Document Type : Research Paper
Authors
1 Assistant professor, Department of Biology, Rasht Branch, Islamic Azad university, Rasht, Iran.
2 Department of biology, Rasht Branch, Islamic Azad University, Rasht, Iran
Abstract
Background and Aim: The spread of antibiotic resistance, especially in gram-negative bacteria, has led to the use of the combined effect of antibiotics and obtaining herbal compounds in the treatment of bacterial infections. The aim of the present study was to investigate the effect of the active ingredients allicin and cinnamon on β-lactam-resistant Pseudomonas aeruginosa.
Materials and Methods: The herbal active ingredients allicin and cinnamon were purchased from Sigma. 20 clinical strains of P.aeruginosa prepared from the Pathobiology Laboratory of in Rasht were subjected to biochemical tests to confirm the diagnosis and purification of the bacteria, including the ability to produce hemolysin, oxidase, catalase, urease, the ability to grow at 42 degrees Celsius, the growth profile in MCA, TSI, and SIM media, the growth profile in mueller-hinton agar, and gram staining. To investigate the antibacterial effects of the active ingredients, cinnamon at a concentration of 0.5 mg/ml and allicin at a concentration of 0.004 mg/ml, the antibiogram test (Kirby Bauer) and the quantitative minimum inhibitory concentration test using the MIC and MBC serial dilution methods were used, and finally the diameter of the growth inhibition zones around the discs impregnated with the active ingredient was measured and recorded. In the following in-silico test, for the preparation of ligand and protein, the three-dimensional structure of allicin and cinnamon and the proteins of both were obtained from the database (https://pubchem.ncbi.nlm.nih.gov) and www.rcbs.org, respectively. Then, using the Discover Studio software, the rotatable charges and the center of gravity of the molecule in all bands were determined and the molecular docking for the protein was repeated 200 times independently. In this study, the Lamarckian GA genetic algorithm was used and the obtained data was analyzed. RNA extraction, cDNA synthesis and RT-PCR were performed to determine the effect of the active ingredients on the expression level of the blaIMP gene.
Results: The 20 clinical strains studied were identified as P.aeruginosa based on colony morphology, colony odor, pigment formation in Mueller Hinton agar, hemolysin production on blood agar, Gram stain, positivity in oxidase test, growth at 42°C on nutrient agar, motility test positivity, and lack of sugar fermentation in MCA and TSI media. The mean diameter of the growth inhibition zone in the disk diffusion test of Pseudomonas aeruginosa bacteria in the intervention with 0.004 mg/ml allicin was 16 mm and 0.5 mg/ml cinnamon was 17 mm. The active ingredient allicin was reported to be MIC 0.001 and MBC 0.002 mg/ml in one strain, and MIC and MBC were reported to be 0.002 and 0.004 mg/ml in the other strains, respectively. The active ingredient of cinnamon in one strain had a MIC of 0.125 and an MBC of 0.25 mg/ml, and in other strains, the MIC was 0.25 mg/ml and the MBC was 0.5 mg/ml. The results of molecular docking between cinnamon and the beta-lactamase protein showed that the lowest minimum free energy of binding (ΔG) was found (- 4.83 kcal.mol-1), while the binding energy between allicin and the β-lactamase protein was - 4.124 kcal.mol-1. According to the data, it can be concluded that the active ingredient of cinnamon has the greatest effect on the β-lactamase protein and cinnamon has a favorable effect on the weakening of the function and activity of the β-lactamase protein. The small difference in the minimum free energy of binding (ΔG) of the two effective substances cinnamon and allicin on β-lactamase, with the interaction of the control drug sulfonamide and β-lactamase (-5.34 kcal.mol-1), shows that the effect and interaction of cinnamon and allicin on the β-lactamase protein is positive. Cinnamon and allicin both led to a decrease in the expression of the blaIMP gene in the bacteria Pseudomonas aeruginosa at subMIC concentrations.
Conclusion: Today, one of the main problems in relation to pathogenic microorganisms is their increased resistance to antibiotics. β-lactam-resistant P.aeruginosa strains treated with cinnamon and allicin showed a decrease in the expression of the β-lactam resistance gene compared to the untreated strain, and the result is that treatment with cinnamon and allicin leads to a decrease in beta-lactam resistance. It is suggested that the two active ingredients, cinnamon and allicin, be used alongside chemical drugs to combat infections caused by the bacteria P.aeruginosa after conducting standard clinical trials.
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