甲硝唑耐药和逆转过程中幽门螺杆菌全基因组转录水平的调控作用

Helicobacter pylori (H. pylori) is an important human pathogen causing a variety of diseases including gastric cancer. The prevalence rate of H. pylori infection in our population is high and range between 60-70%. Antibiotic resistance to clarithromycin and metronidazole plays an important role in treatment failure. Our clinical study showed that addition bismuth, increasing the dosage and prolonging treatment duration can overcome H.pylori resistance to clarithromycin and achieved higher cure rate. From our primary study, the transcriptional level from the whole genome was 28% lower in the metronidazole resistant strain compared to the parental strain and included significant down-regulation of rdxA, sodB, and HP0947. We plan to 1) collect antibiotic resistant H. pylori and its parental susceptible strain, 2) establish in vitro induced the resistant standard strains challenged by antibiotic, 3) examine transcriptional levels of the whole H. pylori genome (1590 ORFs) between antibiotic resistant H. pylori and its parental susceptible strain,in vitro induced the resistance to antibiotic of standard strains. The purpose of our project is to analyse and compare the genome-scale transcriptional change and modulation in response to one or two antibiotics (amoxicillin, clarithromycin, metronidazole,tetracycline,and quinolone) stress, the interaction between two different antibiotics and the targets genes of antibiotics, to elucidate the mechanism of antibiotic resistance/susceptibility and antibiotics compatibility to overcome H.pylori resistance to antibiotics.

幽门螺杆菌（Hp）是一种能够引起慢性胃炎、消化性溃疡和胃癌瘤等严重病变的细菌，我国人群的感染率高达60％，Hp对抗生素（甲硝唑和克拉霉素等）耐药是抗生素联合根除治疗失败的主要原因。我们的临床试验证实，调整甲硝唑配伍、延长疗程和改变剂量能够克服耐药，获得高根除率。我们拟对分离自患者的甲硝唑敏感/耐药菌株和体外诱导耐药标准菌株进行全基因组ORF芯片分析，以发现甲硝唑耐药相关的Hp全基因转录水平的变化，识别潜在目的基因的功能，用RNA和免疫印迹杂交及等位基因突变方法确定目的基因突变及其蛋白功能失活；将配伍的阿莫西林或四环素作用于相应的甲硝唑耐药菌株，比较耐药菌株全基因组转录水平改变和调控，分析配伍抗生素配伍对耐药目的基因的作用，并进行临床验证，为克服H.pylori对甲硝唑耐药和提高疗效提供依据，也为含甲硝唑方案的重新使用和抗生素组合应用于幽门螺杆菌的治疗提供依据。

随着抗生素耐药率的增加，幽门螺杆菌治疗越发困难。甲硝唑是目前根除 H.pylori 治疗中最常用的抗生素之一，虽然H. pylori对甲硝唑耐药率很高，但临床上可以通过增加甲硝唑剂量，延长治疗时间及有效的抗生素配伍等来克服耐药。我们通过使用标准菌株ATCC700392 体外诱导实验，比较了对甲硝唑敏感和耐药的幽门螺旋杆菌在全基因组的转录水平改变，发现与对照菌株相比，甲硝唑耐药株其整体基因表达下降28％，其中包括rdxA的显著下调。在甲硝唑敏感和耐药的临床分离配对菌株中得出了类似的结果，这表明整体基因表达水平下降可能是对甲硝唑环境下的一种适应性反应。甲硝唑耐药菌株在加入甲硝唑干预作用下，以及耐甲硝唑菌株中（临床菌株和标准菌株），其转录调控的水平73 个基因显著（>3倍）和中度上调，294 个基因下调，其中编码超氧化物歧化酶的sodB显著下调，sodB对氧代谢产生的超氧化物进行解毒，其显著下调可能与羟自由基的减少、与甲硝唑的耐药发生相关。而且sodB的下调与rdx表达存在相关性。