Clarithromycin

Erythromycin, clarithromycin, roxithromycin, diltiazem, ritonavir, ketoconazole, fluconazole, cimetidine, ticlopidine, omeprazole, fluvoxamine, paroxetine, fluoxetine, quinidine, aflatoxin B1, and sterigmatocystin were obtained from Wako, Japan. Gestodene and mifepristone were obtained from Tokyo Chemical Industry, Japan. G418 was obtained from Funakoshi, Japan. Ethinylestradiol, rifampicin, tienilic acid, terbinafine, hypoxanthine, aminopterin, and thymidine (HAT) and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma Aldrich, USA. All other chemicals were of the highest grade commercially available.

Antibiotic susceptibility was detected using a serial two-fold agar dilution assay to determine the minimum inhibitory concentrations (MICs) of amoxicillin, clarithromycin, metronidazole, levofloxacin, and minocycline (Wako Pure Chemical Industry, Osaka, Japan) according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Wayne, PA, USA). Briefly, bacteria were subcultured on Mueller-Hinton II Agar medium (Becton Dickinson, Sparks, MD, USA) supplemented with 5% defibrinated horse blood. The bacterial suspension was adjusted to OD₆₀₀ = 0.1, and a 48-pin inoculator was used to inoculate the culture plate (1 μL per spot, approximately 10⁴ colony forming units [CFU] of bacteria). H. pylori strain 26695 was used as a control strain. MICs were judged according to the presence or absence of growth at the spots at the lowest concentration of antibiotic, followed by checking growth at the spots using 1:1 dilutions after 72-h incubation. Resistance or sensitivity to antibiotics was judged according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST; http://www.eucast.org/). The clinical breakpoints of MICs indicating antibiotic resistance are >0.125 mg/L, amoxicillin; >0.5 mg/L, clarithromycin; >8 mg/L, metronidazole; >1 mg/L, levofloxacin; and >1 mg/L, minocycline. Duplicate agar dilution assays were repeated 2–3 times.