Document Type : Research Paper
Authors
1 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 increasing spread of antibiotic resistance, particularly among Gram-negative bacteria, has encouraged the exploration of combined therapeutic approaches involving antibiotics and plant-derived compounds for the treatment of bacterial infections. Therefore, the present study aimed to investigate the effects of the bioactive compounds allicin and cinnamon on β-lactam-resistant Pseudomonas aeruginosa.
Materials and Methods: The herbal active compounds allicin and cinnamon were purchased from Sigma‑Aldrich. Twenty clinical strains of Pseudomonas aeruginosa obtained from the Pathobiology Laboratory of Razi Hospital were used in this study. The isolates were subjected to standard biochemical tests to confirm identification and ensure bacterial purity. These tests included hemolysin production, oxidase, catalase, and urease activities, growth at 42 °C, and growth characteristics on MacConkey agar (MCA), triple sugar iron agar (TSI), sulfide–indole–motility (SIM) medium, and Mueller–Hinton agar, as well as Gram staining. To evaluate the antibacterial effects of the active compounds, cinnamon at a concentration of 0.5 mg mL⁻¹ and allicin at a concentration of 0.004 mg mL⁻¹ were tested. Antibacterial activity was assessed using the antibiogram method (Kirby–Bauer disk diffusion test) and quantitative determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the serial dilution method. The diameter of the bacterial growth inhibition zones around discs impregnated with the active compounds was measured and recorded. For the in-silico analysis, the three-dimensional structures of allicin, cinnamon, and the target proteins were obtained from the PubChem database and the RCSB Protein Data Bank, respectively. Using Discovery Studio software, rotatable bonds, atomic charges, and the center of gravity of the molecules were determined. Molecular docking simulations were performed for the target proteins and repeated independently 200 times. In this study, the Lamarckian genetic algorithm (LGA) was applied, and the resulting data were analyzed. Finally, RNA extraction, cDNA synthesis, and RT-PCR were carried out to evaluate the effects of the active compounds on the expression level of the blaIMP gene.
Results: The 20 clinical strains were confirmed as Pseudomonas aeruginosa based on colony morphology, characteristic odor, pigment production on Mueller–Hinton agar, hemolysin production on blood agar, Gram staining (Gram-negative bacilli), positive oxidase test, growth
at 42 °C on nutrient agar, motility, and lack of sugar fermentation on MacConkey agar and TSI media. In the disk diffusion assay, the mean diameter of the growth inhibition zone was 16 mm for 0.004 mg·mL⁻¹ allicin and 17 mm for 0.5 mg·mL⁻¹ cinnamon. The MIC and MBC of allicin were 0.001 and 0.002 mg·mL⁻¹ for one strain, and 0.002 and 0.004 mg·mL⁻¹ for the remaining strains, respectively. For cinnamon, the MIC and MBC were 0.125 and 0.25 mg·mL⁻¹ for one strain, and 0.25 and 0.5 mg·mL⁻¹ for the others. Molecular docking analysis revealed that the minimum free binding energy (ΔG) between cinnamon and β-lactamase was –4.83 kcal·mol⁻¹, whereas that for allicin was –4.12 kcal·mol⁻¹. These results indicate that cinnamon has a stronger inhibitory interaction with β-lactamase and effectively reduces its activity. Compared with the control drug sulfonamide (ΔG = –5.34 kcal·mol⁻¹), the small difference in binding energies suggests that both cinnamon and allicin positively interact with β-lactamase. Furthermore, treatment with sub-MIC concentrations of both compounds resulted in decreased expression of the blaIMP gene in P. aeruginosa.
Conclusion: One of the major challenges in combating pathogenic microorganisms is the increasing prevalence of antibiotic resistance. In this study, β-lactam-resistant Pseudomonas aeruginosa strains treated with cinnamon and allicin exhibited reduced expression of the β-lactam resistance gene compared to untreated strains, indicating that these compounds can decrease β-lactam resistance. Based on these findings, it is suggested that cinnamon and allicin could be used in combination with conventional antibiotics to treat P. aeruginosa infections, following validation in standard clinical trials.
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