Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, Korea.
Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea.
J Gastroenterol Hepatol. 2018 Mar;33(3):681-688. doi: 10.1111/jgh.13906.
rdxA and frxA mutations and enhancement of efflux pump have been suggested as the cause of metronidazole resistance in Helicobacter pylori. This study was performed to investigate the resistance mechanisms related to clinical eradication outcome, and to examine direct involvement of hefA in metronidazole-resistant isolates with intact rdxA and frxA.
A total of 53 H. pylori-positive patients who were treated with metronidazole-containing sequential or quadruple therapy from 2011 to 2015 were enrolled. The metronidazole susceptibility of H. pylori isolates was examined by agar dilution test. Mutations in rdxA and frxA, were analyzed with DNA sequencing, and impact of hefA on metronidazole resistance was examined with quantitative real-time reverse transcription polymerase chain reaction, knockout and genetic complementation test for hefA.
Seven mutation types of rdxA and/or frxA were found in H. pylori isolated from non-eradicated subjects. rdxA mutation was associated with eradication failure (P = 0.002), and nonsense mutation in rdxA reduced eradication efficacy (P = 0.009). hefA expression was significantly higher in resistant isolates (P < 0.001), especially in rdxA(-)frxA(-) as compared to rdxA(+)frxA(+) (P = 0.027). Resistant isolates with no mutation in rdxA and frxA became susceptible after hefA knockout. Genetic complementation for hefA recovered metronidazole resistance in all of three hefA knockout mutants.
These results suggest that rdxA mutations play a critical role in metronidazole resistance as well as the outcomes of eradication therapy. In addition, hefA seems to be directly involved in metronidazole resistance, which explains the resistance in clinical isolates with intact rdxA and frxA.
rdxA 和 frxA 突变以及外排泵的增强被认为是幽门螺杆菌甲硝唑耐药的原因。本研究旨在探讨与临床根除结果相关的耐药机制，并研究 hefA 在 rdxA 和 frxA 完整的甲硝唑耐药分离株中的直接作用。
2011 年至 2015 年，共招募了 53 名接受甲硝唑含序贯或四联疗法治疗的 H. pylori 阳性患者。采用琼脂稀释法检测 H. pylori 分离株对甲硝唑的敏感性。用 DNA 测序分析 rdxA 和 frxA 的突变情况，用实时定量逆转录聚合酶链反应、hefA 敲除和遗传互补试验检测 hefA 对甲硝唑耐药的影响。
在未根除患者的 H. pylori 分离株中发现了 7 种 rdxA 和/或 frxA 的突变类型。rdxA 突变与根除失败相关（P = 0.002），rdxA 无义突变降低了根除疗效（P = 0.009）。耐药分离株 hefA 表达明显升高（P < 0.001），尤其是 rdxA(-)frxA(-)比 rdxA(+)frxA(+)（P = 0.027）。无 rdxA 和 frxA 突变的耐药分离株在 hefA 敲除后变得敏感。hefA 基因的遗传互补恢复了所有 3 个 hefA 敲除突变株对甲硝唑的耐药性。
这些结果表明，rdxA 突变在甲硝唑耐药以及根除治疗的结果中起着关键作用。此外，hefA 似乎直接参与甲硝唑耐药，这解释了临床分离株中 rdxA 和 frxA 完整但甲硝唑耐药的原因。
