title: Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan Department of Endoscopy, Cho Ray Hospital, Ho Chi Minh, Vietnam.
authors.forename: Tran Thanh
authors.initials: TT
authors.lastname: Binh
authors.affiliation: Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Shiota
authors.affiliation: Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan.
authors.forename: Rumiko
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Matsuda
authors.affiliation: Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan.
authors.forename: Tran Thi Huyen
authors.initials: TT
authors.lastname: Trang
authors.affiliation: Biology Department, Long Island University, Brooklyn, NY, USA.
authors.forename: Dong Hyeon
authors.initials: DH
authors.lastname: Kwon
authors.affiliation: Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan Department of Medicine-Gastroenterology, Baylor College of Medicine and Michael E. DeBakey Veterans Affairs Medical Center, Houston, TX, USA.
authors.forename: Shun
authors.initials: S
authors.lastname: Iwatani
authors.affiliation: Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan Department of Medicine-Gastroenterology, Baylor College of Medicine and Michael E. DeBakey Veterans Affairs Medical Center, Houston, TX, USA yyamaoka@oita-u.ac.jp.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yamaoka
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017291:Clarithromycin / Q000494:pharmacology*; D016000:Cluster Analysis; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012641:Selection, Genetic; D012692:Serial Passage; D014169:Transformation, Bacterial
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin
keywords: 23S rRNA; in vitro; infB; natural transformation; rpl22