Brucella agar plates

Forty male BALB/c mice (free of Helicobacter spp. parasites and exogenous murine viral pathogens) were bred and maintained in a specific pathogen-free facility. The animal experiments were conducted in line with China Laboratory Regulation Act (2017) under a Project License [SYXK(SU)2017-0044] authorized by Jiangsu Provincial Science and Technology Department and approved by Institutional Animal Care and Use Committee of Yangzhou University. Mice with free access to food and water were maintained in a room at 22–26°C with 40–60% relative humidity under a 12-h:12-h light-to-dark cycle. H. hepaticus 3B1 (ATCC 51449) was cultured on Brucella agar plates (BD, United States) supplemented with 5% sheep blood and antibiotics (amphotericin B, vancomycin, and cefoperazone) for 4–5 days under microaerobic conditions (85% N₂, 10% CO₂, and 5% O₂) at 37°C.

Tissue specimens were placed in sterile 0.9% sodium chloride solution in the operating theater and immediately transferred to the laboratory. Upon receipt, the specimens were minced using an IKA Ultra-Turrax tissue homogenizer (Sigma-Aldrich, Buchs, Switzerland) and culture media were inoculated and incubated under aerobic and anaerobic conditions. Sheep agar plates without antibiotics (COS; bioMérieux, Marcy l'Etoile, France), and with colistin-nalidixic acid (CNA) were used for the detection of Gram-positive bacteria (bioMérieux), while MacConkey (MCK) agar plates (bioMérieux) were used for Gram-negative bacteria and Sabouraud agar plates for fungi. In addition, thioglycolate broth, aerobic selective chocolate agar plates (HAE2; bioMérieux), anaerobic sheep blood agar plates with hemin and vitamin K1 (Brucella agar; BD), laked sheep blood Brucella agar plates with kanamycin and vancomycin (BD), and phenylethylalcohol agar plates with vitamin K1 (BD) were used. Growth on agar plates was examined at 24, 48, and 72 h as well as after one week; the thioglycolate broth was examined up to day 10 and the Sabouraud agar plates up to day 20 (12 ). Antibiotic susceptibility testing was performed according to EUCAST recommendations (13 ).

H. pylori reference strains of ATCC 49503, ATCC 43504, ATCC 51932, and ATCC 700392 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). H. pylori SS1 strain was obtained from the Korean Type Culture Collection at Gyeongsang National University (Jinju, Korea). H. pylori were incubated on Brucella agar plates (BD Biosciences, Braintree, MA, USA) supplemented with 10% bovine serum (BRL Life Technologies, Grand Island, NY, USA) at 37 °C for 72 h under a humidified condition with 5% CO₂. The number of bacterial particles in the H. pylori suspension was set to McFarland 0.33 and cultured in Mueller–Hinton broth supplemented with 10% bovine serum at 37 °C for 72 h under a humidified condition with 5% CO₂. Gastric biopsy specimens for the isolation of H. pylori were collected at Yong-In Severance Hospital, Korea. H. pylori clinical strains were isolated from 46 patients undergoing gastroscopic examinations to confirm the infection of H. pylori.

Two well-characterized cagA⁺, vacA s1/m1 H. pylori strains, originally isolated from human patients, were used in our experiments: the reference strain 60190 (kind gift from Dr. G. Perez-Perez, New York University, ATCC #49503 [65 (link)]), and strain PMSS1 (kind gift from Dr. K. Wilson, Vanderbilt University), which is widely used in murine infection experiments [66 (link)]. H. pylori strain 60190 was shown to be susceptible to metronidazole, amoxicillin, clarithromycin, levofloxacin, rifampicin and tetracycline [67 ], and strain PMSS1 was confirmed to be susceptible to metronidazole, amoxicillin, clarithromycin, and tetracycline in previous studies [68 (link),69 (link)]. For the experiments, bacteria were grown at 37 °C under microaerophilic conditions on Brucella agar plates, 5% sheep blood (Becton Dickinson) for 3 days. Colonies were harvested into warm Brucella broth supplemented with 10% FBS and were then cultured in a shaking incubator for a further 18 h period prior to use in the experiments.

One hundred and nine H. pylori clinical strains were isolated from 220 patients undergoing upper gastro-duodenal endoscopy between August 2017 and October 2018 in four hospitals (the Cinquantenaire’s Hospital, Astryd Clinics, Marie-Yvette Clinics, and The Presidential Hospital of the African United City) located in Kinshasa, the capital city of the DRC. Gastric biopsy specimens sampled from the antrum were cultured following a previously established standard procedure [20 (link)]. Briefly, biopsy specimens were transitorily stored at −80 °C at the National Institute of Biomedical Research (INRB) in Kinshasa. Then, they were shipped to Oita University in Japan, where all analyses were performed. Upon arrival, each specimen was inoculated onto a Helicobacter selective Agar medium (Nissui Pharmaceutical co., Ltd., Tokyo, Japan) and incubated for up to 10 days. H. pylori colonies growing on the selective plates were identified and sub-cultured for three to four days on Brucella Agar plates (Becton Dickinson, Sparks, MD, USA) supplemented with 7% horse blood (Nippon Biotest Laboratories Inc., Tokyo, Japan). Bacterial cultures were maintained at 37 °C in microaerophilic conditions (10% CO₂, 5% O₂, and 85% N₂).

H. pylori ATCC 49503 strain (East-asian type: CagA⁺/VacA⁺, American Type Culture Collection, Manassas, VA, USA) was grown on Brucella agar plates (Becton-Dickinson, Braintree, MA, USA), supplemented with 10% bovine serum (BRL Life Technologies, Grand Island, NY, USA) at 37 °C for 72 h under a humidified atmosphere with 5% CO₂. For disc diffusion method, the number of bacteria in the H. pylori suspension was adjusted to a McFarland scale 2 (6 × 10⁸ cells/mL) on Mueller-Hinton agar (Becton-Dickinson), supplemented with 10% bovine serum and then incubated for 72 h. To determine the minimum inhibitory concentration (MIC) of β-caryophyllene against H. pylori, the number of bacterial particles in the H. pylori suspension was set to McFarland scale 0.5 (1.5 × 10⁸ cells/mL). Various concentrations of β-caryophyllene (7.81–4000 μg/mL) were treated and the bacteria were incubated for 72 h and final optical density (600 nm) of the bacterial suspension was measured by using NanoQuant spectrophotometer (infinite M200, TECAN, Männedorf, Switzerland). For normal control, the same volume of ethanol was administrated to culture media. β-caryophyllene was provided by SFC BIO Co., Ltd. Seoul, in republic of Korea.