Pylori agar

H. pylori strains for antibiotic susceptibility testing were isolated from gastric mucosal samples (one from gastric antrum and one from gastric corpus) obtained during an upper endoscopy. They were cultured in the Department of Microbiology and Parasitology, University hospital of Split on Pylori agar (bioMerieux, Marcy l’Etoile, France) after incubation for 3–5 days, at 37 °C, in the microaerophilic atmosphere. The susceptibility of H. pylori isolates to amoxicillin, clarithromycin, tetracycline, levofloxacin and metronidazole was determined by an E-test (AB Biodisk, Solna, Sweden). E-tests were performed on Columbia agar plates with 7% horse blood without supplement. Plates were inoculated with bacterial suspension (turbidity of 3–4 McFarland) and incubated at 37 °C for 72 h under a microaerophilic atmosphere. The antibiotic breakpoints were >0.125 mg/L for amoxicillin, >0.5 mg/L for clarithromycin, >1 mg/L for tetracycline, >1 mg/L for levofloxacin, and >8 mg/L for metronidazole.

Six strains of H. pylori (Hp44, Hp48, Hp53, Hp58, Hp59, and Hp61) were isolated in the Microbiology Department of Hospital La Princesa (Madrid, Spain) from gastric biopsies. Selective (Pylori agar) (BioMerieux, Madrid, Spain) and nonselective media (Blood-supplemented Columbia Agar) (BioMerieux) were used for culturing biopsies. H. pylori strains were stored at −80 °C in Brucella Broth (BB) (Becton, Dickinson, & Co., Madrid, Spain) plus 20% glycerol. Müeller-Hinton agar supplemented with 5% defibrinated sheep blood (MHB) (Becton, Dickinson, & Co, Franklin, NJ, USA) was used as agar-plating medium. BB supplemented with 10% horse serum (HS) (Biowest, Barcelona, Spain) was the liquid medium. The inoculum for H. pylori strains were prepared as follows: 200 μL of frozen strains were inoculated in a MHB plate and incubated in a variable atmosphere incubator (VAIN) (85% N2, 10% CO₂, and 5% O₂) (MACS-VA500, Don Whitley Scientific, Bingley, UK) at 37 °C for 72 h. Bacterial inoculum for the different assays was preparing suspended H. pylori colonies grown in a MHB plate in 2 mL of BB + 10% HS or culture medium cell (~1 × 10⁸ colony forming units (CFU/mL).

H. pylori strains for antibiotic susceptibility testing were cultured from biopsy specimens of gastric mucosa on Pylori agar (bioMerieux, Marcy l’Etoile, France) after incubation for 3–5 days at 37 °C in a microaerophilic atmosphere. Susceptibility testing of H. pylori isolates to amoxicillin, CAM, tetracycline, LVX, and metronidazole was determined by E-test (AB Biodisk, Solna, Sweden). E-tests were performed on Columbia agar plates containing 7% horse blood without supplement. Plates were inoculated with a bacterial suspension (turbidity of 3–4 McFarland) and incubated for 72 h at 37 °C in a microaerophilic atmosphere. Antibiotic cut-off points were determined following the European Committee on Antimicrobial Susceptibility Testing (EUCAST; Clinical Breakpoint Table v. 5.0) criteria: >0.125 mg/L for amoxicillin, >0.5 mg/L for CAM, >1 mg/L for tetracycline, >1mg/L for LVX, and >8 mg/L for metronidazole [55 ]. Gastric tissue samples were collected during upper endoscopy from patients diagnosed with gastrointestinal disease. Cultivation of H. pylori strains was performed at the University Hospital Split in the Department of Clinical Microbiology and Parasitology.

H. pylori strains were isolated from gastric mucosal biopsies obtained from symptomatic patients from the Microbiology Department, Hospital Universitario La Princesa (Madrid, Spain). Biopsies were cultured in selective (Pylori agar, BioMerieux, Madrid, Spain) and non-selective media (Blood-supplemented Columbia Agar, BioMerieux, Madrid, Spain). H. pylori strains were stored at −80 °C in Brucella Broth (BB) (Becton, Dickinson, & Co., Madrid, Spain) with 20% glycerol. The agar-plating medium consisted of Müeller-Hinton agar supplemented with 5% defibrinated sheep blood (MHB) (Becton, Dickinson, & Co., Madrid, Spain), and the liquid growth medium consisted of BB supplemented with 10% horse serum (Biowest, Barcelona, Spain). H. pylori inoculum strains were prepared as follows: the frozen stored strains were reactivated by inoculation (200 μL) in an MHB plate and incubation in a microaerophilic atmosphere using a Variable Atmosphere Incubator (VAIN) (85% N₂, 10% CO₂, 5% O₂) (MACS-VA500, Don Whitley Scientific, Bingley, UK) at 37 °C for 72 h. Bacterial biomass grown in one MHB plate was collected with a sterile cotton swab and suspended in 2 mL of BB or culture medium cell (~1 × 10⁸ colony forming units (CFU/mL)), and was then used as an experimental bacterial inoculum in the different experimental assays.