Increasing the antibacterial activity of gentamicin in combination with extracted polyphosphate from Bacillus megaterium

Authors


Correspondence

Giti Emtiazi, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran. E-mail: emtiazi@yahoo.com

Abstract

Aims

The aim of this research was production of polyphosphate (poly P) and study on its antibacterial effects.

Methods and Results

Poly P granules in the cells were observed with the help of Albert staining and extracted by Mussig-Zufika method. Thin layer chromatography and nuclear magnetic resonance spectroscopy (31P NMR) were used to characterize properties of these granules. Relation of phosphorus consumption and poly P production with growth was determined by the vanado-molybdate colorimetric method. Among the 60 strains of bacteria isolated from the environmental samples, strain G11 showed ability for the formation of high levels of poly P. Phylogenetic analysis showed that this isolate had 98% similarity with Bacillus megaterium. 16S rRNA sequence of isolate was deposited in GenBank with accession number JX115009. The average poly P chain length was 10·5 in this bacterium. The antimicrobial activity of bacterial extracted poly P was much better than chemical poly P, and its interaction with gentamicin increased the activity of this drug. The best synergistic activity of this interaction was observed for Corynebacterium glutamicum and Pseudomonas aeruginosa species. The highest adsorption of phosphorus occurred in stationary phase of growth curve, and then the amount of phosphorus increased in medium by degradation of stored poly P.

Conclusions

In this study, we isolated a high-level producer bacterium of poly P and extracted poly P by chemical treatment. In addition, we compared antimicrobial activity of chemical poly P with bacterial poly P and its interaction with gentamicin against both Gram-positive and Gram-negative bacteria.

Significance and Impact of the Study

Many studies have shown that bacteria are becoming resistant to gentamicin sulphate. In this study, we approved that Acinetobacter baumannii, a pathogenic gentamicin-resistant bacterium, is sensitive to bacterial poly P, and thus, this poly P can be substituted for gentamicin in treatment.

Introduction

Polyphosphate (poly P) was observed as the metachromatic granules in yeast for the first time by Lieberman (1888). Later, an enzyme in Escherichia coli that formed the polymer of inorganic phosphate from ATP and readily converted the polymer back to ATP was found and identified as poly P kinase (PPK; Kornberg et al. 1956). Intracellular polymers stored by poly P-accumulating micro-organisms include poly P, polyhydroxyalkanoates (PHAs) and glycogen (Serafim et al. 2002). The main enzymes associated with poly P metabolism in bacteria are poly P kinase (PPK1; responsible for poly P synthesis), poly P kinase (PPK2), poly P–AMP–phosphotransferase (PAP), exo-polyphosphatase (PPX) and endo-polyphosphatase (PPN; responsible for Poly P degradation; Brown and Kornberg 2008; Hooley et al. 2008). Inorganic poly P is a linear polymer of inorganic phosphate (Pi) connected with high-energy bonds (Brown and Kornberg 2008). The anhydride bond energy and Pi of poly P are possible sources for nucleoside triphosphates that form the building blocks of RNA and DNA (Waehneldt and Fox 1967; Kulaev and Skryabin 1974). Poly P has been found in all kinds of living cells and is considered to have various biological functions, for example, Pi reservoir, an alternative source of high-energy bonds and a buffer against alkali or metals. In addition, it has been known that poly P has some regulatory functions in prokaryotic cells, such as in competence for transformation, motility, biofilm formation, gene expression in stressed conditions (that is, physical, chemical, predation and virulence extremes) and protein degradation in amino acid starvation (Brown and Kornberg 2008).

Many heterotrophic bacteria and phytoplanktonic organisms accumulate high amounts of poly Ps under sufficient external phosphorus supply and store them internally as granules. Four different methods can be used for the identification of poly P granules of intact cells: (i) staining of granules followed by light and electron microscopy, (ii) extraction of granules followed by chemical analysis as orthophosphate equivalents, (iii) nuclear magnetic resonance spectroscopy (31P-NMR) analysis in vivo or after chemical extraction (Eixler et al. 2005) and (iv) a direct fluorescence-based DAPI assay system that removes the requirement for prior poly P extraction before quantification (Kulakova et al. 2011).

The oldest and most extensively used methods to document the presence of poly P is the staining of cells with toluidine blue, neutral red or methylene blue. However, a quantification of poly P granules is not possible using these protocols, and the available dyes are not specific. An advantage using 31P-NMR is that the signal of poly P can be distinguished from the signal of other phosphorus-containing compounds (Eixler et al. 2005). NMR spectroscopy is a useful tool in analytical chemistry for the detection, identification and structure elucidation of compounds.

Phosphorus compounds of living cells include phosphates, phosphonates and various esters of phosphates and phosphonates. The chemical shift of 31P atoms in these compounds can span over a 30 ppm range, thus making 31P-NMR spectroscopy an attractive tool for examining phosphorus metabolites in micro-organisms, plants and animal tissues. In addition, the method has no problem of solvent suppression, as no water signal appears in the 31P resonance region (Kulaev et al. 2005). Poly P-accumulating organisms can actively take up soluble ‘p’ from wastewater system and accumulate it in the form of poly P granules (Mino 2000). Several studies showed biological phosphate removal is the best method for ‘p’ uptake in wastewater treatment (Nakamura et al. 1991; Seviour et al. 2003). In addition, poly P extracted from these bacteria has many applications in food industry, dairy industry, medicine and other industries. Inorganic poly P are generally recognized as safe and are widely used as food additives in the meat and dairy industry to enhance major functional properties (i.e. to protect flavour, increase yield due to their water-binding capacity and emulsification properties, retard oxidative rancidity and colour deterioration and to enhance cured-colour development). Some studies demonstrate the antimicrobial properties of poly P on Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium sporogenes, Clostridium tyrobutyricum, Clostridium pasteurianum and Clostridium botulinum, where poly P manifested inhibitory effects on bacterial growth at concentrations commonly used in the food industry (Lee et al. 1994; Loessner et al. 1997; Maier et al. 1999; Akhtar et al. 2008).

In this study, we extracted poly P from a bacterium isolated from soil, identified with 31P NMR and studied the antibacterial activity of poly P on growth of Staph. aureus, Corynebacterium glutamicum, B. cereus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella sp. and E. coli species. In addition, this study describes the synergistic effect of poly P on antibacterial properties of gentamicin.

Materials and methods

Cultivation and isolation of poly P-producing bacteria

Water and soil samples were used as source of samples for screening poly P-accumulating bacteria in this study. Bacterial strains with a capacity of poly P accumulation were isolated using phosphorus-rich medium.

Medium

The phosphorus-rich medium is used to screen and isolate poly P-accumulating bacteria, containing 0·0407 g l−1 MgCl2·6H2O, 0·0059 g l−1 MnC12, 0·010 g l−1 FeSO4·7H2O, 0·014 g l−1 CaCl2, 0·00082 g l−1 ZnCl2, 0·001 g l−1 (NH4)6Mo7O24·4H2O, 2·456 g l−1 K2HPO4 and 10 g l−1 glucose. The pH of the medium was adjusted to 7·2 with addition of 1 N NaOH and HCl (Merck, Darmstadt, Germany; Clark et al. 1986).

Inoculum preparation

Samples (1 g) powdered by mortar were suspended in 9 ml sterile saline solution and vortexed for 1 h (Barton et al. 2001). The phosphorus-rich medium, but without any phosphate, was inoculated by 1% filtrated suspension and incubated at 30°C and 120 rpm for 18 h. After this period, 1 ml of the culture was transferred into 100 ml from the phosphorus-rich medium in a 250-ml Erlen-Mayer flask (1% inoculums v/v). After inoculation, the flasks were incubated at 30°C and 120 rpm for 72 h (Dasan 2002; Vagabov et al. 2008).

Screening of the cultures for phosphate uptake and poly P accumulation

All the isolates obtained were screened for their ability to uptake inorganic phosphate (Pi) from environment and to store the phosphate in an osmotically inert form of poly P granules in the cell. The best strain was selected by microscopic observation with the help of Albert staining method (Mukherjee and Ghosh 1988).

Estimation of the residual phosphate in the medium

Five millilitre of the culture broth was isolated at 6, 12 … 48 and 54 h of culture, and cell growth was monitored by measuring the absorbance of samples at 600 nm. This medium was centrifuged at 3300 g at 5°C for 15 min (Clark et al. 1986), and the supernatant obtained was used for estimation of the residual phosphate in the medium. Colorimetric determination of phosphate is based on the reaction with ammonium vanadate and ammonium molybdate resulting in a bright yellow-coloured complex of phosphovanadomolybdate (λmax = 450 nm; Mussing-Zufika et al. 1994).

Determination of poly P

Cells were harvested at 6, 12 … 48 and 54 h of culture and washed twice in wash buffer (1·5 mol l−1 NaCl containing 0·01 mol l−1 EDTA and 1 mmol l−1 NaF). The suspended cell pellet in 1·5 ml of wash buffer was sonicated on ice for 10-min period with 2-min interval at 16 KHz. After removal of cell debris from the homogenate by centrifugation at 12 000 g (13 500 rpm) for 5 min, total intracellular poly P was determined by acid hydrolysis in concentrated HCl at 100°C for 45 min (100 μl of HCl was added to 0·5 ml of cell extract). The liberated inorganic phosphate was measured by the spectrophotometry at 450 nm. The poly P concentrations were expressed in grams of phosphate per gram of biomass (De Lima et al. 2003).

Extraction and purification of poly P

In the best time of poly P production, cells were collected by centrifugation at 5°C and 2000 g for 15 min, and the pellet used for poly P extraction. In extraction process, all centrifugations were carried out at 11 000 rpm. Extraction was performed in an ultrasonic bath within 20 min, followed by centrifugation. Extraction consists of following stages:

  • Fraction I: After the initial centrifugation, the pellet (approximately 0·1 g wet weight) was successively extracted with 0·5 ml 2 mmol l−1 EDTA (pH 7·0), 0·3 ml 2% TCA (w/v) and 1 ml TCA/acetone (0·7% TCA in 67% acetone/water, w/v). The pellet was washed with 67% acetone (v/v) and centrifuged. The supernatants were combined together as fraction I. It is important to keep the temperature below 5°C during all steps of poly P extraction, especially the first step.
  • Fraction II: The remaining pellet was resuspended with 0·8 ml EDTA. The 0·04 ml of sodium hypochlorite (12%) was added and the pH adjusted to 7–8 (NaOH, 0·2 mmol l−1). The sample was stored overnight at −20°C after the addition of 0·3 ml of a methanol/chloroform mixture (1/1 (v/v), saturated with 0·1 mol l−1 (NH4)2SO4, pH 6·5). The next day the sample was centrifuged, and the supernatant was transferred into a new centrifugation tube. The residue was extracted twice with 0·8 ml 2 mmol l−1 EDTA (pH 7·0) and 0·3 ml CH3OH/CHC13. These supernatants were combined as fraction II (Clark et al. 1986; Mussing-Zufika et al. 1994). Fractions I and II were separately concentrated using a Freeze-drier machine (Hetosicc, Heto, Denmark) and stored at −20°C until further analyses with Thin Layer Chromatography (TLC) and 31P-NMR spectrum. The remaining pellets were not used for further extraction.

Poly P characterization by 31P NMR

After extraction and preliminary study by TLC, samples were dissolved in D2O and analysed in a Bruker AC400 at 400 MHz, using a 15° pulse, 65k data points, a 7·0 s repetition rate and 3072 scans. The poly P signal was expected at a chemical shift of −20 ppm. From the signal intensities of the terminal and middle phosphate groups, the chain length of the poly P can be determined according to eqn (1), where n represents the average chain length, [PP1] the terminal phosphate groups signal intensity and [PP4] the middle phosphate groups signal intensity, respectively (Kulaev et al. 2005):

display math(1)

Identification of isolated strain

DNA extraction

DNA from culture was prepared by boiling. The samples were centrifuged at 9600 g (12 000 rpm) for 10 min. The supernatant was eliminated, and the pellet was suspended in biology-grade water and centrifuged at 9600 g (12 000 rpm) for 10 min. Cells are washed three times by this water. The pellet was twice freeze-thawed and resuspended in 1 ml of molecular biology-grade water and boiled at 100°C in a water bath for 15 min, centrifuged at 6700 g (10 000 rpm) for 5 min, and supernatant was stored at −20°C (Yang et al. 2008).

PCR

PCR was initially carried out on DNA extracts using the universal primers RW01, 5′-AAC TGG AGG AAG GTG GGG AT-3′ and DG74, 5′-AGG AGG TGA TCC AAC CGC A-3′, which are highly conserved among all bacteria. The amplified PCR product is approximately 370 bp in length. Amplification reactions were carried out in a 25-μl reaction volume. Reaction tubes contained 1 μl of each primer pair (0·3 μg μl−1), 0·5 μl dNTPs (0·2 mmol l−1), 2·5 μl ×10 PCR buffer, 0·75 μl MgCl2 (25 mmol l−1), 17 μl PCR H2O, 2 μl template DNA extracted from bacterium and 0·25 μl Taq polymerase (5 U μl−1). The PCR was performed in an Eppendorf Thermal Cycler using appropriate programmes optimized for this primer, after denaturation of DNA by heating for 2 min at 94°C. The PCR programme involved 30 cycles, each cycle consisted of denaturation at 94°C for 2 min, annealing at 55°C for 1 min and extension at 72°C for 1 min. This was followed by a final elongation step for 2 min at 72°C. The PCR products were separated on a 1·7% agarose gel containing ethidium bromide in 1× TBE buffer, run at 100 V for 1·5 h, and the gel was visualized on a UV Transilluminator. The purified PCR product was sequenced in both directions using an automated sequencer by Macrogen (Seoul, Korea). The sequences were edited using Finch TV V.1.4.0., and the blastn program was used for homology searches with the standard programme default (Uh et al. 1998).

Study of interaction of poly P with gentamicin through a qualitative colorimetric method

Interaction of poly P with gentamicin was approved by qualitative colorimetric assay. Gentamicin is a white, water-soluble and poly amine drug, which produces yellow dye in the presence of Nessler reagent. Attachment and interaction of poly P with gentamicin cause abolition of yellow dye (Royds et al. 2005).

Disc diffusion test

In this study, antibacterial effect of chemical poly P, bacterial poly P, commercial gentamicin and a mixture of bacterial poly P and gentamicin (for studying of poly P and gentamicin interaction) was compared by the standardized Bauer-Kirby disc diffusion method (Baur et al. 1966). We tested the antibacterial effect of gentamicin on seven strains with 10-, 20-, 30-, 40-, 50- and 60-μg gentamicin discs. Diameter of the inhibition zone increased from 10 to 50 and then remained constant, and therefore, 50 μg was chosen. Similar experiments were performed for poly P and eventually 5 mg poly P was chosen as the minimum amount to inhibit bacterial growth. Lower concentrations had an inhibition zone diameter <8 mm on all tested bacteria. The bacterial inoculums (0·5 McFarland units) were uniformly spread evenly on Mueller Hinton agar (Merck) using a glass L-rod. In this study, we used aerobic bacteria isolated from clinical specimens of patients. These bacteria included E. coli, Klebsiella sp., Ps. aeruginosa, Staph. aureus and B. cereus (because Enterobacteriaceae, Pseudomonas spp., Enterococcus spp. and Staphylococcus spp. have developed resistance to gentamicin, USP to varying degree and also antibacterial activity of chemical poly P on these bacteria has been studied in several investigation; Lee et al. 1994; Loessner et al. 1997; Maier et al. 1999; Akhtar et al. 2008), Ac. baumannii (because it is an opportunistic human pathogen and key source of infection in debilitated patients in the hospital and usually resistant to gentamicin; Chang et al. 2007; Al Jarousha et al. 2008) and Coryne. glutamicum (because the permeability of the cell envelope of members of Corynebacterineae is particularly low; Bayan et al. 2003).

Blank sterile disc (0·5 cm diameter; Hi-Media) was saturated with 50 μl of chemical poly P (8%), bacterial poly Ps (8%), commercial gentamicin (0·1%) and a mixture of bacterial poly P and gentamicin with former concentrations, allowed to dry and was introduced on the upper layer of the seeded agar plate. After holding the plates at room temperature for 2 h, they were incubated for 24–48 h at 37°C, and the diameter of growth inhibition zone was determined. Methanol/chloroform mixture [1/1 (v/v)] and filter-sterilized distilled water were used as negative controls in this experiment. Freeze-drier machine was used for the evaporation of methanol/chloroform from disc similar to last stage of poly P extraction.

Results

Morphological observation of poly P granules

After staining the bacteria, the presence of poly P granules was observed by light microscope (Fig. 1). The poly Ps were showed dark green after staining by Albert, while others light green. Comparing the culture of bacteria in phosphorus enrichment and phosphorus-deficient media showed that bacteria in phosphorus-rich medium produce poly P granules, while that in phosphorus-deficient medium did not produce these granules. The best poly P producer strain was selected according to the microscopic observations.

Figure 1.

Albert staining of strain G11 on phosphorus-enriched medium: Dark green intracellular granules in the green stained bacteria.

Characterization and identification of strain G11

The bacterium under investigation was isolated from soil sample collected from phosphate fertilizer factory of Mahshahr City in Khuzestan province, Iran. Morphological and physiological characteristics and 16S rRNA gene sequencing were used to identify strain G11. This strain was shown to be a Gram-positive, endospore-forming, rod-shaped, phosphatase-negative and catalase-positive bacterium. PCR gel electrophoresis is shown in Fig. 2. The blast analysis showed that partial 16S rRNA of strain G11 is more than 98% identical to Bacillus megaterium. The accession number of G11 that is submitted to the database of GenBank is JX115009.

Figure 2.

Agarose gel electrophoresis of 16S rRNA gene PCR-amplified region: (Lane 1) strain G11, (Lane 2) Negative control (no added DNA) and (Lane ladder) 100-bp DNA ladder.

Relation of phosphorus consumption and poly P production with growth in Bacillus megaterium

As shown in Fig. 3, the maximum Pi consumption and poly P synthesis occur, respectively, after 30 and 36 h of incubation and growth. The highest adsorption of phosphorus occurred in stationary phase of growth curve, and then, the amount of phosphorus increased in medium by degradation of stored poly P. Actually, bacterial cell lysis is a main factor in the release of phosphorus into the medium.

Figure 3.

Profiles of (♦) growth, (■) phosphorus consumption and (▲) poly P production in Bacillus megaterium strain G11.

31P-NMR spectroscopy characterization of poly Ps

Figure 4 shows 31P NMR spectrums of chemical tripoly P, fraction I and fraction II achieved from extraction of poly P from B. megaterium. Fraction II like positive control contains PP1 and PP4 which indicate fraction II have poly P. The average poly P chain length was 10·5 in B. megaterium.

Figure 4.

The 31P NMR spectrum of different poly P solutions at 400 MHz. (a) Chemical tripoly P, (b) Fraction I and (c) Fraction II PP1 (approximately −7) describes pyrophosphate and terminal phosphate group of poly P with only one oxygen bridging; PP4 (approximately −20 to −22) describes an internal phosphate of poly P and Pi (chemical phase shift approximately 5 to −1) corresponds to an orthophosphate group having no bridging oxygen. The resonances of 4·25, 4·01, 3·78, 3·69 and 3·61 ppm are in the sugar phosphate (SP) region. Extracellular Pi (Piex) gives resonance at 0·64 ppm. The resonance at −1·32 ppm is from phosphomannan. NMR, nuclear magnetic resonance.

Study of interaction of poly P with gentamicin through a qualitative colorimetric method

Ammonium in gentamicin forms a yellow-brownish coloured substituted ammonium salt with Nessler reagent. The amount of colour is a function of the concentration of ammonium. The yellow colour disappeared by the addition of poly P to a gentamicin solution that indicates that poly P can be bonded to gentamicin by ammonia.

Disc diffusion test

Gentamicin is an aminoglycoside antibiotic, used to treat many types of bacterial infections, particularly those caused by Gram-negative organisms, including Pseudomonas, Proteus and the Gram-positive Staphylococcus. This study showed that poly P has antimicrobial activity on Staphaureus, Coryne. glutamicum, B. cereus, Psaeruginosa, Ac. baumannii, Klebsiella sp. and E. coli species and approved the increased antibacterial activity of gentamicin in interaction with bacterial extracted poly P. Table 1 shows the diameter of inhibition zones caused by gentamicin, bacterial extracted poly P, chemical poly P and combination of gentamicin and poly P. The highest antibacterial activity of this aggregation was seen on Coryne. glutamicum and Psaeruginosa species (Fig. 5).

Table 1. Compression of antibacterial effects of gentamicin, chemical poly P and bacterial extracted poly P
BacteriaDiameter of inhibition zones (mm)
GentamicinChemical poly PExtracted poly PGentamicin + extracted poly P
Acinetobacter baumannii 0112222
Klebsiella sp.3171438
Pseudomonas aeruginosa 2171132
Escherichia coli 761012
Corynebacterium glutamicum 38223848
Bacillus cereus 28122032
Staphylococcus aureus 2191821
Figure 5.

Antibacterial effect of gentamicin, extracted poly P and combination of gentamicin and extracted poly P on several bacteria: (a) Staphylococcus aureus, (b) Klebsiella sp., (c) Pseudomonas aeruginosa, (d) Bacillus cereus, (e) Acinetobacter baumannii, (f) Corynebacterium glutamicum.

Discussion

It has been reported that poly P is present in every cell in nature including bacterial, fungal, plant and animal cells (Kulaev and Vagabov 1983; Brown and Kornberg 2008). In this article, we demonstrated the presence of poly P in B. megaterium isolated from soil by several methods (TLC and NMR).

The Clark method (Clark et al. 1986), which contains a cold TCA–acetone extraction step, causes the least poly P hydrolysis in Gram-negative bacteria, but it is not suitable for Gram-positive bacteria, for example, Bacillus due to cell wall. Mussing-Zufika et al. (1994) described a modified extraction method which is a combination of various extraction methods, including chemical treatment and mechanical disruptions. This method does not hydrolyse poly P and produces intact poly P chains that can be analysed further. Poly P with chain length of 10·5 has the inhibitory effects on growth of Ac. baumannii, E. coli, Ps. aeruginosa, Klebsiella sp., Coryne. glutamicum, B. cereus and Staph. aureus. The novelty of this work is a comparative study on the antibacterial activity of chemical poly P, bacterial extracted poly P and its interaction with gentamicin. Previous studies have investigated antibacterial activity of chemical poly P (Lee et al. 1994; Loessner et al. 1997; Maier et al. 1999; Akhtar et al. 2008) and its interaction with various drugs as carriers to bind to drug molecules covalently (Schofield et al. 2006; Liu et al. 2010) but not bacterial poly P. This study showed that the activity of bacterial poly P is much more than chemical poly P, and its aggregation with gentamicin increases the antimicrobial activity of this drug. In contrast to previous researches (Post et al. 1963; Chen et al. 1973) that have suggested that Gram-negative bacteria are generally resistant to poly P, we described the antibacterial properties of bacterial poly P against four Gram-negative bacteria. Importantly, for this study, Ac. baumannii, a pathogenic gentamicin-resistant bacterium, was sensitive to bacterial poly P, and thus, this poly P can be substituted for gentamicin. Bacterial-extracted poly P not only has antimicrobial effects, but its interaction with gentamicin showed high efficiency of this polymer in increasing drug effects.

The nature of growth inhibition mechanism is still unknown, but the presence of negative binding sites in condensed poly Ps may favour chelation of essential metal ions (Mg2+ and Ca2+) and leakage of cell membrane and therefore cause growth inhibition and cell lysis. Actually, bactericidal effect of poly P is dependent on the logarithmic phase (Maier et al. 1999; Nereus and Gunther 2010). Chelation of divalent metals is the only one of the proposed biological roles of poly P. It seems that interaction of poly P with gentamicin can facilitate adhesion of gentamicin to 30-s subunit ribosome and so enhance its antibacterial effects. Therefore, the antimicrobial potency of gentamicin mostly was markedly enhanced by the incorporation with bacterial poly P (Mcinerney et al. 2006). Bacterial poly P as a biological material is better than chemical poly P in several respects. Biological materials form complex arrays and hierarchical structures and are also tools or machines that perform very specific operations. They have many distinctive features, such as being the result of evolution and being multifunctional (one material has more than one function), whereas evolution is not considered in synthetic materials and multifunctionality still needs further investigation. Moreover, these materials do not react specifically with their target (Meyers et al. 2008). These can be interpreted as reasons for the more antibacterial activity of bacterial-extracted poly P than chemical poly P.

Acknowledgements

This study was supported by grant from the University of Isfahan to Sara Ghashghaei for obtaining M.Sc. degree. In addition, we would like to thank Gh. R. Ghezelbash and Dr. M. H. Habibi from University of Isfahan and E. Ghashghaei from Shahid Chamran University of Ahvaz for helping.

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