2.3. In Vitro Anti-H. pylori Effect of PEAME

To obtain H. pylori clinical isolates for testing the biological effect of PEAME, 25 leftover samples of gastric mucosal biopsies were used to isolate H. pylori. As a diagnostic procedure, the samples were collected from the antrum using endoscopy obtained from children presented with dyspepsia (Age: 7–10 years), referred to the teaching hospitals of Tanta University. Biopsies were kindly provided by the staff members in the Department of Pediatrics Internal Medicine, Faculty of Medicine, Tanta University. Biopsies positive for H. pylori following urease test were transferred to sterile McCartney bottles containing Brain Heart Infusion (BHI) broth with 20% glycerol and 0.2 g/L cysteine. Bottles were transferred on ice to the microbiology laboratory within 60 min after collection for microbiological culture and analysis.

Gastric biopsies were subjected to homogenization at 5000 rpm for 30 s to get a homogenous mixture of the gastric tissue in BHI broth. Each homogenized test sample was inoculated onto Mueller–Hinton agar supplemented with defibrinated 7% blood and antimicrobials (amphotericin B 3 mg/L, vancomycin 6 mg/L, polymyxin B 2500 IU/L). Plates were then incubated under microaerobic conditions at 37 °C for 3–7 days. Microscopy, as well as appropriate biochemical tests (urease, oxidase, and catalase), were performed for identification of H. pylori. Identification was carried out based on the Bergey’s Manual of Systematic Bacteriology [21].

To select the resistant strains for biological testing, susceptibility of H. pylori isolates to common therapeutic antimicrobials (clarithromycin, metronidazole, and amoxicillin) was performed according to the Clinical and Laboratory Standards Institute (CLSI, standard M7-A5, informational supplement M100-S10, 2000) [22]. Antimicrobials were tested in a concentration ranged between 0.016–256 µg/mL, then the percentage of test pathogens resistance to antimicrobials was calculated. The effect of pomegranate extract on H. pylori isolates was also investigated, and the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were recorded.

To investigate the antimicrobial effect of PEAME in vitro, time course viability assay of the test pathogen was performed in the absence or presence of pomegranate extract at 1× MBC or 2× MBC as described by Ali et al. (2005). Samples were withdrawn at different time intervals (2, 4, 6, 8, 24, 48 h) to determine the viable count. Data were presented as the number of survivors (CFU/mL) against time (h). The experiment was repeated thrice, and the standard deviation (SD) was calculated.