Background: Growing antibiotic resistance has made treating otitis externa (OE) increasingly challenging. On the other hand, local antimicrobial treatments, especially those that combine essential oils (EOs) with nanoparticles, tend to be preferred over systemic ones. It was investigated whether Ajwain (Trachyspermum ammi) EO, combined with chitosan nanoparticles modified by cholesterol, could inhibit the growth of bacterial pathogens isolated from OE cases in dogs. In total, 57 dogs with clinical signs of OE were examined and bacteriologically tested. Hydrogels of Chitosan were synthesized by self-assembly and investigated. EO was extracted (Clevenger machine), and its ingredients were checked (GC-MS analysis) and encapsulated in chitosan-cholesterol nanoparticles. Disc-diffusion and broth Micro-dilution (MIC and MBC) examined its antimicrobial and therapeutic properties. Results: Staphylococcus pseudintermedius (49.3%) was the most common bacteria isolated from OE cases, followed by Pseudomonas aeruginosa (14.7%), Escherichia coli (13.3%), Streptococcus canis (9.3%), Corynebacterium auriscanis (6.7%), Klebsiella pneumoniae (2.7%), Proteus mirabilis (2.7%), and Bacillus cereus (1.3%). The investigation into the antimicrobial properties of Ajwain EO encapsulated in chitosan nanoparticles revealed that it exhibited a more pronounced antimicrobial effect against the pathogens responsible for OE. Conclusions: Using chitosan nanoparticles encapsulated with EO presents an effective treatment approach for dogs with OE that conventional antimicrobial treatments have not cured. This approach not only enhances antibacterial effects but also reduces the required dosage of antimicrobials, potentially preventing the emergence of antimicrobial resistance.
Keywords: Essential oil; Trachyspermum ammi; Otitis externa; Nanoparticles; Chitosan; Hydrogels
Ajwain, scientifically known as Trachyspermum ammi (T. ammi), belongs to the Apiaceae family and the Apiales order. The well-known T. ammi fruit or dried seeds are regarded as a highly nutrient-dense or therapeutically enhanced component of the plant [[
Numerous studies have extensively investigated the antimicrobial properties of EOs and their constituents. However, EOs are volatile and delicate compounds prone to enzymatic reactions and susceptible to degradation when exposed to oxygen, light, moisture, and heat. These factors can compromise their biological properties, leading to decreased activity and increased toxicity, which limits their traditional use [[
Chitosan (CS) and its derivatives have found remarkable applications in the biomedical field, particularly in the controlled release of active substances. CS is commonly modified to form a hydrogel (HG) for this purpose [[
Canine Otitis Externa (OE) is a common skin disease affecting dogs, accounting for approximately 20% of small-animal counseling cases [[
Topical antimicrobial therapy is the preferred treatment approach for OE, as systemic medications are less effective. However, conventional drugs often encounter bacterial resistance. Hence, EOs are being explored as alternative therapies, and further studies are needed to determine their efficacy against the bacteria that cause OE [[
Based on the results of cytological analysis of swabs obtained from dogs suffering from OE, the frequency of gram-positive cocci, gram-negative rods, gram-positive rods, and gram-positive cocci/rods were 58.66%, 33.33%, 1.33%, and 6.66%, respectively.
A total of 75 bacterial species were isolated from dogs with OE, indicating their involvement in causing the condition. Among these, Staphylococcus pseudintermedius was the most frequently observed bacterium, with a frequency of 37 (49.3%). Following S. pseudintermedius, the next most commonly identified bacteria were Pseudomonas aeruginosa (11 (14.7%)), Escherichia coli (10 (13.3%)), Streptococcus canis (7 (9.3%)), Corynebacterium auriscanis (5 (6.7%)), Klebsiella pneumoniae (2 (2.7%)), Proteus Mirabilis (2 (2.7%)), and Bacillus cereus (1 (1.3%)). The isolated bacterial species were identified based on several criteria, including Gram morphology, culture morphology, and specific biochemical tests associated with each species. The identification process followed the guidelines and references provided by Markey et al. and Quinn, widely recognized references in bacterial identification [[
No antibiotic can provide 100% effectiveness against all isolated bacteria. However, the isolated bacteria in this study demonstrated satisfactory susceptibility to certain antibiotics. Specifically, they showed sensitivity to Amikacin, Gentamicin, Cephalothin, and Ceftriaxone. S. pseudintermedius exhibited high sensitivity to Amikacin, Cephalothin, and Gentamicin while showing notable resistance to Penicillin G, Ampicillin, and Oxytetracycline. In general, Penicillin G, Erythromycin, and Ampicillin demonstrated the highest levels of antibiotic resistance among the bacteria examined (Table 1). The quality control strains used in the study yielded results following the guidelines set by the CLSI, indicating that the test conditions were appropriate and reliable [[
Table 1 Antimicrobial resistance percentage of isolated bacteria from dogs with OE
Bacteria Percentage of Resistance for Each Antibiotic P5 Enr5 Kf30 Amp2 Ot30 Rd5 E15 Ak30 Cn10 Cro30 64.86 40.54 2.7 56.8 37.8 21.6 29.7 0 8.1 5.4 80 70 40 80 30 50 20 10 10 20 54.5 81.8 36.4 63.6 72.7 45.4 9.1 9.1 27.3 18.2 100 100 50 50 50 50 50 0 50 50 28.6 42.9 0 14.3 57.1 14.3 28.6 0 14.3 28.6 100 100 40 100 40 60 40 0 0 0 100 50 0 0 0 100 50 0 0 0 100 100 0 100 0 0 100 0 0 0
P5, Penicillin G; E15, Erythromycin; Kf30, Cephalothin; Amp2, Ampicillin; Ot30, Oxytetracycline; Rd5, Rifampcin; Enr5, Enrofloxacin; Ak30, Amikacin; Cn10, Gentamicin; Cro30, Ceftriaxone
Using gas chromatography/mass spectrometry (GC/MS) analysis, the study identified 11 compounds present in T. ammi EO, which was extracted in the present study (Table 2). Among these compounds, Thymol was present in a notably high percentage, accounting for 48% of the EO composition.
Table 2 Chemical analysis of EO of T. ammi by GC/MS
SN. Components Percentage Formula 'RT* 1. Thymol 48 C10H14O 78.23 2. 23 C10H14 45.17 3. γ-Terpinene 17 C10H16 43.43 4. 1.3 C10H16 17.26 5. 0.6 C10H16 13.26 6. Sabinene 0.8 C10H16 12.19 7. 0.5 C10H16 10.38 8. 0.4 C10H16 11.62 9. Terpinene 0.4 C10H16 10.23 10. 0.3 C10H16 7.84 11. 0.2 C10H16 6.78
SEM analysis revealed that the produced Nano-gels exhibited a diameter of less than 100 nm, indicating their nanoscale size. Furthermore, the Nano-gels displayed a uniform and spherical morphology (Fig. 1). The encapsulation efficiency of the EO was determined to be 67.23%, indicating a substantial accumulation of the EO within the Nano-gels.
Graph: Fig. 1Scanning electron microscopy (SEM) image of CS-CHOL Nano-gel
The findings indicate that the antibacterial effects of T. ammi EO encapsulated in CS-CHOL Nano-gel are more pronounced than those of the free form of T. ammi EO (Fig. 2). Negative control discs containing CS-CHOL Nano-gel alone did not affect the six bacterial strains tested. The most significant inhibitory effect of T. ammi EO was observed against S. pseudintermedius. On the other hand, the lowest inhibitory effect of T. ammi EO encapsulated in CS-CHOL Nano-gel was observed against P. aeruginosa (Fig. 2). The results were considered significant when the p-value was less than 0.05 for the T. ammi EO and T. ammi EO encapsulated in CS-CHOL Nano-gel groups.
Graph: Fig. 2The diagram of the inhibition zone diameter of the selected microbial strains against the disc containing T. ammi EO encapsulated in CS-CHOL Nano-gel (a) and a disc containing T. ammi EO (b). P, P-value; Staph, S. pseudintermedius ; Strep, S. canis , Cor, C. auriscanis ; Pseudo, P. aeruginosa ; Pro, P. Mirabilis
The results of MIC tests showed a more significant inhibitory effect of T. ammi EO encapsulated in CS-CHOL Nano-gel compared to CS-CHOL Nano-gel without T. ammi EO (P < 0.05) (Fig. 3-a) and T. ammi EO (P < 0.05) (Table 3). In both repetitions of the experiment, the results were the same. By comparing the MIC of T. ammi EO and CS-CHOL Nano-gel with T. ammi EO, with S. pseudintermedius, S. canis, C. auriscanis, E. coli, and P. Mirabilis, the P-value was less than 0.05, and significant results were reported. Comparison MBC of T. ammi EO and T. ammi EO encapsulated in CS-CHOL Nano-gel for S. pseudintermedius, S. canis, and E. coli bacteria reported significant results (Table 3).
Graph: Fig. 3The MIC of T. ammi EO encapsulated in CS-CHOL Nano-gel (Name of bacteria-Nano CCL) and CS-CHOL Nano-gel without T. ammi EO (Name of bacteria-CCL) on the selected bacteria (a). The MBC of T. ammi EO encapsulated in CS-CHOL Nano-gel (Name of bacteria-Nano CCL) and CS-CHOL Nano-gel without T. ammi EO (Name of bacteria-CCL) on the selected bacteria (b)
T. ammi EO encapsulated in CS-CHOL Nano-gel had an excellent inhibitory effect on S. pseudintermedius, S. canis, P. aeruginosa, and P. Mirabilis, so the mean MIC for these bacteria was 112.5 ppm. The mean MIC for C. auriscanis and E. coli was 137.5 ppm, which indicates that higher amounts of T. ammi EO encapsulated in CS-CHOL Nano-gel are needed. Among the tested bacterial samples, S. pseudintermedius and S. canis were the most sensitive to T. ammi EO encapsulated in CS-CHOL Nano-gel (Table 3), and their mean MIC was 68.75 ppm. CS-CHOL Nano-gel with T. ammi EO shows a satisfactory bactericidal effect (MBC) for S. pseudintermedius and S. canis (Fig. 3-b). In general, there is little difference between MIC and MBC values.
Table 3 Statistical analysis and comparison of MIC and MBC of T. ammi EO and T. ammi EO encapsulated in CS-CHOL Nano-gel with each selected bacteria separately by T-test
Indicator S. pseudintermedius S. canis C. auriscanis E. coli P. Mirabilis P. aeruginosa MIC EO* 0.002 0.002 0.001 0.003 0.001 1 CS-EO** 0.004 0.004 0.001 0.003 0.002 1 MBC EO 0.002 0.002 1 0.003 1 1 CS-EO 0.004 0.004 1 0.003 1 1
Various studies have employed different cross-linking methods to synthesize Nano-gels to microencapsulate EOs. These methods include self-assembly, ionic cross-linking, cross-linking polymerization, crystallization, radiation cross-linking, and functional group cross-linking. Each method offers unique advantages and can be tailored to meet specific requirements regarding the desired properties and applications of the Nano-gels [[
The hydrophilic nature of chitosan (CS) is a crucial characteristic that allows for creation of self-assembled nanoparticles by incorporating hydrophobic components into CS. This property makes CS well-suited for applications in drug delivery. The hydrophobic cavities within CS can serve as storage compartments for various bioactive materials. The drug's therapeutic efficiency can be improved by binding targeted components to the drug-loaded nanoparticles' surface [[
The present study confirmed the shape and size of the Nano-gels through SEM imaging, which revealed well-separated spherical nanoparticles without any aggregation. This lack of aggregation can be attributed to the positive charges on the CS, which prevent the Nano-gels from sticking together. Previous research by Kosaraju et al. (2006) has reported that interactions between polyphenols and matrix polymers can result in smooth surface particles. Therefore, intermolecular or intramolecular interactions may contribute to the relatively smooth surface of the nanoparticles [[
The antimicrobial properties of T. ammi EO have been demonstrated in various studies [[
In the current study, the loading capacity of T. ammi EO in the Nano-gel was determined to be 67.23%, indicating successful storage of the EO in the Nano-gel. The structure of the Nano-gel was also found to protect the EO and control its release effectively. The durability of EO-containing nanoparticles can be attributed to the slow and stable release of active components within the nanoparticles [[
The free and encapsulated T. ammi EO demonstrated excellent inhibitory activity against S. pseudintermedius and S. canis. The growth-inhibiting activity of the encapsulated T. ammi EO was more pronounced than that of the free EO against P. Mirabilis and C. auriscanis. Statistical analysis of the MIC and MBC results in the present study indicated that the encapsulated T. ammi EO exhibited a strong antibacterial effect against Gram-positive bacteria, such as S. pseudintermedius and S. canis, as well as Gram-negative E. coli. Furthermore, no significant difference was observed in the MIC and MBC results between the CS-CHOL Nano-gel without T. ammi EO, and T. ammi EO encapsulated in CS-CHOL Nano-gel in P. Mirabilis species. Similar findings were reported by Zarenejad et al. in 2022, where no substantial difference in effectiveness was observed in Pseudomonas spp. when evaluating the efficacy of EOs. The reduced antibacterial effect of T. ammi EO against Gram-negative bacteria can be attributed to the unique cell walls of these bacteria, which provide them with greater resistance to the antimicrobial activity of the EO [[
Several studies conducted on OE have reported findings similar to the current research regarding the isolation of infectious agents and their antibiotic resistance patterns. In 2023, Abani et al. isolated Staphylococcus spp., Streptococcus spp., Pseudomonas spp., and Enterobacteriaceae spp. from cases of OE in dogs, and these isolates showed evident antibiotic resistance, similar to the findings of the present study [[
In order to mitigate the adverse effects and potentially reverse antimicrobial resistance, one effective treatment strategy is to reduce the usage of antimicrobials. Additionally, the development of drug combinations has emerged as a novel approach to controlling resistant pathogens. Combining antimicrobials with EOs against resistant bacteria can broaden the antimicrobial spectrum, thereby reducing the emergence of resistant variants and minimizing the reliance on a single antimicrobial agent [[
There is an undeniable and urgent need for alternative treatment options for infections caused by antibiotic-resistant bacteria. Addressing this issue requires proactive initiatives and efforts from the scientific and medical communities. Developing novel therapies, exploring alternative antimicrobial agents, promoting antimicrobial stewardship programs, and implementing infection prevention and control measures are some of the key strategies that can help combat the challenge of antibiotic resistance. It is crucial to prioritize research and funding in this area to ensure the availability of effective treatment options for patients affected by antibiotic-resistant infections. The present study's findings support the effectiveness of T. ammi EO in HGs against the pathogens responsible for OE. The superior antibacterial effects observed with EO encapsulated in HGs, compared to free EOs and CS-CHOL Nano-gel, indicate the potential of such HGs in combination with other commonly used antimicrobials for treating OE. This approach offers a way to reintroduce EOs and antimicrobials that may have been abandoned due to widespread resistance. Combining these agents in HGs can enhance the antibacterial effects, and the required dosage of antimicrobials may be reduced, potentially preventing the emergence of antimicrobial resistance.
Polysaccharide Chitosan (Sigma, USA) with low molecular weight and about 8% degree of deacetylation was purchased; Brain heart infusion (BHI), mannitol salt agar (MSA) and Mc-Conkey (Merck, Germany); Mueller-Hinton agar (MHA) (Oxoid, UK); API-Coryne system (BioMerieux, France); 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Cholesterol, Acetic acid, Dimethyl sulfoxide (DMSO), blank paper discs were obtained from Padtan-Teb Company, Iran; Commercial antibiotic disks (Oxoid Ltd, UK); and fruit of T. ammi plant was purchased from Pakan Seed Company (Isfahan, Iran) with herbarium stock number, 293-0303-1.
Investigations were conducted into fifty-seven dogs with OE referred to the Veterinary Hospital of Small Animals. The most important signs in the history of affected animals included itching of the ear, abnormal secretions, and malodorous ears. In clinical examination, symptoms such as redness of the auricle and external ear canal, pain in palpation of the ear, abnormal secretion, and malodorous ear were observed. OE was confirmed in all cases by otoscope and cytological examination. Animals with any history of other diseases or antibiotic therapy within the last two months were excluded from this study. If several cases from the same housing were referred with OE, sampling was conducted from only one.
Samples were collected from dogs that exhibited signs of OE, including ear itching, abnormal secretions, and odor. The sampling procedure involved using sterile swabs to collect samples from the end of the vertical part of the external ear. Three sterile swabs were taken to prevent contamination before conducting an otoscope examination. Each swab was used for different analyses, including cytological and bacterial cultures. A third swab was replaced if any swabs became contaminated or damaged during the procedure. Care was taken to ensure that the swab made minimal contact with the outer portion of the ear canal and the surrounding hairs when entering and exiting during the sampling process. The swab was rotated 360° inside the ear canal for approximately five seconds. Following sample collection, the swabs were rolled onto cytological slides and fixed with heat. Gram staining was performed on the slides and was subsequently evaluated microscopically. The swabs were sent to a laboratory within two hours for further analysis.
The process related to bacteriology, including aerobic cultivation in blood agar (Columbia agar with 5% sheep blood), BHI, MSA, and MacConkey. The cultures were incubated at 37 °C for 24 to 48 h. Bacterial species were identified by Gram morphological and biochemical characteristics (TSI, Urease test, IMViC, Motility test, Lecithinase Test, oxidase, Catalase, Nitrate reduction, and Coagulase.), as described by Murray et al. and Markey et al. [[
The fruit of the T. ammi plant was purchased, and after confirming the purity of the seed, the EO was extracted from the milled powder of the fruit by steam distillation using a Clevenger machine. EO was dehydrated with sodium sulfate. It was kept in a dark glass container with a closed lid, away from light, and at a cool temperature [[
The EO compounds were identified using GC/MS. The system consisted of an Agilent 6890 gas chromatograph with an Agilent 5973 mass-selective detector (Agilent, USA). Since the compounds in EOs are known as volatile substances in terms of molecular weight and polarity, the process of separating and identifying the components of EO was carried out by gas chromatography combined with mass spectrometry.
The Nano-gel was prepared using a self-assembly method involving CS and cholesterol (CHOL) with the assistance of an EDC cross-linker [[
To incorporate CHOL into the CS solution, 215 mg of CHOL was initially dissolved in 10 µl of ethanol. Subsequently, 178 µl of EDC was added to the CHOL solution, and the mixture was kept in darkness for a short period. To the formation of amid linkages between the carboxyl groups of CHOL (C
The resulting Nano-gel particles were dispersed in a 1% acetic acid solution. The dispersion was filtered using a filter with a pore size of 0.2 mm to obtain a uniform particle size. This filtration step helped to remove any larger particles or aggregates from the Nano-gel suspension. Subsequently, the filtered Nano-gel particles were dried thoroughly using a vacuum dryer. The dried Nano-gel was then stored in a dark glass container at a cool temperature.
SEM was employed to investigate the morphology and size of the CS-CHOL Nano-gel. The SEM analysis was conducted at an accelerating voltage of 70 kV to obtain detailed information about the structure and size distribution of the Nano-gel particles.
A total of 0.5 µl of T. ammi EO was dissolved in 1 µl of DMSO, then 0.5 µl of the solution was diluted with deionized water to reach a final volume of 10 µl. This solution was named Solution Number 1. CS-CHOL Nano-gel (1 mg) was added to 1 µl of Solution Number 1 and kept for 24 h at room temperature. It was centrifuged for 15 min at 12,000 rpm and 20 °C to separate the particles. In the end, two separate phases were created; the supernatant was discarded, and the sediment containing CS-CHOL Nano-gel encapsulated with T. ammi EO was used for microbial tests. The optical absorbance of the supernatant was taken by UV-Vis spectrophotometry using а Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA) at a wavelength of 280 nm to calculate the Loading efficiency of the EO according to the formula [[
1
Graph
Six bacterial strains that exhibited higher abundance in dogs affected by OE were selected for the next steps: S. pseudintermedius, S. canis, P. aeruginosa, E. coli, C. auriscanis, and P. Mirabilis. These strains were preserved in glycerol broth and stored at -80 °C. The selected strains were utilized to examine the impact of free EO and encapsulated EO.
In order to assess the antibacterial activity, 30 µl of T. ammi EO and T. ammi EO in CS-CHOL Nano-gel (100 µg ml
Graph: Fig. 4Determination of the antimicrobial sensitivity of the EO by the disc diffusion method on Pseudomonas. K: CS-CHOL Nano-gel as a negative control disk; Z: free T. ammi EO; Z + N: T. ammi EO in CS-CHOL Nano-gel
The antimicrobial susceptibility patterns of the isolated bacterial strains were determined using the disk diffusion test on MHA. The test was performed following the recommendations of the Clinical Laboratory Standards Institute (CLSI) as described in the 33rd Edition of the M100 guidelines (2023) [[
After incubation for 24 h at 37 °C, the diameters of the inhibitory zones around the antibiotic disks were measured and evaluated according to CLSI guidelines. The strains were classified as sensitive or resistant to the drug, with intermediate susceptibility considered resistant. Only the percentage of resistance is reported in the results. Strains obtained from the American Type Culture Collection (ATCC), such as E. coli 25,922, S. aureus 25,923, S. pneumoniae 49,619, and P. aeruginosa 27,853, were used for quality control.
The MIC of encapsulated T. ammi EO in CS-CHOL Nano-gel against the bacteria isolated from cases of OE in dogs was determined using the in vitro broth microdilution method. The experimental protocol used in the present study was based on the methods described by Sharifi and Nayeri Fasaei [[
Each bacterial isolate was subjected to individual testing in a separate experiment, and each experiment was repeated twice. Two wells were utilized in each microplate for the positive and negative controls. The positive control consisted of 100 µl of BHI broth and 100 µl of microbial suspension. Conversely, the negative control contained 100 µl of BHI broth and 100 µl T. ammi EO in CS-CHOL Nano-gel. The microplates were then incubated aerobically at 37 °C for 24 h. After incubation, the wells were visually examined, and the optical absorbance was measured using an ELISA reader. The MIC was determined to be the lowest concentration of T. ammi EO in CS-CHOL Nano-gel, at which no visible growth was detected in the wells. To assess the effects of the CS-CHOL Nano-gel without T. ammi EO, the MIC was determined individually for each bacterium.
The MIC of the EO alone was also determined to compare the effects of T. ammi EO alone with the effects of the EO encapsulated in CS-CHOL Nano-gel. To enhance the solubility of T. ammi EO, it was diluted 1:10 with a 1.5% DMSO solution. DMSO is widely recognized as non-toxic when used at concentrations below 10% (v/v) [[
The collected data was analyzed using SPSS 25.0 software (SPSS Inc., Chicago, IL). A comparison between T. ammi EO, CS-CHOL Nano-gel encapsulated with T. ammi EO, and CS-CHOL Nano-gel without T. ammi EO was conducted using an independent t-test with four repetitions.
The authors are sincerely grateful to the Laboratory of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, and University of Tehran's Veterinary Hospital of Small Animals.
Conceptualization, SJ, and BNF; methodology, BNF; software, NJN and SMJ; validation, SJ, and BNF; formal analysis, NJN; investigation, NJN; resources, NJN; data curation, NJN and SMJ; writing—original draft preparation, SMJ; writing—review and editing, SMJ and BNF; supervision, SJ and BNF; project administration, BNF; funding acquisition, SJ All authors have read and agreed to the published version of the manuscript.
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
All relevant data is contained within the manuscript. The data generated and analyzed during the current study are available from the corresponding author on request.
Due to obtaining the consent of the animal owners and their presence, as well as respecting the patient's rights and humane principles during sampling, the need for ethics approval by the ethics committee of the Faculty of Veterinary Medicine of the University of Tehran, Iran, was waived. Verbal informed consent was obtained from all owners to collect samples from the dogs. The ethics committee approved verbal informed consent. The researchers dated and signed the consent form indicating that "I have read and explained this consent form to the participant before receiving the participant's consent, and the participant had knowledge of its contents and understood it.
Not applicable.
The authors declare that they have no competing interests.
- T. ammi
- Trachyspermum ammi
- EO
- Essential Oil
• HG
- Hydrogel
• CS
- Chitosan
• OE
- Otitis Externa [inflammation of the external ear canal
• DMSO
- Dimethyl sulfoxide
• CHOL
- Cholesterol
• EDC
- Ethylcarbodiimide hydrochloride
• SEM
- Scanning Electron Microscopy
• GC/MS
- Gas Chromatography-Mass Spectrometry
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By Niloofar Jelokhani Niaraki; Shahram Jamshidi; Bahar Nayeri Fasaei and Seyed Mehdi Joghataei
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