某肾炎患者幽门螺杆菌感染菌株微进化及相关分析

发布时间：2018-04-08 19:05

本文选题：幽门螺杆菌　切入点：微进化　出处：《中国疾病预防控制中心》2015年博士论文

【摘要】：幽门螺杆菌(Helicobacter pylori,简称H. pylori或HP)是引起慢性胃炎、消化性溃疡和胃癌的主要病原体。HP与特发性血小板减少性紫癜、缺血性贫血等胃肠道外疾病的相关性也日益受到重视。前期研究发现肾小球肾炎患者的肾脏组织存在可以与HP抗体结合的抗原,HP感染可能参与肾炎的发生发展。由于人群中存在较多比例的多株HP混合感染,获得真正的关联菌株用于致病性研究非常重要。目前,HP根除率显著下降,特别是在包含阿莫西林(amoxicillin, AMO)的治疗方案中,不能完全用质子泵抑制剂(proton pump inhibitor, PPI)选择和克拉霉素等抗生素的耐药性升高来解释。虽然AMO的总体耐药率较低,但AMO用药对HP根除治疗中常用抗生素的总体敏感性的影响缺乏研究。第一部分：某肾炎患者幽门螺杆菌感染菌株微进化及相关分析本研究利用多位点序列分析技术、基因组测序以及耐药表型的检测,对来自1例慢性肾炎患者的18个HP克隆的遗传特征、群体结构关系及微进化过程进行了详细分析。研究结果显示该患者感染的HP是源于不同祖先菌株的两个克隆谱系的混合感染。两个祖先菌株至少在12年前就开始了共感染过程,结果显示出了在患者胃内分别经历的2.8年和4.2年的微进化,并形成各自遗传多样性特征的两个克隆谱系的过程。克隆谱系间连续的重组导致一定程度的趋同进化。两个克隆谱系重组率相差10倍,并带有明显不同的限制-修饰系统。首次直接在临床分离株中检测到含有原受体菌的散在序列(interspersed sequences of the recipient, ISR)的重组片段,并发现了噬菌体频繁感染导致的重组事件,提示噬菌体在HP进化中发挥重要作用。基因组比较发现两个克隆谱系虽然均具有完整的Cag毒力岛和相同序列的Vac A,但两克隆系间存在50 kb差异,该区编码完整的2型转座子TnPZ,为进一步开展2型TnPZ与HP致病相关性,特别是与患者肾炎发病关系的研究提供了重要线索。第二部分：幽门螺杆菌阿莫西林耐药相关基因表达及序列分析HP AMO耐药分为不稳定的表型耐药和稳定的遗传耐药两种形式。本研究选择AMO敏感株HP 26695作为出发菌株实验室诱导获得AMO耐药HP。分析不同AMO浓度下,5种青霉素结合蛋白(PBP1-PBP5)、2种孔蛋白(HopB和HopC)和一个外排泵蛋白(HefA)编码基因的序列变化及mRNA水平的表达差异。qRT-PCR结果显示,在不同浓度AMO的选择压力下,HP各检测基因的mRNA表达水平不相同。AMO 4.0 mg/1以下(0.5、1.0、2.0 mg/1), PBP1和PBP2的mRNA表达水平均高于敏感株2倍以上,外排泵蛋白HefA的mRNA表达在AMO 0.5和1.0 mg/l浓度也呈2倍以上上调；在4.0 mg/l的AMO压力下,则主要表现为PBP1～PBP3的mRNA表达上调,孔蛋白HopB和HopC的mRNA表达下调；而AMO达到8.0 mg/l浓度时,HopC mRNA明显下调。以上结果说明HP可以通过增加PBP的表达、促进细胞壁合成,提高外排泵蛋白表达、增加药物外排或者降低孔蛋白表达量、减少药物摄取等多种机制提高AMO耐药水平。本研究还获得了AMO遗传耐药株,并在PBP1编码基因新发现T593K和594G+两个AMO耐药相关的突变位点。本研究还发现,单纯的AMO选择压力,可同时造成不同程度的左氧氟沙星,莫西沙星耐药以及高水平四环素的耐药。以上结果表明,尽管目前AMO耐药率低,临床分离到稳定耐药株很少,但是AMO表型耐药的存在以及因为使用AMO导致的多重耐药的发生,可能是HP根除率下降不可忽视的因素。以上现象尚未见相关报道。本研究通过上述宿主内HP菌群特征和微进化分析以及HP AMO表型耐药和遗传耐药机制的研究,加深了对HP进化和遗传多样性产生机制以及AMO耐药机制的认识,对后续HP致病相关性研究及HP的防治具有一定的意义。
[Abstract]:Helicobacter pylori (Helicobacter pylori, referred to as H. pylori or HP) is the main pathogens of.HP cause chronic gastritis, peptic ulcer and gastric cancer and idiopathic thrombocytopenic purpura, anemia and gastrointestinal related ischemic disease has received increasing attention. Previous study found that can bind with the antibody of HP antigen in patients with glomerulonephritis kidney tissue, HP infection may be involved in the development of nephritis. Due to the presence of a higher percentage among the strains of HP mixed infection, get really very important for related strains of pathogenic research. At present, the eradication rate of HP decreased significantly, especially in containing amoxicillin (amoxicillin, AMO) of the treatment, not completely by proton pump inhibitor (proton pump inhibitor, PPI) and clarithromycin resistance selection of antibiotics increased to explain. Although the overall resistance rate of AMO was low, but AMO The lack of research on the effects of HP eradication of drug commonly used antibiotics in the treatment of the overall sensitivity. The first part: the evolution of micro strain and related multilocus sequence analysis using the analysis technology of a nephritis patients with Helicobacter pylori infection, genome sequencing and detection of drug resistance phenotype, the genetic characteristics from 18 HP clones, 1 cases of chronic nephritis patients the detailed analysis of relationship between population structure and micro evolution. The results showed that the infection of patients with HP mixed infection of two clones derived from different ancestral lineage strains. Two strains of ancestors at least 12 years before the start of the co infection process, the results show that the experience in patients with stomach 2.8 years and 4.2 years in the process of micro evolution, two clones and their pedigree forms characteristic. The genetic diversity between successive clonal lineage recombination leads to convergent evolution to a certain extent. Two grams Long lineage recombination rate is 10 times, and with different restriction modification system. For the first time in clinical isolates detected with the original receptor bacteria scattered in the sequence (interspersed sequences of the recipient, ISR) of the recombinant fragments, and found frequent recombination events caused by phage infection, suggesting that phages play an important role in the evolution of HP. The comparison between genomes showed that two clones had complete lineage while Cag pathogenicity island and the same sequence of Vac A, but the two clones are 50 KB difference, the complete encoding type 2 transposon TnPZ, for the further development of the type 2 TnPZ and HP pathogenic relevance, especially provides an important study on the relationship between patients with nephritis and clues. The second part: the expression of related gene of Helicobacter pylori resistant to amoxicillin and sequence analysis of HP AMO resistance divided into genetic resistance phenotype of resistance unstable and stable Two forms. This research chooses AMO HP 26695 as the starting strain sensitive strain induced by different concentrations of AMO AMO laboratory analysis of resistance HP., penicillin binding protein 5 (PBP1-PBP5), 2 holes (HopB and HopC) protein and an efflux pump protein (HefA) expression of.QRT-PCR encoding gene sequence variation results and the level of mRNA showed that the pressure in different concentration of AMO, HP detection of mRNA gene expression level is not the same.AMO below 4 mg/1 (0.5,1.0,2.0 mg/1), the expression level of PBP1 and PBP2 mRNA were higher than that of sensitive strains were more than 2 times, efflux pump protein HefA mRNA expression in AMO 0.5 and mg/l 1 concentration it was more than 2 times increase; at 4 mg/l under the pressure of AMO, mainly from PBP1 to PBP3 the expression of mRNA, HopB and HopC pore protein down regulated expression of mRNA and AMO reached 8; the concentration of mg/l, HopC and mRNA were down regulated. These results suggest that HP By increasing the expression of PBP, promote the synthesis of cell wall, increased efflux pump protein expression, increased drug efflux or reduce the pore proteins, reduce drug intake and other mechanisms to improve AMO resistance level. This study also obtained AMO genetic resistant strains, and PBP1 gene encoding newly discovered mutation sites related to T593K two 594G+ and AMO resistance. The study also found that the AMO pressure alone, can also cause varying degrees of resistance to levofloxacin, moxifloxacin resistance and high level tetracycline. The above results show that although the AMO resistance rate is low, stable resistant strains isolated from clinical rare, but AMO phenotypic resistance exists and because multiple use of AMO resulted in the occurrence of drug resistance, may be a factor in the declining rate of HP eradication can not be ignored. This phenomenon has not been reported. This study by the host HP and micro flora characteristics in Chemical analysis and the mechanism of phenotypic resistance and genetic resistance of HP AMO have deepened the understanding of HP evolution and genetic diversity, and the mechanism of AMO resistance, which has a certain significance for the follow-up HP pathogenic correlation research and HP prevention and treatment.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R692.3;R446.5

【共引文献】