Effect of Moringa oleifera Seed Oil on Antimicrobial Activity of some Antibiotics against some Pathogenic Gram Negative Bacteria

Abstract

Several reports had focused on the antimicrobial activity of Moringa oleifera oil against pathogenic microorganisms but none of these reports had studied the antimicrobial activity of combinations between M. oleifera oil and antibiotic against Gram negative bacteria. In the present study, antimicrobial efficacy of M. oleifera oil alone and combined with antibiotic is studied by agar diffusion method. The results revealed that the antibacterial activity of M. oleifera oil against E. coil, Klebsiella sp. Pseudomonas sp. and Proteus sp. is weak or not existed. In addition, the sensitivity of tested bacteria to some tested antibiotics had increased in medium contained M. oleifera oil depending on tested bacteria and antibiotic, as well as the concentration of M. oleifera oil. In addition, the sensitivity of all tested bacteria to imipenem (IMP) had significantly increasedin medium contained M. oleifera oil. While, the sensitivity of Klebsiella sp. and Proteus sp. to chloramphenicol (C) had significantly decreased in medium contained M. oleifera oil, but it had significantly increased with E. coli. Moreover, addition of M. oleifera oil to the medium had significantly increased the sensitivity of E. coli & Klebsiella; E. coil & Pseudomonas and E. coil & Proteus tomeropenem (MEM), cefixime (CFM) and ertapenem (ETP) & doxycycline (DO), respectively. Furthermore, the antimicrobial activity of amikacin (AK) & gentamicin (EN) in medium contained M. oleifera oil had significantly increased only against Proteus & Pseudomonas. Thus, M. oleifera oil could be used as antibiotic resistant modifying agent against multi-drug resistant Gram negative bacteria.

Full text

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
140
Original Research Article
Effect of Moringa oleifera Seed Oil on Antimicrobial Activity of some
Antibiotics against some Pathogenic Gram Negative Bacteria
Wael Mohamed Abu El-Wafa* and Walaa Said Mohamed Abd El-All
National Organization for Drug Control and Research, Giza, Egypt
*Corresponding author
A B S T R A C T
Introduction
During the last decades, the limit of
microbial diseases and infections has been
exceeded dramatically. A major problem in
antimicrobial chemotherapy is the increasing
occurrence of resistance to antibiotics,
which leads to the insufficiency of
antimicrobial treatment.
The overuse of antibiotics and consequent
antibiotic selection pressure is thought to be
the most important factor contributing to the
appearance of different kinds of resistant
microbes (Ang et al., 2004; Sokovi et al.,
2010; Bajpai et al., 2013).
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 140-151
http://www.ijcmas.com
Several reports had focused on the antimicrobial activity of Moringa oleifera oil
against pathogenic microorganisms but none of these reports had studied the
antimicrobial activity of combinations between M. oleifera oil and antibiotic
against Gram negative bacteria. In the present study, antimicrobial efficacy of M.
oleifera oil alone and combined with antibiotic is studied by agar diffusion method.
The results revealed that the antibacterial activity of M. oleifera oil against E. coil,
Klebsiella sp. Pseudomonas sp. and Proteus sp. is weak or not existed. In addition,
the sensitivity of tested bacteria to some tested antibiotics had increased in medium
contained M. oleifera oil depending on tested bacteria and antibiotic, as well as the
concentration of M. oleifera oil. In addition, the sensitivity of all tested bacteria to
imipenem (IMP) had significantly increasedin medium contained M. oleifera oil.
While, the sensitivity of Klebsiella sp. and Proteus sp. to chloramphenicol (C) had
significantly decreased in medium contained M. oleifera oil, but it had significantly
increased with E. coli. Moreover, addition of M. oleifera oil to the medium had
significantly increased the sensitivity of E. coli & Klebsiella; E. coil &
Pseudomonas and E. coil & Proteus tomeropenem (MEM), cefixime (CFM) and
ertapenem (ETP) & doxycycline (DO), respectively. Furthermore, the antimicrobial
activity of amikacin (AK) & gentamicin (EN) in medium contained M. oleifera oil
had significantly increased only against Proteus & Pseudomonas. Thus, M. oleifera
oil could be used as antibiotic resistant modifying agent against multi-drug resistant
Gram negative bacteria.
Ke yw ords
M. oleifera,
Gram negative,
resistance,
imipenem,
chloramphenicol

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
141
Natural products isolated from various
medicinal plants have traditionally been the
most common source of drugs and still
represent more than 30% of the current
pharmaceutical markets (Jabar and Al-
Mossawi, 2007; Fakurazi et al., 2012 and
Kumar et al., 2012). Moringa oliefera is an
ancient tree that is historically known to
possess numerous medicinal qualities
(Posmontier, 2011) and it’s a native to the
sub-Himalayan parts of India, Pakistan,
Bangladesh and Afghanistan. This rapidly-
growing drumstick tree was utilized ancient
Romans, Greeks and Egyptians and has
become widely cultivated and naturalized in
many locations in the tropics and sub
tropics, West, East and South Africa, Latin
America, the Caribbean, Florida and the
Pacific Islands. Recently, many
investigations pointed to the antimicrobial
properties of the various parts of M. oleifera
roots, flowers, bark, stem and seeds against
various pathogenic microorganisms,
especially Gram negative bacteria (Lockett
et al., 2000; Ghebremichael et al., 2005;
Anwar and Rashid, 2007; Rahman et al.,
2009; Walter et al., 2011). Present study was
planned to detect the effect of M. oleifera oil
on antimicrobial activity of some antibiotics
against pathogenic Gram negative bacteria.
Materials and Methods
Microorganisms and plant material
Clinical strains of Gram negative bacteria
including Escherichia coli, Klebsiella sp.
Proteus sp. and Pseudomonas aeruginosa
were obtained from Al Borg Laboratories,
Mohandeseen, Giza, Egypt during
November, 2013. Tested strains were
confirmed their identification before study
using the key proposed by Barrow and
Feltham (2003). Tested bacterial cultures
were maintained on nutrient agar slants at
4oC throughout the study and used as stock
cultures. M. oleifera oil was purchased from
Pure Life Company for Agricultural
Investment, Giza, Egypt.
Media and antimicrobial agents
Muller-Hinton agar medium (MHA),
Nutrient agar medium (NA), antimicrobial
agent disks including: Ampicillin
(AMP)10µg, Cefepime (FEP) 30µg,
Cefixime (CFM) 5µg, Ceftriaxone (CRO)
30µg, Ertapenem (ETP) 10µg, Imipenem
(IPM) 10µg, Meropenem (MEM)10µg,
Amikacin (AK) 30µg, Gentamicin (EN)
10µg, Doxycycline (DO) 30 µg,
Ciprofloxacin (CIP) 5µg, Levofloxacin
(LVX) 5µg, Norfloxacin (NOR) 10µg,
Nalidixic acid (NA) 30 µg and
Chloramphenicol (C) 30µg/disk were
purchased from Oxoid Ltd. Co. and Tween
20 was purchased from Sigma Chemicals
Company (St. Louis, Mo, USA).
Preparation of bacterial inoculum
Bacterial suspension of various tested
clinical strains was prepared by direct
colony suspension method as follow:
appropriate number of separated colonies
were picked up from NA fresh culture plate
(previously inoculated with single colony of
tested strain and incubated for 24h at 37oC),
suspended with sterile saline solution and
adjusted their inoculum to a turbidity
equivalent to 0.5 McFarland standard.
Antibacterial activity of M. oleifera oil
Antibacterial activity of M. oleifera oil
against various tested clinical bacterial
isolates was studied by agar well diffusion
method according to Parez et al. (1990)
using 200µL of M. oleifera oil for each well.
After 24h of incubation at 37ºC, all plates
were observed for zones of growth
inhibition, and the diameter of these zones

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
142
was measured in millimeters. All tests were
performed in triplicate and the antibacterial
activity was expressed as the mean of
inhibition diameters (mm) produced.
Detection of synergetic interaction
between M. oleifera oil and antibiotics
Sterile Mueller-Hinton agar plates
containing 0.125,0.25, 0.5, 1.0 and
2.0ml/100 ml of M. oleifera oil were
prepared by adding M. oleifera oil to melted
MHA, cold to 45–55oC and supplemented
with 0.5% (v/v) Tween 20. In addition, the
same previous agar plates without M.
oleifera oil were used in the present study as
control.
A sterile cotton wool swab dipped into the
bacterial suspension was spread evenly on
the surface of previous MHA plates. The
inoculated plates were allowed to dry before
placing the diffusion antibiotic disks.
Susceptibility of 4 tested isolates to various
tested antibiotics was performed by disk
diffusion method as described by Clinical
and Laboratory Standards Institute (CLSI,
2011). Using commercially available
antibiotic disks containing AMP (10µg),FEP
(30µg), CFM (5µg), CRO (30µg), ETP
(10µg), IPM (10µg), MEM (10µg), AK
(30µg), EN (10µg), DO (30µg), CIP (5µg),
LVX (5µg), NOR (10µg), NA (30µg) and C
(30µg) were placed on the surface of the
inoculated MHA plates with Escherichia
coli, Klebsiella sp. or Proteus sp. while FEP
(30µg), IPM (10µg), MEM (10µg), AK
(30µg), EN (10µg), CIP (5µg), LVX (5µg)
and NOR (10µg) were placed on the
surface of the inoculated MHA plates with
P. aeruginosa. The inoculated plates were
then incubated at 37 °C for 24 h. Inhibition
zone diameters were measured inclusive of
the diameter of the disks (three replicates
were applied for each test). Results were
expressed as sensitive, intermediate and
resistant according to CLSI, (2011). Data
were subjected to analysis of variance
(ANOVA) using SPSS at 5% level of
significance and means values were
compared using a least significant difference
(LSD).
Result and Discussion
Antibacterial of M. oleifera oil against
various clinical tested strains was weak or
not existed against various tested strains.
The efficacy of M. oleifera oil combined
with antibiotics was studied by agar
diffusion method. Data presented in Table 1
showed that the effect of Moringa oleifera
oilon antibacterial activity of various tested
antibiotics against E. coli was divers
depending on the antibiotic used and the
concentration of M. oleifera oil. In the case
of beta-lactam and cephlosporin antibiotics,
the antibacterial activity of variuos tested
beta-lactam and cephlosporin antibiotics had
significantly increased with in
mediumcontaned M. oleiferaoil, except
AMP and CFM antibiotics. In addition,
0.125% (v/v) of M. oleifera oil was the most
suitable concentration for significant
increasing of antimicrobial activity of CRO
and IPM antibiotics against tested strain
compared to control (Table 1), which
increased their antimicrobial activities to
25.7 and 6%, respectively.
Also, 1.0 % (v/v) of the tested oil was the
most suitable concentration for significant
increasing of antimicrobial activity of ETP
and MEM antibiotics against tested strain
compared to control, which exceeded the
antimicrobial activity of control by 20.3 and
8.6%, respectively. In addition, 2.0% (v/v)
of the tested oil was the most suitable
concentration for significant increasing of
antibacterial activity of FEP antibiotic
against tested strain compared to control,

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
143
which exceeded the antimicrobial activity of
control by 29.5% (Table 1 and Fig. 2).
In the case of chloramphenicol and
doxycycline, the antimicrobial activity of
each tested antibiotics had significantly
increased in medium contained 0.5 and 1.0%
(v/v) of M. oleifera oil, respectively, which
exceeded the antimicrobial activity of
control by 41.9 and 13.41%, respectively.
On the other hand, addition of M. oleifera
oil in tested medium did not give any
significant change on antimicrobial activity
of tested aminoglycoside (AK and EN) or
fluoroquinolone antibiotics (CIP, LVX,
NOR and NA) compared to control (Table 1
and Fig. 1).
From previous results, it could be concluded
that the sensitivity of E. coli to
chloramphenicol, doxycycline and most of
cephalosporin antibiotics had significantly
increased in medium contained M. oleifera
oil, but the level of sensitivity was
influenced by the type of tested antibiotic
and the concentration M. oleifera oil. In
contrast, the sensitivity of E. coli to
aminoglycoside, fluoroquinolone and some
beta lactam antibiotics (AMP and CFM) had
not significantly changed in medium
contained M. oleifera oil compared to
control.
Data presented in Table 2 detect that the
effect of M. oleifera oil on antibacterial
activity of various tested antibiotics against
Klebsiella sp. was divers depending on the
antibiotic used and the concentration of M.
oleifera oil. In the case of beta-lactam and
cephalosporin antibiotics, the antibacterial
activity of MEM and IMP against tested
strain had significantly increased in medium
contained 1.0 and 2.0 % (v/v), respectively,
which exceeds the antimicrobial activity of
control by 33.34 and 32.23%, respectively
(Fig. 2). While, the antimicrobial activity of
other tested beta-lactam and cephalosporin
antibiotics against tested strain had not
changed in medium contained M. oleifera
oil.
In the case of aminoglycoside antibiotics,
addition of M. oleifera oil 1% (v/v) in tested
medium gave a significant increase of
antibacterial activity against tested strain
with AK, which exceeded the antimicrobial
activity of control by 18.68%. While, the
antimicrobial activity of EN against tested
strain had not changed in medium contained
M. oleifera oil (Table 2 and Fig. 2).
In the case of fluoroquinolone antibiotics,
addition of M. oleifera oil (0.125, 1.0 and
2.0 %, v/v) to the medium gave a significant
increase in the antimicrobial activity against
tested strain with LVX, NA and NOR,
respectively, which exceeded the
antimicrobial activity of control by 31.46,
35.46 and 46.65%, respectively. On the
other hand, the sensitivity of tested strain to
chloramphenicol had significantly decreased
in medium contained 0.5% (v/v) of M.
oleifera, which reduced the antimicrobial
activity to 16.1 % compared to control.
While, the antimicrobial activity of DO
against tested strain had not changed in
medium contained M. oleifera oil (Table 2
and Fig. 2).
From previous results, it could be
summarized that the sensitivity of Klebsiella
sp. to most tested antibiotics in medium
contained M. oleifera oil was not changed.
While, the sensitivity of tested strain to
fluoroquinolone (except CIP) and some
cephalosporins including MEM and IMP
had significantly increased in medium
contained M. oleifera oil. Furthermore,
addition of M. oleifera oil to the medium
had significantly reduced the sensitivity of
tested strain to chloramphenicol.

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
144
Data recorded in Table 3 showed that the
change of antibacterial activity of various
tested antibiotics against Proteus sp. was
divers depending on the antibiotic used and
the concentration of M. oleifera oil. In the
case of beta-lactam and cephalosporin
antibiotics, the antibacterial activity of IMP
and ETP against tested strain had
significantly increased in medium contained
0.125 and 2.0 % (v/v), respectively, which
exceeded the antimicrobial activity of
control by 97.80 and 54.19%, respectively
(Fig. 3). While, the antimicrobial activity of
other tested beta-lactam and cephalosporin
antibiotics against tested strain had not
changed in medium contained M. oleifera
oil.
In the case of aminoglycoside antibiotics,
addition of M. oleifera oil (1.0 and 2.0 %,
v/v) in tested medium gave a significant
increasing of bacterial sensitivity to EN and
AK, respectively, which exceeded the
antibacterial activity of control by 4.84 and
56.25%, respectively. Also, addition of
1.0ml of M. oleifera oil to the medium had
significantly increased the bacterial
sensitivity to doxycycline against tested
strain, which exceeded the antibacterial
activity of control by17.17% (Table 3 and
Fig. 3). On the other hand, the sensitivity of
tested strain to chloramphenicol had
significantly decrease in medium contained
0.125 % (v/v) M. oleifera, which reduced
the antibacterial activity to 12.48 %
compared to control. While, the antibacterial
activity of fluoroquinolone antibiotics
against tested strain had not changed in
medium contained M. oleifera oil (Table 3
and Fig. 3).
From previous data, it could be concluded
that the addition of M. oleifera to the
medium had increased the sensitivity of
Proteus sp. to tested aminoglycosides,
doxycycline and some cephalosporins (IMP
and ETP). While, the sensitivity of tested
bacteria to fluoroquinolones, most of beta-
lactam and cephalosporin antibiotics had not
changed. Furthermore, addition of M.
oleifera oil to the medium had significantly
reduced the sensitivity of tested strain to
chloramphenicol.
Data current in Table 4 show that the
sensitivity of Pseudomonas sp. to most
tested antibiotics had significantly increased
but with different levels based on the type of
antibiotic used and the concentration of M.
oleifera oil. In case of cephalosporin
antibiotics, the sensitivity of tested bacteria
to IPM and FEP had significantly increased
in medium contained 0.125 and 1.0% (v/v),
which increased the antimicrobial activities
of tested antibiotics against tested strain to
46.31 and 77.4%, respectively (Fig.
4).While, the antimicrobial activity of MEM
against tested strain had not changed in
medium contained M. oleifera oil.
In the case of aminoglycoside antibiotics,
the sensitivity of tested bacteria to AK and
EN had significantly increased in medium
contained 0.25 and 1% (v/v), which
increased the antimicrobial activities of
tested antibiotics to 46.31 and 77.4%,
respectively, the sensitivity of tested bacteria
to NOR had significantly increased in
medium contained 0.5% (v/v), which
increased the antimicrobial activity of tested
antibiotics to 77.4%. While, the
antimicrobial activity of CIP and LVX
against tested strain had not changed in
medium contained M. oleifera oil (Table 4
and Fig. 4).
From all abovementioned results, it could be
concluded that the sensitivity of tested
bacteria to various tested antibiotics might
be changed in medium contained M. oleifera
oil depending on tested bacteria and
antibiotic, as well as the concentration of M.

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
145
oleifera oil. In addition, the sensitivity of all
tested bacteria to IPM had significantly
increased in medium contained M. oleifera
oil. Also, the sensitivity of Klebsiella and
Proteus to chloramphenicol has significantly
decreased in medium contained M. oleifera
oil, but it has significantly increased with E.
coli.
In addition, addition of M. oleifera oil to the
medium has significantly increased the
sensitivity of E. coli & Klebsiella; E. coil &
Pseudomonas and E. coil & Proteus to
MEM, FEB and ETP & DO, respectively.
Furthermore, the sensitivity to AK & EN in
medium contained M. oleifera oil had
significantly increased only against Proteus
& Pseudomonas.
Essential oils are valuable natural products
used as raw materials in many fields,
including perfumes, cosmetics,
aromatherapy, phyto-therapy, spices and
nutrition. This has recently attracted the
attention of many scientists and encouraged
them to screen plants to study the biological
activities of their oils from chemical and
pharmacological investigations to
therapeutic aspects (Prashith Kekuda et al.,
2010).
Table.1 Antibacterial1 response to combinations between antibiotics and
M. oleifera oil against E. coli
Antibiotic disks
(Concentration)
M. oleifera oil concentrations % (v/v)
Control2
0.125
0.25
0.5
1.0
2.0
Inhibition zone means3 ±SD (mm)
AMP(10µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
FEP(30µg)
20.33±0.6a
20.33±0.6a
20.33±1.5a
20.00±1.0a
20.67±0.6a
26.33±0.6b
CFM(5µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
CRO(30µg)
24.67±0.6a
29.67±1.7b
31.00±0.0 b
30.67±1.2b
29.00±1.7b
29.00±0.6b
ETP(10µg)
24.67±0.6a
25.33±1.2 a
25.33±0.6 a
25.67±1.2 a
29.67±0.6ab
30.67±0.6 ab
IPM (10µg)
33.33±1.5 a
35.33±0.6 b
35.33±0.6 b
35.33±0.6 b
35.33±0.6b
35.33±0.6b
MEM(10µg)
31.00±1.7a
30.67±1.0a
31.67±0.6a
31.00±1.7a
33.67±1.2b
34.00±1.2b
AK(30µg)
21.67±1.5a
21.33±1.7a
21.33±0.6 a
21.33±1.5 a
22.00±0.6a
22.00±0.6a
EN(10µg)
10.33±0.6a
10.00±0.6a
10.00±0.6 a
10.00±0.00 a
10.33±0.0a
10.33±0.0a
DO(30 µg)
22.33±0.6a
21.33±0.6a
21.00±0.6a
23.33±0.6a
24.67±1.7b
25.33±0.6b
CIP( 5µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
LVX( 5µg)
10.67±0.6a
9.67±0.6a
9.67±1.2a
10.33±0.6a
10.67±0.6a
10.33±0.6a
NOR(10µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0 a
0.00±0.0a
0.00±0.0a
NA (30 µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
C (30µg)
20.67±1.2a
25.00±1.2b
24.67±1.2b
29.33±0.6c
29.33±0.6c
29.33±1.0c
1: Studied by disk diffusion method (CLSI, 2011) using Mueller Hinton agar supplement with M. oleifera oil and
Tween 20 (0.5%, v/v), 2: without M. oleifera, 3: Values in the same raw followed by same letter are not significantly
different according to ANOVA (L.S.D. p ≤ 0.5), SD: Standard division.

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
146
Table.2 Antibacterial1 response to combinations between antibiotics and M. oleifera oil against Klebsiella sp
1: Studied by disk diffusion method (CLSI, 2011) using Mueller Hinton agar supplement with M. oleifera oil and Tween 20 (0.5%, v/v), 2: without M. oleifera,
3: Values in the same raw followed by same letter are not significantly different according to ANOVA (L.S.D. p ≤ 0.5). SD: Standard division.
Antibiotic disks
(Concentration)
M. oleifera oil concentrations % (v/v)
Control2
0.125
0.25
0.5
1.0
2.0
Inhibition zone mean3 ±SD (mm)
AMP(10µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
FEP(30µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
CFM(5µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
CRO(30µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
ETP(10µg)
20.2±0.6a
20.2±1.2a
19.33±0.0a
19.67±0.6a
20.00±1.5a
19.33±0.6a
IPM (10µg)
30.00±1.0a
33.67±0.6b
33.67±1.5b
33.33±1.5b
33.67±0.6b
39.67±0.6c
MEM(10µg)
29.00±1.0a
32.67±1.0b
32.33±0.6b
34±1.7b
38.67±0.6c
38.00±0.6c
AK(30µg)
25.00±1.0a
24.33±1.0a
23.67±0.6a
24.00±1.0a
29.67±0.6b
28.00±1.2b
EN(10µg)
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
0.00±0.0a
DO(30 µg)
13.67±1.2a
13.67±1.0a
14.67±0.6a
15.00±0.0a
14.33±0.6a
13.00±0.6a
CIP(5µg)
20.67±0.6a
21.67±1.2a
20.33±0.6a
20.67±1.2a
20.67±0.6a
20.67±0.6a
LVX(5µg)
23.33±0.6a
30.67±0.6b
29.67±0.6b
29.33±0.6b
29.67±0.6b
29.33±0.6b
NOR(10µg)
20.00±1.0 a
24.33±1.23b
25.33±1.23c
26.23±1.0d
26.33±0.58a
29.33±1.15a
NA (30 µg)
20.6±1.2a
20.67±1.2a
21.00±1.7a
21.33±1.5a
28.00±1.7b
27.67±1.2b
C (30µg)
29.00±1.0a
30.00±0.6a
30.33±1.2a
26.00±1.7b
24.33±1.5b
25.67±1.7 b

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
147
Table.3 Antibacterial response to combinations between antibiotics and M. oleifera oil against Proteus sp
1: Studied by disk diffusion method (CLSI, 2011) using Mueller Hinton agar supplement with M. oleifera and Tween 20 (0.5%, v/v), 2: without M. oleifera, 3:
Values in the same raw followed by same letter are not significantly different according to ANOVA (L.S.D. p ≤ 0.5). SD: Standard division.
Antibiotic disks
(Concentration)
M. oleifera oil concentrations % (v/v)
Control2
0.125
0.25
0.5
1.0
2.0
Inhibition zone mean3 ±SD (mm)
AMP(10µg)
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
19.33±0.0
0.00±0.0 a
FEP(30µg)
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
CFM(5µg)
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
CRO(30µg)
0.00±0.0a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
ETP(10µg)
16.00±1.0 a
24.67±1.0b
24.67±0.6 b
24.33±1.2 b
24.67±0.6 b
26.00±0.6 c
IPM (10µg)
15.0±0.0 a
29.67±0.6 b
29.33±0.6 b
29.33±1.2 b
29.67±1.2b
28.67±0.6 b
MEM(10µg)
29.33±1.2 a
29.33±0.6 a
29.33±1.2 a
29.67±o.6 a
29.33±1.2 a
29.33±1.2 a
AK(30µg)
16.00±1.0 a
16.67±1.0 a
19.00±1.2 b
19.33±1.2 b
19.33±1.7 b
25.00±1.5c
EN(10µg)
20.67±1.2a
20.67±0.0a
20.67±1.5a
20.33±0.6a
21.67±0.6b
21.00±1.2 b
DO(30 µg)
9.67±0.6a
10.33±1.5 a
10.33±0.6 a
10.33±0.6 a
11.33±0.6 b
11.67±0.6 b
CIP(5µg)
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
LVX(5µg)
9.67±0.6 a
10.33±0.6 a
9.67±0.6 a
9.67±o.6 a
9.67±0.6 a
9.67±0.6 a
NOR(10µg)
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
NA (30 µg)
0.00±0.0a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
0.00±0.0 a
C (30µg)
29.33±1.2a
25.67±0.6b
25.67±1.0b
25.33±o.6b
25.33±1.2b
25.33±1.2b

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
148
Table.4 Antibacterial response to combinations between antibiotics and M. oleifera oil against
Pseudomonas sp
1: Studied by disk diffusion method (CLSI, 2011) using Mueller Hinton agar supplement with M. oleifera and
Tween 20 (0.5%, v/v), 2: without M. oleifera, 3: Values in the same raw followed by same letter are not significantly
different according to ANOVA (L.S.D. p ≤ 0.5). SD: Standard division.
Fig.1 Effect of M. oleifera oil on antimicrobial activity of some antibiotic against E. coli
Fig.2 Effect of Moringa oleifera oil on antimicrobial activity of some antibiotic against
Klebsiella sp
Antibiotic disks
(Concentration)
M. oleifera oil concentrations % (v/v)
Control2
0.125
0.25
0.5
1.0
2.0
Inhibition zone mean ±SD (mm)
FEP (30µg)
10.33±0.0a
10.33±0.0a
10.33±0.0a
10.33±0.0a
10.33±0.0a
18.33±0.0b
IPM(10µg)
22.33±1.2 a
32.67±1.5 b
32.67±1.5 b
31.67±1.5b
31.33±0.6b
31.67±0.6b
MEM(10µg)
29.67±0.6 a
30.67±1.2 a
30.33±1.7 a
30.67±0.6 a
31.00±1.5 a
31.33±1.2 a
AK(30µg)
21.67±1.5a
22.33±1.2a
24.67±1.0 b
24.00±1.2b
24.00±0.6b
24.33±1.5b
EN(10µg)
18.33±1.5a
19.33±0.6a
19.33±1.2a
21.00±1.7b
35.33±1.2c
35.67±1.2c
CIP(5µg)
28.33±0.6a
28.67±1.2a
28.67±1.2a
29.33±0.6a
29.33±0.6a
29.33±0.6a
LVX(5µg)
30.67±1.5 a
31.00±1.2 a
30.33±1.5 a
30.33±0.6 a
31.67±1.2 a
30.67±1.7 a
NOR(10µg)
27.00±0.0a
27.67±1.2a
29.33±1.5a
32.67±0.6b
32.67±1.2b
32.33±1.2b

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
149
Fig.3 Effect of M. oleifera oil on antimicrobial activity of some antibiotic against Proteus sp.
Fig.4 Effect of M. oleifera oil on antimicrobial activity of some antibiotic against
Pseudomonas sp.
Although, there are many investigations
revealed the antimicrobial activity of M.
oleifera oil against bacteria and fungi
(Prashith Kekuda et al., 2010 and Marrufo et
al., 2013), but in the present study the
antimicrobial activity of M. oleifera oil
against various tested clinical strains is weak
or not existed and that may be due to the
highly resistant of tested strains to the
contents of M. oleifera oil, while the
sensitivity of various tested bacteria to some
antibiotics was increased in medium
contained M. oleifera oil compared to
control. Obtained results revealed that
antibacterial activity of antibiotics against
some pathogenic bacteria could be increased
in case it combined with other materiel even
it has antibacterial activity or not.
Marrufo et al. (2013) revealed that the
antimicrobial effectiveness of most essential
oil against Gram negative is due to the
phenol compounds. In addition, the
composition of outer membrane of gram
negative bacteria, essential oil can alter not
only such structures but penetrate within the
cell, leading to those alterations, such as the
denaturation of proteins and enzymes, the
“unbalance” of the K + and H + ion
concentration, until the modification of the
entire cell morphology, which can lead
to the death of the microorganisms.

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
150
Furthermore, The molecular mechanism of
action of the essential oil of M. oleifera
is unknown, but the essential oil can
probably inhibit the generation of
adenosine triphosphate from dextrose and
disrupt the cell membrane (Gill and
Holley, 2004). Thus, combination between
M. oleifera oil and antibiotics could increase
the antibiotic activity against resistant
bacteria.
This is the first report concerning the
synergistic effects of M. oleifera oil in
combination with different traditional
antibiotics against most common pathogenic
Gram negative bacteria, which has
emphasized that M. oleifera oil is one of the
most promising natural compounds that can
be used as antibiotic resistance modifying
agent in microorganisms. Further studies are
focused on the active phytochemicals of M.
oleifera oil and their interaction with IMP
and C antibiotics against resistant
pathogenic Gram negative bacteria.
M. oleifera oil could be used as antibiotic
resistant modifying agent against multi-drug
resistant Gram negative bacteria, which can
contribute in some way to overcome of
bacterial resistance to many traditional
antibiotics and therefore can be reused again
especially in developing countries, such as
Egypt.
References
Ang, J.Y., Ezike, E., Asmar, B.I. 2004.
Antibacterial resistance. Indian J.
Pediatr, 71: 229–239.
Anwar, F., Rashid, U. 2007. Physico-
chemical characteristics of Moringa
oleifera seed and oil from a wild
provenance of Pakistan. Pak. J. Bot.,
39(5): 1443–1453.
Bajpai, V.K., Shukla, S., Sharma, A. 2013.
Essential oils as antimicrobial agents.
Natural Prod., Pp. 3975–3988.
Barrow, G.I., Feltham, R.K.A. 2003. Cowan
and steel's manual for the identification
of medical bacteria. Cambridge
University Press, Cambridge. Pp. 50–
150.
CLSI (Clinical And Laboratory Standards
Institute), 2011. Performance standards
for antimicrobial susceptibility testing,
twenty-first informational supplement.
CLSI document M100-S21. Vol. 31,
No.1. Wayne, Pennsylvania, USA. Pp.
42–87.
Fakurazi, S., Sharifudin, S.A., Arulselvan, P.
2012. Moringa oleifera hydroethanolic
extracts effectively alleviate
acetaminophen-induced hepatotoxicity
in experimental rats through their
antioxidant nature. Molecules, 17:
8334–8350.
Ghebremichael, K.A., Gunaratna, K.R.,
Henriksson, H., Brumer, H.,
Dalhammar, G.A. 2005. Simple
purification and activity assay of the
coagulant protein from Moringa
oleifera seed. Water Res., 39(11):
2338–44.
Gill, A.O., Holley, R.A. 2004. Mechanisms
of bactericidal action of
cinnamaldehyde against Listeria.
monocytogenes and of eugenol against
L. monocytogenes and Lactobacillus
sakei. Appl. Environm. Microbiol., 70:
5750–5755.
Jabar, M.A., Al-Mossawi, A. 2007.
Susceptibility of some multiple
resistant bacteria to garlic extract. Afr.
J. Biotechnol., 6(6): 771–776.
Kumar, V., Pandey, N., Mohan, N., Singh,
R.P. 2012. Antibacterial & antioxidant
activity of different extract of Moringa
oleifera leaves- an in vitro study. Int. J.
Pharma. Sci. Rev. & Res., 12(1): 89–
94.
Lockett, C.T., Calvert, C.C., Grivetti, L.E.
2000. Energy and micronutrient
composition of dietary and medicinal

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 140-151
151
wild plants consumed during drought.
Study of rural Fulani, northeastern
Nigeria. Int. J. Food Sci. Nutr., 51(3):
195–208.
Marrufo, T., Nazzaro, F., Mancini, E.,
Fratianni, F., Coppola, R., De Martino,
L., Bela Agostinho, A., De Feo, V.
2013. Chemical composition and
biological activity of the essential oil
from leaves of Moringa oleifera Lam.
cultivated in Mozambique. Molecules,
18: 10989–11000.
Parez, C., Paul, M., Bazerque, P. 1990.
Antibiotic assay by agar well diffusion
method. Acta. Biol. Med. Exp., 15:
113–115.
Posmontier, B. 2011. The medicinal
qualities of Moringa oleifera. Holistic.
Nurs. Pract., 25(2): 80–87.
Prashith Kekuda, T.R., Mallikarjun, N.,
Swathi, D., Nayana, K. V., Aiyar,
M.B., Rohini, T.R. 2010. Antibacterial
and antifungal efficacy of steam
distillate of Moringa oleifera Lam. J.
Pharm. Sci. & Res., 2(1): 34–37.
Rahman, M.M., Sheikh, M.M., Shamima,
A.S., Islam, M.S., Rahman, M.A.,
Rahman, M.M., Alam, M.F. 2009.
Antibacterial activity of leaf juice and
extracts of Moringa oleifera Lam.
Against some human pathogenic
bacteria. CMU. J. Nat. Sci., 8(2): 219.
Sokovi, M., Glamoèlija, J., Marin, P.D.,
Brki, D., Van Griensven, L.J.L.D.
2010. Antibacterial effects of the
essential oils of commonly consumed
medicinal herbs using an in vitro
model. Molecules, 15: 7532–7546.
Walter, A., Samuel, W., Peter, A. Joseph,
O. 2011. Antibacterial activity of
Moringa oleifera and Moringa
stenopetala methanol and n-hexane
seed extracts on bacteria implicated in
water borne diseases. Afri. J.
Microbiol. Res., 5(2): 153–157.