A novel colorimetric broth microdilution method to determine the minimum inhibitory concentration (MIC) of antibiotics and essential oils against Helicobacter pylori

Abstract

Helicobacter pylori infections have been associated with the pathogenesis of a number of stomach and gastroduodenal diseases. In order to find alternative drugs for their treatment the search is increasingly focused on new antimicrobial products. However, no standardized methods are available to test the anti-Helicobacter pylori activity in particular of natural substances. Therefore we developed a broth microdilution assay to investigate the susceptibility of this fastidious slow growing bacterium against 15 essential oils widely used to treat disorders of the gastrointestinal tract. The MIC values were determined colorimetrically using p-iodonitrophenyltetrazolium violet (INT) as an indicator for bacterial cell viability. The test sytem was evaluated with three common antibiotics: amoxicillin, ampicillin and levofloxacin. The antibiotic MICs were controlled by Etest®. The Helicobacter reference strain was remarkably susceptible to both the antibiotics (amoxicillin MIC: 0.02 μg/ml, ampicillin MIC: 0.064 μg/ml, levofloxacin MIC: 0.39 μg/ml) and the essential oils. Most of their MICs ranged from 0.015 to 0.064% (v/v) and about 140.0 to 280.0 μg/ml, respectively. Interestingly, chamomile oil, orange flower oil and ginger oil inhibited the bacterial growth in extraordinarily low concentrations of 0.0075% (v/v) and about 65 μg/ml, respectively. The bactericidal concentrations were generally one to two dilution steps higher. In conclusion, we could develop an innovative assay for the MIC determination of essential oils and antibiotics against Helicobacter pylori, which is simple to handle, accurate, reproducible and not as time- and material-consuming as traditional agar dilution techniques.