Karmali agar base

A total of 515 H. pylori strains preserved in our laboratory were obtained from the following regions: Fujian (n = 83), Shandong (n = 90), Guangxi (n = 79), Yunnan (n = 73), Heilongjiang (n = 56), Hunan (n = 46), Neimenggu (n = 33), Qinghai (n = 33), Zhejiang (n = 7), Beijing (n = 4), Ningxia (n = 4), Taiwan (n = 4), Shanxi (n = 2), and Xizang (n = 1). H. pylori was streaked onto the Karmali agar plate supplemented with Karmali Agar base (CM 0935, Oxoid) containing 5% defibrinated sheep blood, and the plate was incubated at 37 °C for 3–5 days in a microaerobic atmosphere (5% O₂, 10% CO₂ and 85% N₂). Bacteria were identified as H. pylori based on its external morphology, negative Gram staining and positive for catalase, oxidase and urease. The confirmed isolates were frozen at -80 °C until the genomic DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted DNA was stored at -20℃ and used directly for PCR. This study was approved by Ethical Committee of National Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and Prevention (approval No. ICDC-2013001).

H. pylori type strain NCTC 11637 (Lock et al., 2001 (link)) was used for this study and 16S rRNA identification was used to verify the strains. Bacteria was cultured on Karmali Agar Base (CM0935, Oxoid, United Kingdom) supplemented with 5% sterile defibrinated sheep blood (Beijing XLF Medical Sales Co. Ltd, China) for 3–5 days at 37°C, under microaerophilic conditions: microaerobic gas mixture composed of 5% oxygen, 10% carbon dioxide, and 85% nitrogen (GEN bag microaer, BioMérieux, France). H. pylori were subcultured three times before each experiment.

Gastric biopsy specimens were homogenized thoroughly in brain heart infusion (BHI) broth and then streaked onto the Karmali blood agar base plates under a biological safety cabinet (Thermo Scientific). The Karmali Agar base (Oxoid, CM 0935) was supplemented with 5% defibrinated sheep blood and 1% combined antibiotics comprising of trimethoprim (150 mg/L), vancomycin (125 mg/L), amphotericin B (100 mg/L) and polymyxin B (100 mg/L). The plates were incubated at 37 °C under microaerobic conditions (5% O₂, 10% CO₂ and 85% N₂) for 3–5 days. H. pylori colonies were identified according to its morphological characteristics, negative Gram staining and positive for catalase, oxidase, and urease. The identified H. pylori was subcultured to single colonies and then preserved in sterile BHI broth with 20% glycerol and frozen at − 80 °C until the genomic DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted DNA was stored at − 20℃ and used directly for PCR.