different groups
used in this study indicates that this bacterium
is a multi-drugs resistance (MDR). Such high
antimicrobial resistance is probably promoted
due to selective pressure exerted on bacteria
due to numerous reasons, e.g. an excessive
and indiscriminate use of broad-spectrum
antibiotics (Garba et al., 2012). The inordinate
accessibility of antibiotics in shops and open
markets from different origins of poor quality
and ineffective for treatment as well as
consumption of drugs without proper medical
prescription certainly will increase the risk of
emergence of MDR bacteria. Furthermore,
misuse of antibiotics and relaxation in general
hygienic measures are associated with
increasing infections with these bacteria
(Saleh, 2012; Bowler et al., 2001).
3.2 Susceptibility to Argan oil-H2O2 compound
The results Regarding the Anti-pseudomonal
effect of Argan oil indicated that there was no
any considerable effect for Argan oil alone and
Hydrogen Peroxide alone as well compared
with the control, while when these two

4. Ph ton 273
substances combined with each other
properly, a potential compounds can be
obtained (figure 3.2). Combination of Argan oil:
Hydrogen Peroxide in 1:1, 2:1 and 1:2 resulted
in an inhibition zone of 23.8 mm, 24.6 mm.
and 23.1 mm. diameter as shown in figure 3.2.
All isolates of P. aeruginosa tested in this
study exhibited a remarkable sensitivity to
Argan oil-H2O2 compound although the
inhibition zones were close with each other for
all concentrations.
Results shown in figure 3 indicate the Argan
oil-H2O2 compounds efficiently defeat the
action of Ampicillin, Carbenicillin, Co-
trimoxazole, Tetracyclin and Cefuroxime, while
strongly compete other potent antibiotics
represented by Gentamycin, Norfloxacin,
Aztreonam, Ceftaxidime, Tobramycin and
Amicacin as illustrated in figure 4.
In conclusion, this study settles the activity of
Argan oil as anti-pseudomonal agent when
used in combination with Hydrogen Peroxide
which can be used externally as cream to treat
the wounds and burns infections with some
additional studies can be carried out in future.
Figure 1: Percentage of Antibiotics Resistance of P. aeruginosa isolates towards a group of antibiotics
Figure 2: Mean of inhibition zone (mm) of Argan oil-H2O2 mixture on P. aeruginosa

5. Ph ton 274
Figure 3: Comparison the activity of Argan oil – H2O2 compound with five traditional antibiotics against P.
aeruginosa
Figure 4: Sensitivity values P. aeruginosa towards of Argan oil mixture on P. aeruginosa isolates in compares
with high level sensitivity antibiotics
Conclusion
In conclusion, this study settles the activity of
Argan oil as anti-pseudomonal agent when
used in combination with Hydrogen Peroxide
(H2O2) which can be used externally as cream
to treat the wounds and burns infections.
Research Highlights
The idea of this project revolves around the
following:
1. Detection a novel agent for killing/inhibition
the multi-drugs resistant Pseudomonas
aeruginosa.
2. Substitution the traditional chemical drugs
causing side effects by natural products and
safe for human use.

6. Ph ton 275
3. Most bacterial types developed high
resistance to almost all antibiotics during the
repeated use, therefore, it is of great
importance to develop new antibacterial
compounds from new sources.
4. The economic value is taken into
consideration in this study as antibacterial
agents of plants/herbs sources cheap and
widely available all around the world.
Limitations
We aspire to investigate the effects of Argan
oil in an in vitro and in vivo but it was quite
difficult to obtain animals (rabbits), the reason
which prevents the completion of the in vivo
experiments. Obtaining Argan oil from
Morocco took a long time. There was no
enough time for following up the expiration
date of the compound.
Recommendations
1. Workout the Argan oil on other
microorganisms rather Pseudomonas.
2. Using other additive regents synergistically
react with Argan oil rather than Hydrogen
Peroxide.
3. Using Lab. animals to detect the effects of
Argan oil in an in vivo. Toxicity of this
substance can be also detected in such
experiments.
4. The expiration date of the compound can be
followed up.
Acknowledgement
We are thankful to Prof. Dr. Ali Khair Alla
(dean), College of Medicine, University of
Babylon for his financial support. We are also
grateful to Assistant Prof. Dr. Kadgim H.
Chaloop (surgeon) for his valuable guidance in
obtaining bacterial isolates. We express our
sincere gratitude to Prof. Dr. Mohammed Sabri
(head), Department of Microbiology,College of
Medicine, Babylon University, Iraq for
providing the necessary laboratory facilities.
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