Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing
Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing
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DOI：
10.1093/jac/dku050
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发表时间：
2014-07-01
影响因子：
5.2
通讯作者：
Yamaoka, Yoshio
中科院分区：
文献类型：
--
作者：
Tran Thanh Binh;Shiota, Seiji;Yamaoka, Yoshio
克拉霉素耐药是导致幽门螺杆菌根除失败的最重要因素。虽然克拉霉素耐药主要与23 S rRNA基因的3个点突变有关，但尚不清楚是否有其他突变与这种耐药有关。幽门螺杆菌暴露于低浓度克拉霉素。用下一代测序仪测定基因组序列。使用自然转化将候选突变引入菌株26695中。Etest法和琼脂稀释法测定MIC，高耐药株23 S rRNA基因突变为A2143 G，低耐药株23 S rRNA基因突变为A2143 G。在23 SrRNA基因以外的6个基因中有7个候选突变。将hp 1048（infB）、hp 1314（rpl 22）和23 S rRNA基因中的突变序列成功地转化到菌株26695中，转化子显示出对克拉霉素的MIC增加和低抗性。在infB或rpl 22中含有单一突变的转化体（9 bp插入或3bp缺失）或23 S rRNA基因显示低MIC（0.5、2.0、4.0和32毫克/升，而含有双突变的转化体（23 S rRNA基因突变和infB或rpl 22突变）显示出更高的MIC（> 256 mg/L）。我们发现了与克拉霉素耐药相关的新突变。pylori（infB和rpl 22），其与23 S rRNA具有协同作用，导致更高的MIC。
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.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.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).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.