Molecular characterization of Helicobacter pylori resistance to rifamycins

IntroductionAntibiotic resistance is a major contributing factor in treatment failure of Helicobacter pylori eradication. Rifabutin (RB) is a rescue treatment and rifampicin (RP) is used to screen RB resistance in vitro. The aim of this study was to evaluate the rate of rifamycins resistance and to determine the mutations in the rpoB gene conferring resistance to discuss the current break point. MethodsAntimicrobial susceptibility to RP was first determined by E-test for 1015 H.pylori isolates. RP and RB MICs were then determined by agar dilution method for strains with MIC of RP>1mg/L, and the rpoB gene was sequenced. ResultsOverall, 54 of 1015 strains exhibited a RP MIC>1mg/L by agar dilution method. Among these 54 strains, 10 had MICs of RP>4mg/L and RB1mg/L. They all carried at least one mutation in the rpoB gene at codons 530, 538, 540, 525 in the RP resistance-determining region (RRDR). Implication of the mutation L547F was confirmed by site-directed mutagenesis experiment. In contrast, among the 44 H.pylori isolates with a MIC of RP comprised between 2 and 4mg/L, only 4 of 44 (9%) strains exhibited a mutation in rpoB, but outside RRDR (codons 470, 499, 636, or 657). For 31 of 44 tested strains, the RB MICs were 0.064mg/L. ConclusionThese results suggest that H.pylori isolates should be classified as resistant to RP for MICs>4mg/L. We considered that the optimal cut off for RB was 0.125mg/L. We report a new mutation responsible for rifamycins, resistance, L547F.