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International Journal of Pharmaceutical Applications ISSN 0976-2639.Online ISSN 2278 – 6023
Vol 3, Issue 3, 2012, pp 387-398 http://www.bipublication.com
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND
AMOXICILLIN AGAINST SOME CLINICAL ISOLATES OF METHICILLIN – RESISTANT Staphylococcus Aureus (MRSA)
Hanan. H. Abd El-Kalek and Eman A. Mohamed
Microbiology Department, National Center for Radiation Research and Technology,
Atomic Energy Authority, Nasr City, Cairo, Egypt. Corresponding author: [email protected]
[Received-23/05/2012, Accepted-17/08/2012]
A B S T R A C T
Screenings of nine medicinal plants (three essential oils and six methanolic herbal extracts) were evaluated as antimicrobial inhibitors against MRSA. Results revealed that, all of tested plants possess a degree of antibacterial activity toward MRSA strains under study, but each of lemon grass oil (LGEO), Cardmomm oil and thymus vulgaris extract possess highly degree of antibacterial activity towards (MRSA), (LGEO) showed better inhibitory effects than others. To determine the antimicrobial activity of (LGEO) and investigate the synergistic effects of this oil combined with Amoxacillin against (MRSA), the inhibitory and bactericidal activities of (LGEO), alone and in combination with Amoxacillin were studied, the minimum inhibitory concentrations (MICs) and The minimum bactericidal concentrations (MBCs) of (LGEO) and Amoxacilin in combination were equal in range of 1.2- 1.4 µg/ml. Transmission electron microscope clearly demonstrated that the combination of (LGEO) and Amoxicillin caused both outer and inner membrane damage and releasing of all cell materials. The antimicrobial activity of combinations of the (LGEO) and Amoxacillin indicated their synergistic effects against all MRSA under study. As the present work clearly demonstrates that (LGEO) has key role in the elevation of susceptibility to Amoxacillin.
Key words: Medicinal Plants, Essential oils, Amoxacillin, MRSA, Antimicrobial activity, Synergistic Effects INTRODUCTION
The increase of microbial resistance to antibiotics threatens public health on a global scale as it reduces the effectiveness of treatments and increases morbidity, mortality and health care costs. Evolution of highly resistant bacterial
strains has compromised the use of newer generations of antibiotics [1.2]. Bacteria of the genus Staphylococcus are distributed in nature, as well as being part of the normal microbiota of the skin and of the mucosa of animals including birds. Some species of Staphylococcus are
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND AMOXICILLIN AGAINST SOME CLINICAL ISOLATES
Hanan. H. Abd El-Kalek and Eman A. Mohamed 388
frequently recognized as etiologic agents of opportunistic infections in many animals and humans. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus and Staphylococcus haemolyticus are the most important causative species of human and hospital infections. Besides causing different types of in toxications, S. aureus represents the most common etiologic agent of purulent infections (for example, furuncle, carbuncle, abscess, myocarditis, endocarditis, pneumonia, meningitis) [3,4].
Methicillin-resistant Staphylococcus aureus (MRSA) presents a significant threat to public health in many areas in the world and causing a significant morbidity and motility worldwide [5]
The frequencies of infectious and outbreaks due to MRSA have continued to increase. MRSA is often multidrug resistant and therapeutic options are limited. MRSA normally possesses a multidrug-resistant genotype which causes it resistant to beta lactams, aminoglycosides, fluoroquinolones and macrolides There is an urgent need to develop anti -MRSA agents with novel mechanisms of action to address this problem [6,7,2]. With a growing incidence of infections resistant to antibiotics, an arsenal of either new agents of the supplementation of current antibiotics is needed. Natural products from plants could be interesting alternatives [8].
In the last years, there has been great scientific interest in chemical and pharmacological investigations of the biological properties of medicinal plants Medicinal plants have been the source of many medications that are now applied in clinical practice. The use of extracts as antimicrobial agents shows allow risk of increasing resistance to their action, because they are complex mixtures, making microbial adaptability very difficult and it is also reported to have minimal side effects [4].
Many plant extracts or products have been evaluated not only for direct antimicrobial
activity, but also as resistance-modifying agents [7,2]. Several chemical compounds, synthetic or from natural sources, such as the phenothiazines and natural products, show an indirect effect against many species of bacteria, by enhancing the activity of a specific antibiotic, reversing the natural resistance of specific bacteria to given antibiotics, promoting the elimination of plasmids from bacteria such as Escherichia coli, and inhibiting transport functions of the plasma membrane in regard to given antibiotics The inhibition of plasma membrane-based efflux pumps has also been observed. The enhancement of antibiotic activity or the reversal of antibiotic resistance by natural or synthetic non-conventional antibiotics affords the classification of these compounds as modifiers of antibiotic activity. [9].
Essential oils have been searched for their antibacterial, antifungal, antiviral, insecticidal, anticancer and antioxidant properties [10-12]. There has been an increased concern in studying antimicrobial properties of plants EOs after emergence of antibiotic resistance occurred [13,14]. Although the active constituents may occur in lower concentrations, plant extracts may be a better source of antimicrobial compounds than synthetic drugs. The phenomenon of additive or synergistic effects is often crucial to bioactivity [15] in plant extracts and in some cases; the activity is lost in purified fractions. Development of bacterial resistance to synergistic drug combinations, such as those found in plants, may be slower than for single drug therapies [16,17].
The purpose of the present work was to determine the antimicrobial activity of certain medicinal plants and to investigate the synergistic effects of chosen plants combined with Amoxacillin against MRSA, thereby throwing light on the potential role of plants in increasing the effectiveness of antibiotics.
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND AMOXICILLIN AGAINST SOME CLINICAL ISOLATES
Hanan. H. Abd El-Kalek and Eman A. Mohamed 389
Materials and Methods 1-Plant material Preparation of plant extracts and volatile oils.
Plant material, divided into two groups, the first group was plant methanol extracts (Nigella Sativa, Brassica nigro, Trigonella fonaeum gracum, Thymus vulgaris, Visnaga Ammi, Tanacetum parthenium) was ground, extracted with 70 % methanol and filtered after 48 h. The plant residue was re-extracted with addition of 70% methanol, and after 24 h it was filtered again. Combined filtrates were concentrated on a rotary evaporator at 45oC for methanol elimination, and the extracts were kept in sterile bottles under refrigerated conditions until use. The extracts’ dry weight was obtained by the solvent evaporation and used to determine concentration in mg/ml [18]. The second group of plant material was volatile oils (lemon grasse, Anis and Cardamom essential oils), the parts of plants (90 g, in triplicates) were hydrodistillated for 4 h. using a Clevenger-type apparatus to produce essential oils according to the method by [19] The oils were dried over an hydrous sodium sulfate and stored in sealed vials at low temperature (4 0C).
2- Bacterial strains and Antibiotic susceptibility test.
Fifty consecutive of Staphylococcus. aureus strains were obtained over 3 months period from Specialized Ain Shams Hospital, Cairo, Egypt from various clinical specimens (Ear swab, Urine, Skin, Eye swab and Bone fragment and tissue) and subjected to disk diffusion method using 13 different antibiotics according to [20]. The antibiotic discs from (Oxoid) Oxacillin(O, 1µg), Imipenem(IPM, 10µg), Vancomycin (VA, 30 µg), Norofloxacin (NOR, 10 µg), Tetracycline(TE,30µg), Sulphamethoxazole-Trimethoprim (SXT, 25 µg), Gentamycin(CN, 10µg), Methicillin (ME, 5µg), Chloramphenicol ( C, 30 µg), Carbencillin (CB, 100 µg), Linzolid (Ln, 1µg) , and Teicoplanin (TEC, 30 µg) were used. Briefly the inhibition zone in mm
interpreted as sensitive, moderate sensitive and resistant using the interpretation chart supplied by antibiotic discs manufactures (Oxoid). A standard bacterial strain of S.aureus ATCC was also used.
3- Disk diffusion assay of medicinal plants.
The essential oils and methanolic extracts were screened for antibacterial activity using the agar diffusion technique [21,22] against 18 strain of MRSA. Filter paper discs (Whatman No.1, 6 mm diameter) containing 10 µl of each essential oils and methanolic extracts were applied to the surface of agar plates that were previously seeded by spreading of 0.1 ml (containing 4.5x 105 cfu) from overnight culture. The plates were incubated over night at 37 oC and the resulting inhibition zone was measured in mm, 70 % methanol (v/v) impregnated disc was used to check whether it has any inhibitory effect on bacterial growth. All data collected for each assay are the averages of three determinations.
4- Antimicrobial activities of Lemon grass, Cardamom oils, Thymus vulgaris extract alone and in combination with Amoxacillin.
Antibacterial activity of the antibiotic alone and its combinations with (LGEO), Cardamom oils, Thymus vulgaris extract were performed as described above for 18 strain of MRSA. The concentrations of each extract, oils and antibiotic were prepared as follows 1:1 v/v.
5-Determination of minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and modulation of antibiotic activity.
For the evaluation of essential oil as modulator of resistance to amoxicillin, MIC of the antibiotic was determined in the presence or absence of LGEO. MICs values were determined by the microtiter broth method [23] in sterile flat-bottom 96-well polystyrene plates. Serial dilution techniques were used to determine the MICs of Lemon grass oil and amoxicillin. The range of lemon grass essential oil dilutions were in range
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND AMOXICILLIN AGAINST SOME CLINICAL ISOLATES
Hanan. H. Abd El-Kalek and Eman A. Mohamed 390
(0.5-16 µg/ml), Amoxacillin range (0.25-32 µg/ml) and the combinations between them were in range (0.2 - 6.25 µg/ml). A final concentration of 105 cfu/ml of tested bacteria (M 2, M12 and M16) was added to each dilution. The plates were incubated at 37 o C for 48 h. MIC was defined as the lowest concentration of antimicrobial agent that inhibited bacterial growth, as indicated by the absence of turbidity. Each test included two growth controls consisting of the medium with the solvent (DEMSO) and medium with bacterial suspension as well as sterility control. Minimum bactericidal concentrations (MBCs) were determined by inoculating 10 µl of medium from each of the wells from the MIC test which showed no turbidity onto a fresh agar plates after 2 days incubation. MBCs were defined as the lowest concentration of antimicrobial agent where no bacterial growth on the plates. The evaluation of MIC and MBC was carried out in triplicates.
6- Synergy testing.
The checkerboard method, which is commonly used for measurement of interactive inhibition, was used for the determination of synergy between the antibiotics and natural antimicrobials [24]. the checkerboard method is often combined with calculation of a fractional inhibitory concentration (FIC) index to test the antimicrobial potencies of drugs in medical laboratories [25] The Fractional Inhibitory Concentration (FIC) was derived from the lowest concentration of antibiotic and lemon grass essential oil combination permitting no visible growth of the test organisms on the plates. FIC value for each agent was calculated using the formula:
FIC ( Amoxacillin) = MIC of antibiotic in combination/ MIC of antibiotic alone.
FIC (LGEO) = MIC of LGEO in combination/ MIC LGEO alone.
Combinations were classified as synergistic, if the FIC indices were ≤1, additive if the FIC
indices were = 1, indifferent if the FIC indices were between 1 and 2 and antagonistic if the FIC indices were ≥ 2
FIC index= FIC of LGEO + FIC of Amoxacillin [26]
7- Transmission electron microscopy.
Samples of MRSA untreated and treated with LGEO, Amoxacillin and in combination between them) were centrifuged and suspended in 2.5 % glutraldehyde solution for 24h. in refrigerator. Samples were centrifuged and washed twice for 15 min. in 0.1-M sodium cacodylate buffer. The suspension was then centrifuged; the pellets were post-fixed with 1% osmium tetroxide aqueous solution for 2 h at room temperature. After post-fixation, the cells were dehydrated through graded ethanol series 30, 50, and 70% each for 15 min. at room temperature. Then 1.5 % agar was prepared and very thin layer was poured in sterile petri dish. Small grooves were made and by using Pasture pipette, samples were put in it, then another thin layer of agar was poured on it. Samples were dehydrated with ethanol 70, 90 and 100 % absolute each for 15 min, at room temp. After then the samples were embedded, after embedding capsules were ovened at 40 0Cfor 24h., then at 60 0C for 24 h., thin section were cut with glass knives of used ultra microtome. Samples mounted on carbon-coated copper grids, stained with 2% uranyl acetate for 15 min. and then 2% lead citrate for 15 min. These sections were examined by transmission electron microscope (JEOL, JEM-100 cx) [27].
Statistical analysis.
Results obtained were subjected to statistical analysis using one way analysis of variance [28]. All data were the average of three experiments.
RESULTS AND DISCUSSIONS
With the increase in the incidence of resistance to antibiotics alternative natural products of plants could be of interest. Some plants extracts and phytochemicals are known to have antimicrobial
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND AMOXICILLIN AGAINST SOME CLINICAL ISOLATES
Hanan. H. Abd El-Kalek and Eman A. Mohamed 391
properties, which could be importance in therapeutic treatment. In the last few years, various studies have been conducted in different countries demonstrating the efficacy of this type of treatment [29,30]. Many plants have been evaluated not only for direct antimicrobial activity but also as resistance- modifying agents [9].
Antibiotic resistance pattern for the 50 isolates of Staphylococcus aureus testes against 12 antibiotics are shown in table (1), out of them, 18 (36 %) strains are resistant to methicillin and Oxacillin, these strains were classified as methicillin resistant S. aureus (MRSA).
Table (1): Antibiotic resistance pattern of MRSA and MSSA.
MSSA methicillin sensitive S aureus MRSA methicillin resistant S. aureus
Table (2) shows the distribution percentage of isolates of MRSA from different clinical specimens. Ear swab infection recorded highest percentage (38.9 %) followed by skin swab 33%. Urine, Eye swab and Bone fragment &tissue infections recorded 17, 5, and 5% respectively.
Table (2) Distribution of MRSA isolates according to clinical specimen
Preliminary evaluation of antimicrobial activity
Some herbal materials can act as antibiotic resistance inhibitors [31,2]. The results obtained from the analysis of antibacterial activity exhibited by studied plant extracts are summarized in Table 3. Among of the extracts, the (LGEO), showed highest antibacterial activity against MRSA tested, the inhibition zone recorde (30-35 mm) at the tested concentration (10 µl). However, the Cardamom oil, Thymus vulgaris extract exhibited intermediate antibacterial activity (17-20 mm and 16-19 mm), respectively. On the other hand the rest plant extracts and oils showed a little antibacterial activity at the tested concentration. Several studies reported that LGEO possessed a broad spectrum antimicrobial [32,33]. Aiemsaard et al. [12] concluded that, LGEO possessed a potent antimicrobial activity against S. aureus, Streptococcus agalactiae, Bacillus cereus and Escherichia coli at tested concentration (50 µl.). The inhibition zone of Querqus brantii, Peganum harmala, Ziziphus spina-christi and Oliveira decumbens extracts were 11-40 mm, 15-28 mm, 8-26 mm and 10-20 mm, respectively against the MRSA isolated [2].
Table (3): Antibacterial activity of some essential oils and herbal extracts against tested MRSA.
Inhibition zone (mm)
Plants
30-35a LGEO Essential oils
8-11c Anis 7-17b Cardmom
11-15b M.C
7-11c T.f.g Plant extracts 13-17b T.v
7-11c T.p 9-12c VAS 7-10c N.S 7-11c B.N
* Mean values followed by different superscript (within columns) is significantly different at the 5 % level
Antibiotics
◌MRSA no=18
MRSA (%)
MSSA no=32
MRSA (%)
Oxacillin 18 100 10 31.3 Imipenem 4 22.2 3 9.4
Tetracycline 10 55.5 6 18.8 Vancomycin 4 22.2 0 0.0 Carbencillin 16 88.8 18 56.2 Norofloxacin 5 27.8 7 21.9 Gentamycin 6 33.3 8 25.0 Teicoplanin 15 83.3 3 9.4
Sulphamethoxazole-
Trimethoprim
9 50.0 10 31.2
Chloramphenicol
6 33.0 16 50.0
Linzolid 00.0 0 0 0.00 Methicillin 18 100 0 0.00
No. positive (%) Site of infection
7(38.9) Ear swab 6(33) Skin swab 3(17) Urine 1(5) Eye swab 1(5) Bone fragment &tissue
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND AMOXICILLIN AGAINST SOME CLINICAL ISOLATES
Hanan. H. Abd El-Kalek and Eman A. Mohamed 392
Synergy testing (A) Medicinal plants synergism with amoxicillin It is well known that many bacterial strains can easily develop resistance against antibiotics. S. aureus is one of the most persistent infectious microorganisms [34.2]. Thus, the choice of effective and safe drug to be used against S. aureus is shrinking day by day. Therefore, due attention is needed to develop alternative or combination agent from natural products including medicinal plants. This experiment was carried out to screen the effect of (LGEO), Cardamom oils and Thymus vulgaris extract in combination with the antibiotic (Amoxicillin) to identify systems, which might be used to improve the efficiency of the Amoxicillin against tested MRSA strains. The antibiotic (amoxicillin) used in this study which represents one of penicillins group of antibiotics.
The activity of Amoxacillin against MSSA (C.I), MSSA (ATCC) and 15 MRSA strains tested were from 15 to 28 mm (inhibition zone), three strains of MRSA (M2, M16 and M18) showed less susceptibility to amoxicillin. When Amoxacillin was combined with LGEO, Cardamom oil and Thymus vulgaris extracts, the inhibition zones were increased (19 to 57 mm), and (13 to 53 mm) and (12 to 44 mm), respectively. The data also revealed that, M2, M16 and M18 became sensitive to each combination, the data showed significantly increase in activity of Amoxacillin when combined with tested plant material (Table 4). Combinations of two or more compounds are generally superior to the use of a single compound, especially for the treatment of serious infections caused by antibiotic resistant bacteria. The natural plants inhibit ß-lactamase is remarkable, although they were not directly tested against this specific enzyme isolated from MRSA [35]. The antibiotic activity of gentamicin against Pseudomonas aeruginosa was enhanced in the presence of Croton zehntneri essential oil [36]
Table 4 revealed that the greatest synergism was observed in the combination of amoxacillin with LGEO, followed by Amoxacillin with Cardamom oil and the lowest synergism was in combination between Amoxacillin with Thymus vulgaris extract.
Table (4): Antibacterial activity of amoxicillin alone and in combination with Lemon grass, Cardmom oils and thymus vulgais extract against tested MRSA
A+Cardmon
1:1
A+Thymus vulgaris
1:1
A+LGEO 1:1
Antibiotic Amoxicill
in)(
Tested strains
Inhibition zone (mm) 49a
a 35bb 55a
a 25ca MSSA
(C.I) 53a
a 39bb 57a
a 28ca MSSA(A
TCC) 30b
c 26bc 42a
b 17cb M1
14bd 12b
d 19ac 8.0c
c M2 28b
c 32bb 43a
b 18cb M3
32bc 35a
b 40ab 15c
b M4 30b
c 30bb 45a
b 16cb M5
28bc 29b
b 50aa 16c
b M6 31b
c 35bb 43a
b 19cb M7
28bc 32b
b 40ab 18c
b M8 31b
c 29bb 50a
a 17cb M9 40b
b 44ba 55a
a 20ca M10
30bc 31b
b 53aa 16c
b M11 27bc 25b
c 52aa 18c
b M12 30b
c 33bb 40a
b 17cb M13
28bc 31b
b 43ab 17cb M14
23bc 26b
c 40ab 18c
b M15 15b
d 12bd 21a
c 9.0cc M16
25bc 23b
c 40ab 16c
b M17 13b
d 15bd 23a
c 9.0cc M18
* C.I: Clinical isolate Lemon grass essential oil (LGEO) Cardamom essential oils (CEO) Thymus vulgaris extracts (TVE), A: Amoxicillin ** Mean values followed by different superscript (within columns) and different subscripts (within rows) are significantly different at the 5 % level
The improvement in the activity of this antibiotic is probably due to accumulation of inhibitory concentrations at the target sites or due to the additional inhibitory effect of the tested plant material. Survival of MRSA strains in the presence of plant material could probably be due to the cell membrane permeability or other genetic factors [11,2]. The synergistic effects resulting from the combination of antibiotics with plant extracts were documented in the literatures [31]. Darwish et al. [34] reported that the combinations of gentamicin and chloramphenicol
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND AMOXICILLIN AGAINST SOME CLINICAL ISOLATES
Hanan. H. Abd El-Kalek and Eman A. Mohamed 393
could be improved by the use of plant materials against Methicillin resistant S. aureus (MRSA). The plants, E. officinalis and N. odorata alone or in combination, are promising in the development of phytomedicines, which may be used, alone or in combination with the antibiotic, amoxicillin against MRSA infections [24]. Palaniappan and Holley [37] concluded that, A synergistic interaction was found between penicillin and 0.31 mM of eugenol, and with ampicillin and 1.25 mM eugenol against Staphylococcus aureus.
(B) Evaluation of MIC and MBC values
From the data reported in Table (4) which resulted in, (LGEO) was most effective among the tested plants against resistant strains of MRSA (M2, M16 and M18) towards amoxicillin. So the antibacterial activity of the Amoxacillin, LGEO and their effect in combination were quantitatively assessed by determining the MICs and MBCs as given in Table (5). MICs values of LGEO and Amoxacillin were in the range 2.0 to ≥ 2.5 µg ml-1 and 4.25 to 4.50 µg/ ml-1, respectively. MICs were reduction for LGEO and Amoxacillin where became (1.2 to 1.4 µg/ ml-1) with their combination. Several studies reported a synergistic inhibitory between plant materials and antibiotics Coutinho et al. [38] found that the combination of ethanol extract of Momordica charantia (EEMC) with Amykacin, Gentamicin, Kanamycin, Neomycin, Tobramycin and Chlorpromazine at 32 µg/ ml-1 produced a dramatic reduction in the MIC for all aminoglycosides-methicilin-resistant. Some medicinal plant extracts may provide suitable compounds for future development and possible clinical utility as inhibitors of efflux for various Gram-negative bacteria [39].
Table (5): MICs and MBCs values (µg/ml) of amoxicillin, LGEO and in combination against MRSA.
LGEO& Amoxacillin
Amoxacillin
LGEO MRSA
MBC MIC MBC MIC MBC MIC 1.3a
a 1.3aa 4.25a
c 4.25ac ≥2.5a
b ≥2.5ab M2
1.2aa 1.2a
a 4.25ac 4.25a
c 2.5ab 2.5a
b M16 1.4a
a 1.4aa 4.50bc 4.50b
c 2.0bb 2.0b
b M18
** Mean values followed by different superscript (within columns) and different subscripts (within rows) are significantly different at the 5 % level Also the data in Table (5) revealed that the area of concern is that MIC of LGEO and Amoxacillin obtained in this study were equal the MBC values, suggesting that LGEO and Amoxacillin were bacteriostatic and bactericidal at the same concentration tested.
This effect was most intense when LGEO was added to the medium, suggesting that LGEO is a putative inhibitor of an efflux pump, but additional experiments are needed to confirm such mechanisms. Juve et al. [40] and Burt [10] reported that the modification of drug permeability could as a result; they get partitioned into the lipid bilayer of the cell membrane, affecting the respiratory chain and the energy production. Phytochemical in plants probably act on the plasma membrane of bacteria, affecting the efflux pump [41]. Several components of essential oils as thymol and carvacrol may act as membrane permeabilizers, enhancing the intake of antibiotics [42,43], The mechanisms by which essential oils can inhibit microorganisms involve different modes of action, and in part may be due to their hydrophobicity enhance the activity of antibiotics that act within the cell, such as the aminoglycosides [38]. In this study, LGEO showed significant synergistic activity with Amoxacillin and reduction in the MIC of ß-lactam antibiotic (amoxicillin) against tested MRSA. Choi et al. [44] reported that Ecklonia cava extract can lower the MIC of ß-lactam antibiotic (ampicillin) against MRSA. (C) FIC index of LGEO in combination with Amoxicillin:
The potential reduction of antibiotic resistance by the LGEO was examined by calculation of FIC indices (Table 6). The FIC indices for the combination LGEO with Amoxacillin against tested MRSA (M2, M16 and M18) were 0.82 to 0.86, indicating synergistic interactions. In other words, the MIC of Amoxacillin was lowered
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND AMOXICILLIN AGAINST SOME CLINICAL ISOLATES
Hanan. H. Abd El-Kalek and Eman A. Mohamed 394
from 4.25 µg/ml to 1.3 µg/ml in combination with 1.25 µg/ml (equivalent to 1/14 MIC) of LGEO. The activity of Amoxacillin was enhanced 14-fold by combining with LGEO. Muroi et al. [35] reported that the activity of methicillin was enhanced 32-fold by combining with anacardic acids against MRSA ATCC 33591.
Table (6): Fractional inhibitory concentrations (FIC) values for the combination between Amoxicillin and LGEO.
Interaction FIC Index
FIC (LGEO)
FIC (Amoxicil
lin
Test isolate
Synergy 0.82ac 0.52b
b 0.30ca MRSA2
Synergy 0.90ac 0.56b
b 0.35ca MRSA16
Synergy 0.86ac 0.60bb 0.26c
a MRSA18 ** Mean values followed by different superscript (within columns) and different subscripts (within rows) are significantly different at the 5 % level
In this study the interpretations of activity of LGEO combined with Amoxacillin produced a significantly synergistic activity against the tested MRSA (M2, M16 and M18) (Table 6). Similarly, Betoni et al. [45] suggested that the lemongrass oil presented the highest synergism rate with antimicrobial drugs (penicillin, oxacillin, vancomycin, ampicillin, chloramphenicol, gentamicin, netilmicin, tetracycline, erythromycin, cotrimoxazole, and ofloxacin) against Staphylococcus aureus. The antibacterial activity of the combinations of the ethanol extract of rosemary and cefuroxime showed synergistic effects against all tested MRSA [26]. Generally the data revealed that, LGEO had significantly improved the inhibitory effects the Amoxacillin. This might indicate that the plant material allowed better entrance of the drug through the outer layers to the cell wall, which is the target site for this antibiotic. Various components of extracts can permeabilize the cell membrane, increasing the penetration of antibiotics. The interference with bacterial enzyme systems can also be a potential mechanism of action [46]. These mechanisms of action can be obtained by the combination of antibiotic with extract at a sub-inhibitory
concentration applied directly to the culture medium [17].
Transmission electron micrographs:
Transmission electron micrographs of MRSA (M2) untreated and treated with LGEO, Amoxacillin and in combination between them are presented in Fig 1. MRSA (M2) under study (without any treatment) had the typical structure of gram-positive and the cells showing an regular wall (Fig.1 A). Generally, after the bacterial cells were exposed to the LGEO (2.5 µg/ml), Amoxacillin (4.25 µg/ml) and in combination between them (1.4 µg/ml). With the treatment of amoxicillin there was a little remarkable change in the cell morphology and cell content, but in case of LGEO and combination treatment there was significantly shrunken and the cell size was reduced. All treatments caused swelling of the cell wall and distribution of the cytoplasmic membrane. In addition, cells were found to have densely stained cell walls and irregular cytoplasmic membranes and induced empty spaces in the cells (Fig 1 C and D).
Fig. 1 C, D revealed that the LGEO affected the structural organization of the cytoplasm more than the cell wall of treated MRSA (M2). It might be proposed that in the primary phase, the tested oil probably bind the bacterial cell surface and penetrate into the cell wall causing it to swell followed by cytoplasmic membrane damage while the cells shrink as a defense against the tested oil. LGEO is composed of several chemical components. Its antibacterial activity may have several targets of action on the bacterial cell. The sites of action for the LG components would depend on the properties of compounds; some might act on separate targets and some might be a consequence of another mechanism. This study confirmed the mode of antibacterial action of other essential oils components which has been reviewed by Burt [10] that lipophilicity of the oil components plays an important role in penetrating the lipid layer of the bacterial cell membrane and mitochondria
SYNERGISTIC EFFECT OF CERTAIN MEDICINAL PLANTS AND AMOXICILLIN AGAINST SOME CLINICAL ISOLATES
Hanan. H. Abd El-Kalek and Eman A. Mohamed 395
causing loss of structural organization and integrity. The monoterpene alcohol, among components of LGEO may target membrane integrity causing intracellular leakage. This might be due to the solvation of phospholipids of the membrane [47].
Fig. 1 Transmission electron micrographs of MRSA (M2) untreated cells (A) after exposure to Amoxacillin (B), LGEO (C) and in combination (D) This study confirmed the mode of antibacterial action of LGEO has been reviewed by Aeimsaard et al., [12] that transmission electron micrographs demonstrating morphological changes of S. aureus DMST 4745 after exposure to various concentrations of lemongrass and its majors components showed respect to the leakage of cell constituents and the cell contents in many bacteria were lost.
CONCLUSION
This strategy is named ‘‘herbal shotgun’’ or ‘‘Synergistic multi-target effects’’ and refers to
the use of herbals and drugs in a multitargeted approach of mono or multi-extract combinations affecting not only a single target, but several, cooperating in an agonistic synergistic way. The approach is not exclusive for extract combinations, since effective combinations between single natural products, essential oils or extracts with chemosynthetic or antibiotics have been described by [35,26]. Several reports indicate different antibiotic combinations assyed in vitro and applied in the human and veterinary clinics, but combinations of natural products and synthetic drugs are not reported. The results obtained in this investigation suggest that Lemon grass essential oil could be a source of metabolites with antibacterial modifying activity to be used as adjuvants to antibiotic therapy against multidrug-resistant bacteria as the MRSA strains.
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