Dent supplement

A 50 μL aliquot of the samples was streaked onto H. pylori selective medium, containing Columbia Blood Agar base, 7% laked horse blood, and DENT Supplement (all from Oxoid, Ltd., Basingstoke, United Kingdom), prepared according to the manufacturer's instructions. The plates were incubated for seven days at 37°C in a microaerophilic atmosphere (GENbox; bioMérieux, Marcy l'Etoile, France). Colonies with typical morphology (small or very small, round, or translucent colonies) were subcultured and subjected to Gram staining. Gram-negative spiral-shaped rods were subjected to species identification by molecular analysis.

The biopsy sample was macerated, homogenized, and inoculated in Columbia blood agar (BD-DIFCO, Berkshire, UK), supplemented with 7% horse blood and selective antibiotics for H. pylori, such as trimethoprim, amphotericin B, and polymyxin B in concentrations of 5 µg/ml each and vancomycin 10 µg/ml (DENT supplement, OXOID Limited, Hampshire, UK). The incubation period was 5–10 days at 37 °C in microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂, and 90% humidity) using commercial systems (CampyGen, OXOID Limited, Hampshire, UK). The suggestive growth of H. pylori was identified through phenotypic testing of oxidase and catalase and morphological characteristics of the bacterium by Gram staining. The strains suggestive of H. pylori were suspended in Columbia broth with 5% horse serum and 20% glycerol to be stored at −80 °C.

Antrum and corpus biopsy specimens from each patient were kept in sterile saline solution (0.9%) at 4°C. The endoscopic biopsy specimens were smeared on the surface of Columbia chocolate agar plates enriched with Dent supplement (Oxoid, England) and 1% of fetal calf serum (Gibco, USA) and incubated under microaerophilic conditions (Campy Packs, Oxoid) for up to 3 days. H. pylori isolates were identified by typical Gram-staining morphology and positive biochemical urease, oxidase, and catalase [11 (link)]. Biopsies of 68 patients yielded 75 H. pylori isolates; those obtained from two gastric biopsy sites of seven patients were considered independent. Primary cultures of H. pylori were conserved at −80°C in brain heart infusion with 20% glycerol and later on were subcultured as it was described above.
All colonies from the subculture were used for chromosomal DNA extraction by Wizard Genomic DNA Purification Kit (Promega, USA) according to the manufacturer's instructions. DNA content and purity were determined by measuring the absorbance at 260–280 nm (Spectrophotometer MRC, Spain) and by amplification of ureA gene [12 (link)]. Samples were stored at −20°C before polymerase chain reaction (PCR) amplification was performed.