Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing.

OBJECTIVES: Resistance to clarithromycin is the most important factor causing failure of Helicobacter pylori eradication. Although clarithromycin resistance is mainly associated with three point mutations in the 23S rRNA genes, it is unclear whether other mutations are associated with this resistance. METHODS: Two types of clarithromycin-resistant strains (low- and high-resistance strains) were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations. The genome sequences were determined with a next-generation sequencer. Natural transformation was used to introduce the candidate mutations into strain 26695. Etest and an agar dilution method were used to determine the MICs. RESULTS: High-resistance strains contained the mutation A2143G in the 23S rRNA genes, whereas low-resistance strains did not. There were seven candidate mutations in six genes outside of the 23S rRNA genes. The mutated sequences in hp1048 (infB), hp1314 (rpl22) and the 23S rRNA gene were successfully transformed into strain 26695 and the transformants showed an increased MIC of and low resistance to clarithromycin. The transformants containing a single mutation in infB or rpl22 (either a 9 bp insertion or a 3 bp deletion) or the 23S rRNA gene showed low MICs (0.5, 2.0, 4.0 and 32 mg/L, respectively) while the transformants containing double mutations (mutation in the 23S rRNA genes and mutation in infB or rpl22) showed higher MICs (>256 mg/L). CONCLUSIONS: Next-generation sequencing can be a useful tool for screening mutations related to drug resistance. We discovered novel mutations related to clarithromycin resistance in H. pylori (infB and rpl22), which have synergic effects with 23S rRNA resulting in higher MICs.