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Immunomodulatory and antagonistic effect of lactobacillus reuteri and its purified characterized bacteriocin against salmonella enterica and Shigella flexnerii.

INTRODUCTION

Diarrhea is the second-most frequent cause of death in children less than five years old, and annually causes about 760, 000 deaths among children [1]. Shigellosis (i.e. bacillary dysentery) is a major public health trouble in developing countries. Enlarged incidence of antibiotic resistance in Shigella flexneri create a major public health anxiety [2, 3]. Salmonella is an intracellular pathogen can cause disease in both humans and animals ranging from mild diarrhea to typhoid fever [4]. Antibiotic resistance is a problem of deep scientific concern both in hospital and community settings [5,6,7]. Breast milk, a natural source of potentially probiotic Lactic acid bacteria, protect infants against infectious diseases [8]. In microbiology, the antimicrobial peptides are a significant part of the insolence system of probiotic and they are referred to as bacteriocins [9]. L. reuteri is mainly acknowledged probiotic species and has been commonly utilized as a probiotic in humans and animals for several years. Probiotics can prevent the proliferation of pathogenic bacteria or modulate the immune response [10]. Presently, interesting in bacteriocins is vast because of their inhibitory activity against pathogenic bacteria such as L. monocytogenes [11]. Bacteriocins were metabolized products of food--grad, microorganisms and some of which were used as food preservative to enhance the food safety [12]. The aim of this study was to isolation and identification of L. reuteri from Iraqi human breast milk and detected the Reutericin LHS production from the highest producer one. In addition characterization and purification of Reutericin_LHS, and evaluated its ability to inhibit multidrug resistance Shigella flexneri and Salmonella enteric isolates in vitro. Furthermore examined immunomodulatory and antagonistic activity of L. reuteri against Shigella flexneri and Salmonella enterica invivo.

Methodology:

Bacterial strain:

Thirty-three isolates belong to Shigella flexneri and 29 isolates belongs to Salmonella enterica were isolated from infant and obtained from educational laboratories in Baghdad, Baghdad teaching hospital child protection during October 2015 were identified by Vitek 2 according to the manufacturer's instruction.

Antibiotic susceptibility Test:

Kirby-Bauer method were performed as previously reported by Baron and Finegold [13]. Antibiogram test performed by using Muller-Hinton agar (Hi media/ India) and different antibiotic discs (Bioanalyse/Mast and Oxoid) including: Ampicillin-Sulbactam (SAM) (10/10[micro]g), Amoxicillin-clavulanic acid (AUG) (20/10[micro]g), Amikacin (AK) (30[micro]g), Cefepime (CPM) (30 [micro]g), Cefoxitin (FOX) (30 [micro]g), Cefazolin (CZ) (30 [micro]g), Cefotaxime (CTX) (30[micro]g), Ceftazidime (CAZ) ([micro]g), Carbenicillin (CB) (100 [micro]g), Ciprofloxacin (CIP) (5[micro]g), Nalidixic acid (NA) (30 [micro]g), Gentamicin (GM) (10[micro]g), Tobramycin (TN) (10 [micro]g),Temocillin (TEM) (30[micro]g), and Tetracycline (TE) (30[micro]g). Inhibition zones were measured by a ruler and compared with the zones of inhibition determined by Clinical Laboratories Standards Institute [14].

Isolation and Identification of Lactobacillus reuteri strains from human milk samples: Fifteen L. reuteri isolates were isolated from Iraqis human breast milk were obtained from volunteers and identified by the cultural, microscopical, biochemical examinations and API 50 CHL Oxoid.

Detection of L. reuteri bacteriocin Produced by L. reuteri: To screen L. reuteri isolates for their ability to produce bacteriocin, was used the agar well-diffusion assay as described by Lewus et al., [15].

ReutericinLHS A ctivity Assay:

Bacteriocin activity was measured according to Parente et al., [16] and Pilasombut et al., [17].

Extraction and purification of ReutericinLHS:

Crude Reutericin_LHS extracted according to Powell et al., [18] and purified Gel filtration chromatography according to Abo-Amer, [19]. In each step protein concentration was determined by Lowrey method Lowry et al., [20].

Characterization of Reutericin LHS: Determination of the Molecular Weight:

The molecular weight of the Reutericin_LHS was estimated by gel filtration chromatography (Sephadex G100) [21,22].

Effect of pH:

To determine effect of pH on Reutericin_LHS purified preparations of the bacteriocin were adjusted to various pH values in the range of 2 TO 10. The pH-adjusted bacteriocin. Samples were incubated at 37[degrees]C for 20 min and then neutralized to pH 6 and tested for bacteriocin activity by agar well diffusion method [23].

Effect of Temperature:

It was assayed by treating Reutericin_LHS solution with (20, 25, 30,35,40,50, 60, 70, 80, and 100)[degrees]C respectively. Reutericin_LHS activity was assayed after (30) minutes at each of these temperatures. Activity also assayed after 15 minutes at 121[degrees]C [24].

Effect of Enzymes:

Reutericin_LHS solution was incubated for 1 hour at 30[degrees]C in the presence of the enzymes (pepsin, lipase, [alpha]- amylase (Philip Harris Biological Ltd/UK), papain (BDH/England), trypsin (Merck /Germany) at a final concentration of 1mg/ml. After incubation, the enzymes were heat-inactivated (3 minutes at 100[degrees]C) and tested for remaining activity [25].

Effect of ReutericinLHS on Shigella flexneri and Salmonella enterica in vitro:

The agar well diffusion method was used to detect antibacterial activity of crude and purified Reutericin_LHS produced by L. reuteri strain no.9 against Shigella flexneri and Salmonella enterica strains at the concentration (32) Mg/ml according to Lewus et al., [15].

Animals:

The mice were anesthetized by I.P injection of 0.2ml of 0.65% sodium pentobarbital before bacterial inoculation.

Experimental design:

Animals were divided into (6): groups each one containing (5) mice:

Group (1) Positive control:

(Salmonella enterica): mice challenged orally with 0.1ml of Salmonella enterica inoculum. Containing 1*108 cell/ml, after 3 days were administered 0.1 ml (PBS) for 4days.

Group (2) Positive control:

(Shigella flexneri): mice challenged orally with 0.1ml of Shigella flexneri inoculum. Containing 1*108 cell/ml, After 3 day were administered 0.1ml (PBS) for 4days.

Group (3) Treated:

mice were initially challenged with 0.1ml of Salmonella enterica inoculum and after 3 day were administered 0.1ml of L. reuteri 1*108 cell/ml daily for 4 days post infected.

Group (4) Treated:

mice were initially challenged with 0.1ml of Shigella felxneri inoculum and after 3day were administered 0.1ml of L. reuteri 1*108 cell/ml daily for 4 days post infected.

Group (5) Protective:

mice were initially administered 0.1ml of L. reuteri inoculum for 3 days before Salmonella enterica infection then administered daily (for 4 days) with 0.1ml L. reuteri inoculum.

Group (6) Protective:

mice were initially administered 0.1ml of L. reuteri inoculum for 3 days before Shigella flexneri infection then administered daily (for 4 days) with 0.1ml of L. reuteri inoculum.

Animal were observed and evaluated, mice were sacrificed by cervical dislocation at the 4th day post inoculation, blood samples were obtained through cardiac puncture, and portions of gastrointestinal tract (0.1gm of ileum) and 0.1gm of colon were sampled.

Microbial count:

Microbiological quantification of Salmonella enterica and Shigella flexneri were carried out according to Borowsky et al., [26].

Cytokine assay:

The level of cytokine [interleukin-10] in the sera were determination using Duo set ELIZA kits (R&D SYSTEMS, Minneapolis, MN, USA).

Results:

Antibiotic Susceptibility test:

The highest resistance percentages were found for Shigella flexneri to Ampicillin, Carbenicillin (100%), Cefazolin (90.9%), Cefotaxime (81.81%), Cefoxitin (78.7%) and Gentamicin, Temocillin, Ceftazidim (75.7%). Moreover, 72.7%, 69.6%, 57.5%, 54.5%, 51.5 the Shigella flexneri isolates were resistant to Amikacin, Amoxicillin and Tobramycin, Nalidixic acid, Cefepime, Ciprofloxacin and Tetracyclin, respectively (Figure1).

On the other hand, highest resistance percentages were found for Salmonella enterica to Cefotaxime (93.1%), Ampicillin, Carbenicillin, Temocillin (68.9%), Cefazolin, Ceftazidim, Tetracyclin (65.5%). Moreover, 62%, 58.6%, 55.1%, 51.7% of the Salmonella enterica isolates wereresistant to Cefepime, Amoxicillin, Amikacin and Gentamicin, Cefoxitin, Nalidixic acid and Ciprofloxacin, respectively (Figure 2).

Whereas Tobramycin was the most effective antibiotics since it recorded the lowest resistance percentage; 48.2%.

Detection of Reutericin LHSproduction:

All L .reuteri isolates were able to produce Reutericin_LHS and L. reuteri No. 9, was the best isolate for Reutericin_LHS production.

Purification of Reutericin LHS:

Reutericin_LHS activity was purified up to 12.9107 purification folds' chromatographic technique, the overall yield and activity are summarized in table (4).

Gel Filtration Chromatography:

In the gel filtration chromatography, the maximum activity of Reutericin_LHS was observed in the fractions (32-38).

Characterization of Reutericin LHS:

1. Determination of Molecular Weight:

When used gel filtration chromatography, the molecular weight of Reutericin_LHS was 10600Dalton (figure 7).

2. pH Stability for ReutericinLHS:

Reutericin_LHS activity was stable at pH values (4-8) but 70% of its activity was lost at (2 and 10) pH values, and 60% of it's activity was lost at pH values (3 and 9) (figure 8).

Thermostability for ReutericinLHS:

Reutericin_LHS showed high thermostability at different temperatures (20- 100)[degrees]C for (30) min, it remained active after being treated with (20-80)[degrees]C for the periods above, but it retained 50% of its activity after treatment at 100[degrees]C for one hour and 40% of it's activity after autoclaving treatment (121[degrees]C/ 15) figure (9).

Sensitivity of ReutericinLHS to Enzymes:

The activity of Reutericin_LHS disappeared when it treated with proteolytic enzymes (pepsin, trypsin and papain), whereas it retained whole activity when treated with lipase, and a-amylase (figure 10).

Antibacterial activity of crude and purified Reutericin_LHS on Shigella flexneri and salmonella enterica in vitro:

Crude and purified bacteriocin of L. reuteri (L. reuteri 9) possesses significant antibacterial activity against Shigella flexneri and Salmonella enterica isolates contrast with control p<0.05, (table5)., in this study crude and purified Reutericin_LHS recorded maximum antibacterial activity against Shigella flexneri (27,34) mm and Salmonella enterica (26,31) mm, respectively (figure 11,12)

Antagonistic Effect of L. reuteri against Shigella flexneri and Salmonella enterica in vivo:

As shown in tables (6,7) our result confirmed that mice from group 1 and group 2 (positive control) challenged with Salmonella enterica and Shigella flexneri respectively, the colon was clearly colonized Salmonella enterica (6.03 [+ or -] 3.12) and Shigella flexneri (5.31 [+ or -] 2.77), also ileum count were (5.78 [+ or -] 1.29) and (4.84 [+ or -] 1.85) for Salmonella enterica and Shigella flexneri respectively.

In group 3 and 5 which were infected with Shigella flexneri after 3 days treated with L. reuteri for 4 days, we observed that statistically decreasing the number of Salmonella enterica and Shigella flexneri in their intestinal portions (either the ileum and colon) P<0.01, in these groups may be the mice eliminated pathogenic bacteria (Salmonella enterica and Shigella flexneri) in their feces.

While in groups 5and 6 (protective groups) which were treated with L. reuteri before and post challenge with pathogenic bacteria, colonization by Shigella flexneri and Salmonella enterica were significantly lower in colon followed by ileum compared with other groups P<0.01. And when comparing results obtained in groups (3and4) with (5and 6), we observed that, the number of Salmonella enterica and Shigella flexneri in the latter groups were lower than in the former, these data were also confirmed statistically P<0.01.

Probiotic have traditionally been thought to have immunomodulating effects. To verify this property L. reuteri No. 9 isolate was orally administered to mice prior to infection with resistant Shigella flexneri and salmonella enterica, in order to evaluate the host resistance against infections microorganisms. L. reuteri significantly increased serum IL-10 of the mice infected with salmonella enterica and Shigella flexneri then orally fed with L. reuteri post challenge with pathogenic groups (3 and 4) compared with control P<0.01. Furthermore, orally administration of L. reuteri to mice pre and post challenged with pathogenic bacteria groups (5 and 6) significantly increased serum IL-10 compared to those in groups 3 and 4 P<0.01 and controls groups 1 and 2. (Figure 15 and 16)

Discussion:

Lactobacillus reuteri strain disposition from human breast milk [28]. In this study Lactobacillus reuteri were disposition from human breast milk in high percentages (35.71)% (n=15) out of lactobacillus isolates. Other report recorded that L. reuteri was create in the breast milk of mothers from around the world, giving an overall occurrence of 15% [29]. The specific activity of Reutericin_LHS recorded 2277.3 AU/mg protein with 2.7910 purification folds and 39.989% yield (table 4). In the gel filtration chromatography, the maximum activity of Reutericin_LHS observed in the fractions (32-38) while the specific activity for these fractions was 5436.7 AU/mg protein with 6.66307 purification folds and 11.863% yield. The three active fractions of the previous step were collected and useful once again on the same column; this step gave a single active peak that was identical with the peak of Reutericin_LHS activity .The specific activity at this step was 10534.5 AU/mg protein with 12.9107 purification folds. In this study, Reutericin_LHS activity stable at pH values (4-8) and showed high thermostability at different temperatures (20-100).

However, the major activity of the Reutericin_LHS was determined at an optimum pH of 7.0 to 8.0 [28]. Heat stability of Reutericin_LHS could be due to the pattern of small globular structures and the incidence of strongly hydrophobic regions and steady cross-linkage [30, 31]. which was confirmed from the bacteriocin activity using the agar well diffusion method against the pathogens However, there have been no literatures regarding the molecular weight of Reutericin_LHS produced by L. reuteri isolated from human milk in this study, the molecular weight of Reutericin_LHS was 10600 Dalton as determined by Gel filtration. When treated with enzymes found that structure of Reutericin_LHS did not contain a lipid or carbohydrate moiety this proving its proteinaceous nature. This great sensitivity of LAB bacteriocins to metabolic proteolytic enzymes is very appealing with respect to food safety, because it means that the ingestion of Reutericin_LHS will not alter digestive tract ecology and appear to be nontoxic [31].

The purified and crude Reutericin_LHS showed the inhibitory spectrum. It is important to highlight the activity against Shigella flexneri and Salmonella enterica isolates which is main human pathogens, in this study crude and purified Reutericin_LHS recorded antibacterial activity against Shigella flexneri and Salmonella enterica but the antibacterial activity of purified Reutericin_LHS was significantly higher than crude Reutericin_LHS P <0.05. Deraz et al., [32] reported, antibacterial activity of bacteriocins is influenced via the following aspects: bacteriocin concentration and purification degree, physiological condition of the indicator bacteria (e.g. growth phase) and experimental conditions (e.g. temperature, pH, presence of agents damaging cell wall integrity and other antimicrobial composites). generally, the antibacterial activity of bacteriocins encompasses the raised permeability of the cellular membrane of the target cells for a wide range of monovalent cations (e.g. Cs+, Na+, Li+, K+, Rb+ and choline) causing the damage of proton motive force via dissipation of the transmembrane pH gradient and lastly to the cell death [33,34].

The fact that the number of Salmonella enterica and Shigella flexneri were lower in experimental groups initially treated with L. reuteri and after 3days infected and then treated with L. reuteri with pathogenic bacteria (group 5 and 6). Suggesting that L. reuteri is able to proliferate in the conditions of the gut and colonize in high population level in the gastrointestinal tract ecosystem.

Demonstrating that, L. reuteri was effective antagonistic against Salmonella enterica and Shigella flexneri.

Emphasizing that the administration of L. reuteri before and after infection with Salmonella enterica and Shigella flexneri showed antagonistic effect against this pathogenic bacteria, suggesting that, L. reuteri can be used as a therapeutic tool against Sh. flexneri and S. enterica. To protect the host from infection with pathogenic bacteria, competitive exclusion of pathogens via the adherence of Lactic acid bacteria on host epithelium, and improvement of the immunomodulatory activity of host via Lactic acid bacteria are two of the main factors 9 [35]. The clinical data expression that the intake of Lactobacillus reuteri significantly reduces colonization of pathogenic bacteria. The ability of L. reuteri to influence the basic immune responses in the human gastrointestinal tract perhaps the basis for animproved protection against different pathogenic infection [36]. Probiotics such as L. reuteri may stimulate innate immune responses by several mechanisms including modulation of pro-inflammatory cytokines [37].

Conclusion:

In the experiments here we characterized the proteinaceous nature, heat resistance, and molecular weight of the Reutericin_LHS. Can be classified as a small, heat stable peptide presumably belonging to class II according to the classification of balciunas [38]. There is little literature available that explain the inhibition activity against important human pathogens in vitro and in vivo. The characteristics of Reutericin_LHS are promising for application of it in the food production processes as food preservative instead of chemicals food preservatives. Our results suggest that L. reuteri No.9 isolate was isolated from Iraqi human breast milk is a potent IL-10 induced and it is useful for prevention and treatment of life-threatening infections with resistant Shigella flexneri and Salmonella enterica that indicated for the role of L. reuteri No.9 as immunomodulator factor.

ACHNOWLEDGEMENTS

The authors would like to thank Al-Mustansiriyah University (WWW.uomustansiriyah.edu.iq) Baghdad. Iraq for it is support in the present work.

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(1) Likaa Mahdi, (2) Sara Abd Alkareem, (3) Hadeel Musafer

(1) Likaa Mahdi, Professor at Biology Dept., Department of Biology, College of sciences, AI-Mustansiriyah University, Baghdad 10001, Iraq,

(2) Sara Abd Alkareem, Researcher at Biology Dept., Department of Biology, College of sciences, AI-Mustansiriyah University, Baghdad 10001, Iraq.

(3) HadeeI Musafer, Professor at Biology Dept., Department of Biology, College of sciences, AI-Mustansiriyah University, Baghdad 10001, Iraq.

Received 2 September 2016; Accepted 2 December 2016; Published 31 December 2016

Address For Correspondence:

Likaa Mahdi, Professor at Biology Department, College of Sciences, Al-Mustansiriyah University, Ministry of higher education and scientific research - Baghdad 10001Iraq.

Caption: Fig. 1: Shigella flexneri isolates resistance towards Antibiotics

Caption: Fig. 2: Salmonella enterica isolates resistance towards Antibiotics

Caption: Fig. 5: Purification of Reutericin_LHS by gel filtration chromatography (StepI), using Sepharose 6B column with dimensions (1.5x64) cm that equilibrated and eluted by 20mM sodium citrate buffer (pH 5), flow rate was 40ml/hour, with 5ml for each fraction.

Caption: Fig. 6: Purification of Reutericin_LHS by gel filtration chromatography (StepII), using Sepharose 6B column with dimensions (1.5x64) cm, that equilibrated and eluted by 20mM sodium citrate buffer (pH 5), flow rate was 40ml/hour, with 5ml for each fraction.

Caption: Fig. 7: Molecular weight of Reutericin_LHS produced by Lactobacillus reuteri

Caption: Fig. 8: Stability of Reutericin_LHS produced by Lactobacillus reuteri at different pH values.

Caption: Fig. 9: Residual activity of purified Reutericin_LHS at different temperatures for 30 minutes.

Caption: Fig. 10: Sensitivity of Reutericin_LHS produced by Lactobacillus reuteri9 towards some enzymes

Caption: Fig. 11: Antibacterial activity of crude and purified Reutericin_LHS against Shigella flexneri. 1: Crude Reutericin_LHS, 2: Purified Reutericin_LHS, 3: Control.

Caption: Fig. 12: Antibacterial activity of Crude and purified Reutericin_LHS against Salmonella enterica.!: Crude Reutericin_LHS, 2: Purified Reutericin_LHS, 3: Control.

Caption: Fig. 15: Concentration of interleukin - 10 in the serum of mice infected with Shigella flexneri Results are expressed as (mean + SD), n = 5 *: Probability compared to group 2 (Control), P < 0.01 a:Probability compared to group 4, P < 0.01

Caption: Fig. 16: Concentration of interleukin - 10 in the serum of mice infected with Salmonella enterica Results are expressed as (mean + SD), n = 5 *: Probability compared to group 1 (Control), P < 0.01 a: Probability compared to group 3, P < 0.01
Table 4: Steps of purification of L. reuteri bacteriocin produced by
Lactobacillus reuteri

Purification steps
                                                  Volume    Activity
                                                  (ml)      (AU/ml)

Crude extract(CRE)               Reutericin_LHS   250       159.48

After heating                                     250       159.46
(80[degrees]C/10min)

Extraction with butanol (1:1)                     26        613.2

Gel filtration                   First step       20        236.5

(Sepharose 6B)                   Second step      15        315.3

Purification steps               Protein         (a) Specific
                                 concentration   activity
                                 (mg/ml)         (AU/mg)

Crude extract(CRE)               0.195           815.9

After heating                    0.158           1,004.4
(80[degrees]C/10min)

Extraction with butanol (1:1)    0.269           2277.3

Gel filtration                   0.0435          5436.7

(Sepharose 6B)                   0.029           10534.5

Purification steps               (b) Total
                                 activity    (C) Purifi.   (d) Yield
                                 (AU)         fold         (%)

Crude extract(CRE)               39870       1             100

After heating                    39865       1.2310        100
(80[degrees]C/10min)

Extraction with butanol (1:1)    15944       2.7910        39.989

Gel filtration                   4730        6.66307       11.863

(Sepharose 6B)                   4730        12.9107       11.863

(a) Specific activity (AU/mg): represents Reutericin_LHS activity
divided by protein concentration.

(b) Total activity (AU): represents Activity (AU/ml) x Volume (ml).

(C) Purification fold: represents specific activity of purified
fraction divided by specific activity of crude extract. (d) Yield (%):
represents (total activity of purified fraction divided by total
activity of crude extract) x 100 [27].

Table 5: antibacterial activity of crude and Purified Reutericin LHS
on Shigella flexneri and Salmonella enterica in vitro

Isolates                (mean [+ or -] SD) Inhibition Zone (mm)

                         Crude Reutericin LHS 32 Mg/
                         ml

Shigella flexneri *      19.6 [+ or -] 1.77
                         (p1)

Salmonella enterica **   15.5 [+ or -] 2.11
                         (p1)

Isolates                (mean [+ or -] SD) Inhibition Zone (mm)

                         Purified Reutericin LHS 32 Mg/
                         ml

Shigella flexneri *      27.7 [+ or -] 2.04
                         (P2)   (P1)

Salmonella enterica **   23.54 [+ or -] 2.44
                         (P2)   (P1)

Isolates                 (mean [+ or -] SD) Inhibition Zone (mm)

                         Control D.W

Shigella flexneri *      0 [+ or -] 0

Salmonella enterica **   0 [+ or -] 0

(P1): Probability Compared to Control P< 0.05

(P2): Probability Compared to crude Reutericin LHS P< 0.05.

* : each value is (mean + SD) of (10) strains of Shigella flexneri

** : each value is (mean + SD) of (10) strains of Salmonella enterica

Table 6: Antagonistic effect of L. reuteri against Salmonella
enterica invivo (mean [+ or -] SD) Log cfu/g tissue

Animals groups        ileum                colon

Group (1) positive    5.78 [+ or -] 1.29   6.03 [+ or -] 3.12
control Salmonella
enterica

Group (3)             4.36 [+ or -] 2.05   4.79 [+ or -] 2.96
                      (a)                  (a)

Group (5)             1.15 [+ or -] 1.37   1.91 [+ or -] 0.89
                      (c)   (a)            (c)   (a)

(a) : significant differences according to group (1).

(c) : significant differences according to group (1and3).

Table 7: Antagonistic effect of L. reuteri against shigella flexneri
invivo (mean [+ or -] SD) Log cfu/g tissue

Animals groups               ileum                Colon

Group (2) positive control   4.84 [+ or -] 1.85   5.31 [+ or -] 2.77
shigella flexneri

Group (4)                    4.25 [+ or -] 1.02   4.86 [+ or -] 2.23
                             (d)                  (d)

Group (6)                    1.92 [+ or -] 0.96   1.75 [+ or -] 1.11
                             (e)   (d)            (e)   (d)

(d) : significant differences according to group (2).

(e) : significant differences according to group (2and4)
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Author:Mahdi, Likaa; Alkareem, Sara Abd; Musafer, Hadeel
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Date:Dec 1, 2016
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