Patients - clinical samples
Four isolates, A, B, C, and D of Pseudomonas aeruginosa, were recovered from burn wound infection in patients in the dedicated burns unit-Shahid Motahari hospital-Tehran. The samples were collected in Nutrient agar containing 0.5% Peptone, 0.3% beef extract, 1.5% agar, and 0.5% Sodium Chloride (37 ℃ / 24 h) and then incubated aerobically at 37 ℃ for overnight in a Mueller-Hinton agar containing 2.0 g beef extract, 17.5 g casein hydrolysate, 1.5 g starch and, 17.0 g agar. Pseudomonas aeruginosa strain ATCC 9027 was selected as a standard (STD) control (RRID:WB-STRAIN:WBStrain00041978).
Diagnostic test of bacteria was performed using Gram staining and biochemical tests, including catalase, oxidase, glucose fermentation test, pigment formation, and growth at 42 °C in cetrimide agar and arginine dehydrogenase.
Plants collection, identification, and preparation of plant extract
The leaves of Plantago major and Plantago lanceolata were collected from Tehran and were identified by the Research Institute of Forests and Rangelands. Then 100 grams of the leaves were separately dried in the shade, pulverized by a mechanical grinder, and stored in airtight glass containers in the dark until extraction. Ethanolic extraction (50%) was performed through the Maceration method. 30 mL of distilled water was added to 6 grams of plant extracts and dissolved well at 40 °C. Then, the volume of the solution was increased to 250 mL in a decanter funnel. Then, 200 mL of Petroleum ether was added to it, and after shaking, the petroleum phase was separated. To the remaining aqueous phase was added 100 mL of chloroform, because of which the chloroform phase was separated from the aqueous phase. As a result of this method, three fractions, including water, chloroform, and petroleum, were isolated and prepared [21,22].
Extraction of flavones apigenin and Luteolin using Isocratic HPLC
Flavones, Apigenin, and luteolin tests were arranged by dissolving the extract in acetonitrile and diluting it with 50% acetonitrile. This solution was analyzed by reverse-phase chromatography on an Altima C8 column with isocratic elution of acetonitrile and orthophosphoric acid (1:1 v/v) with a flow rate of 1 mL.min1, a column temperature of 33 °C, and UV location at 204 nm.
Antibacterial susceptibility of P. major and P. lanceolata extract materials was done using the agar disc diffusion method described by Rajesh Kumar et al. [23,24]. McFarland 0.5 turbidity standard was prepared (0.5 mL of a 1.175% w/v) barium chloride dehydrates (BaCl2×2H20) solution + 99.5 mL of 1% (v/v) sulfuric acid). The suspension turbidity was made until comparable to 0.5 McFarland standards; then, from the suspension, a volume of 100 µL was added to solidified Muller-Hinton agar plate. Extractions and their fractionated materials were diluted with 800 µL Dw and 200 µL DMSO. For dilution, first, a concentration of 1 gr/mL of the extract was prepared, and then concentrations of 500, 250, 125, and 62.5 mg/mL were made from it, and the results were recorded. After 24 hours, the inhibition zone of all plates was measured with MIP (Microscopic image processing) software. For increasingly correct examination, the micro-dilution method performed MIC and MBC tests. Which was performed according to the CLSI standard, and the results were recorded. In this test, only a total extract was used. MIC is the minimum inhibitory concentration of antimicrobial agents, and minimum bactericidal concentration (MBC) is the least concentration of plant extracts required to kill bacteria evaluated by micro dilutions or broth dilution methods [24, 25]. For this purpose, bacteria streaked onto Müller-Hinton Agar plates without inhibitor and then incubated for 24h at 37 ºC. Colonies were transferred to a glass with 5 ml of MH-Broth medium and incubated for 24 h at 37ºC. At the end of the experiment, the growth of bacteria was measured at 450 nm using an ELISA reader: Milton Roy- Spectronic21D- USA. Every plate was used three times (triplicates) to exclude any errors.
Molecular Docking simulations
To investigate the model of the Luteolin and Apigenin coupling with the active site of Exotoxin-A, the chemical structures of components were obtained from PubChem and RCSB Protein data bank. Luteolin (PubChem CID: 135952), Apigenin (PubChem CID: 79730), and using RCSBPDB, a crystal structure of Pseudomonas aeruginosa exotoxin A (PDB ID: 1LKQ, DOI: 10.2210/pdb1IKQ/pdb) was downloaded to perform molecular modeling and dynamics simulations. Site-specific molecular docking study was conducted using the Auto Dock Vina program (using a Lamarckian genetic algorithm), and the energy minimization was done using the Hyper-Chem software package. Then the results were analyzed utilizing the Molegro Virtual Docking software . Furthermore, the toxicity risk assessment of compounds was performed using the OSIRIS program. The chemical structure characteristics of ligands and proteins are shown in Table1.
Quantitative PCR- Exotoxin-A gene expression study
RNA extraction and cDNA preparation
Following extraction of bacterial RNA and converting it to cDNAs using total RNA extraction commercial kit and easy cDNA synthesis kits respectively purchased from Parstous biotechnology-Iran according to manufactures instructions, a quantitative polymerase chain reaction (qPCR) was used for detection of exotoxin-A expression value [27,28].
A quantitative PCR assay was conducted in a total volume of 20 µL in 0.5 mL microtubes using Rotor-Gene Q's real-time PCR detection system. The primers were designed using primer3 input version4 online software (http://bioinfo.ut.ee/primer3-0.4.0/), and the efficacy of primers was assessed using Beacon Designer and Mfold software. In this research study, the primer sequences were served as F-5′TTC GTG GAT GAA CAC CTT GA 3′, R-5′TGC TGC ACT ACT CCA TGG TC3′ for Exotoxin A gene, and F-5′CTG GAG AGA CTA AGC CCT CC 3′, R-5′ATT ACT GAC GCT GAT TGT GC3′ for 16s rRNA as the housekeeping gene. PCR-Reaction was performed (using SYBR Green real-time PCR Master kit- Parstous-Iran) in a 20 µL solution having 10µL of CYBR Green Master mix (2X), 2 µL of each primer (10 pmols), and 1 µL of cDNA and 5 µL of distilled water. Amplicon condition was conducted at 94 ºC/10 min for the first denaturation, followed by 32 cycles for 95 ºC/15sec denaturation, 60 ºC/30sec annealing, and 72 ºC/60-sec extension. This was followed by a melting curve analysis for each PCR amplicon, and gene expression was normalized to 16s rRNA as an internal control. The data were analyzed by the ΔΔCt method. Fold changes of Exotoxin the equation 2-ΔΔct calculated a gene . All reactions were done in triplicates.
Statistical analysis of the effect of Bacteria and concentration on gene expression was performed by the 2-way ANOVA method. Furthermore, the ANOVA test calculated the significant difference in expression observed between the groups. Gene expression differences were computed using Gene x6 software, statistical analyzes were performed using SPSS 21 software (RRID:SCR_014598), and graphs were plotted with graph pad prism 8 (RRID:SCR_002798).