Minimum inhibitory concentration of various single agents and the effect of their combinations against Helicobacter pylori, as estimated by a fast and simple in vitro assay method.

Abstract

Fast and simple in vitro methods of accurately assaying anti-Helicobacter pylori (H. pylori) activity are needed to facilitate the selection of optimal drugs for the eradication of H. pylori. With that purpose in mind, we developed a broth microdilution technique that uses a 96-well flat-bottom microplate. Clinical isolates of H. pylori were placed in 25-cm2 tissue culture flasks and were grown in an atmosphere of 5% CO2 in air at 37 degrees C and 100% humidity. Then they were inoculated in 96-well flat-bottom microplates. After 24 h, the bacterial growth was assessed by automatic measurement at A450 with a microplate reader. Minimum inhibitory concentrations (MICs) of 11 drugs against H. pylori and the effect of two-drug combinations were respectively evaluated by the broth microdilution technique and by calculating the fractional inhibitory concentration index according to the checkerboard titration method. The MICs of all drugs tested with this system roughly agreed with those of previous reports in which the agar dilution method was used. The MIC of lansoprazole, a proton pump inhibitor, was low (0.88 microg/ml) and surpassed that of metronidazole (3.15 microg/ml). The effect of the combination of macrolide antibiotics with tetracycline was favorable. The proton pump inhibitor demonstrated a strong additive effect with macrolide antibiotics and fostered the activity of ecabet sodium (mucosal protective agent). This system could estimate the MIC of individual drugs quickly and simply, and could accurately measure the efficacy of two-drug combinations in vitro.