贵州省汉族和布依族幽门螺杆菌感染患者治疗前后胃内菌群变化及其临床意义

The discovery of Helicobacter pylori (Hp) overturned the conventional dogma that the stomach was a sterile organ. During the past three decades, a lot of study focused on Hp and its related diseases got great achievements, and it is realized that the stomach harbors huge number of microorganisms which can cause pathology under some conditions. But there are a limited number of studies reporting the impact of Hp and eradication therapy on the diversity and the composition of the gastric microbiota and also it is still not clear whether non-Hp co-infected with Hp contribute to the gastrointestinal disease or influence the eradication therapy. The aim of this study is to investigate the impact of Hp infection and eradication therapy with antibiotics and proton pump inhibitors on the gastric flora, the contribution of non-Hp in infection and therapy, the influence of non-Hp on the urea breath test and tissue urease test which are diagnostic tests for Hp, and also the differences of gastric flora of patients from Han or Buyi nationality. . Mucosal biopsies will be obtained from patients with gastric disorders during endoscopy examination. Two patients groups include patients of Han nationality from Guiyang city and patients of Buyi nationality from Qiannan autonomous prefecture. Mucosal samples will be obtained before and after eradication therapy. Fresh biopsy tissues will be homogenized and simultaneously inoculated onto non-selective and selective nutrient medium for Hp and non-Hp isolation. Non-Hp will be identified using a combination of colony morphology, Gram staining, microscopic analysis and 16S rDNA sequencing. Urease of non-Hp will be tested. Hp will be identified by Gram staining, microscopic analysis, urease test and Hp specific PCR. Total bacteria DNA will be extracted from the same biopsy samples, and bacterial flora will be determined by PCR amplification and sequencing on the V3-V4 region 16S rDNA using the MiSeq platform of Illumina and be analyzed by QIIME software and the SILVA database. Characteristic bacteria flora and its abundance of different group will be synthetically analyzed by PCA, Metastats, LEfSe analysis and ANOVA. These results will reveal the characterization of the culturable or unculturable bacterial community profile of these patients before and after eradication therapy. . Specific strains isolated from the successfully recovered patients will be used in the following animal experiment to observe whether these strains could inhibit the colonization of Hp. Amoxicillin susceptibility of non-Hp strains isolated from uncured patients will be screened and typical resistant strains will also be used in animal infection experiment to understand the pathogenecity of these non-Hp. SPF C57BL/6N mice will be used in the animal experiment. The pathogenicity will be evaluated by histopathological examination, bacteria isolation and specific qPCR of gastric biopsy and detection of serum IL-6, IL-8 and gastrin by ELISA.. The results should be useful in investigating the pathogenesis and exploring new options for gastric diseases treatment.

幽门螺杆菌（Helicabacte pylori, Hp）的发现颠覆了胃是无菌器官的传统观点。现已证实胃内有多种细菌，但其与Hp感染及治疗效果的相互关系并不清楚。为了解Hp感染及治疗对胃部菌群的影响及其临床意义，本项目拟对Hp根除治疗前后的汉族和布依族患者进行胃黏膜Hp和非Hp的分离培养，对非Hp通过16SrDNA测序鉴定菌种；同时提取黏膜样本总DNA，PCR扩增细菌16SrDNAV3-V4区，采用Illumina MiSeq平台进行高通量测序，结合QIIME软件进行群落微生物多样性生物信息分析和数据的可视化，寻找不同组别黏膜样本的特征菌及其丰度。了解治疗前后、治疗成功和失败者及汉族和布依族患者胃内菌群特征。筛选根治成功者胃内优势非Hp和根治失败者的耐药非Hp进行动物实验，了解优势非Hp是否拮抗Hp定植及耐药非Hp的致病性，为临床研讨治疗方案、合理使用抗生素及筛选有益菌提供实验依据。

现已证实胃内有多种细菌，但胃内菌群与（Helicobacter. Pylori, Hp）感染及治疗效果的相互关系并不清楚。为了解Hp感染及治疗对胃部菌群的影响及其临床意义，本项目在贵州省贵阳市和黔南州分别采集幽门螺杆菌病人在根除治疗前后的胃黏膜进行Hp 和非Hp的分离培养，对非Hp中的细菌和酵母菌分别通过16SrDNA和真菌ITS测序鉴定菌种，并进行Hp对常用根除用抗生素和非Hp对阿莫西林的敏感性检测；同时提取黏膜样本总DNA，PCR扩增细菌16SrDNA V3-V4区后进行高通量测序，进行群落微生物多样性生物信息分析，了解治疗前后、治疗成功和失败者及汉族和布依族病人胃内菌群特征。用根治失败者胃内耐阿莫西林的大肠杆菌和念珠菌进行动物实验，了解其致病性和对Hp感染进展和根除效果的影响。结果表明，胃内存在尿素酶阳性的非Hp，导致快速尿素酶试验和尿素呼气试验阳性，从而影响临床诊断；胃内细菌经需氧培养，链球菌属及奈瑟菌属为培养优势菌属；厌氧条件下，链球菌属及韦荣菌属为优势菌属。经16SrDNA测序发现感染Hp的患者胃内细菌多样性降低，Hp之外的其他细菌种类的相对丰度发生变化。病人胃内存在对包括阿莫西林在内的多种抗生素耐药的细菌以及念珠菌，阿莫西林耐药大肠埃希菌可以在已经感染Hp的小鼠胃内定植，Hp内化的念珠菌也可以在小鼠胃内定植，并且感染这种念珠菌的小鼠大便中可检测到Hp特异性的16SrDNA和cagA，是导致根除失败的原因，也可能导致感染的复发。.本项目未发现入组研究的汉族和布依族幽门螺杆菌感染病人胃内菌群存在差异，由于新冠疫情原因，复查病例数达不到计划要求，也未能经统计学分析找到治疗成功者胃内可能的有益菌群，但我们发现根除失败病人胃内存在尿素酶阳性的非Hp、对阿莫西林耐药的大肠杆菌以及Hp内化的念珠菌，为进一步揭示根除失败和感染复发的原因奠定了良好的基础。同时我们研究了藻蓝蛋白对Hp感染相关性胃炎的抗炎作用，为多重耐药导致抗生素治疗失败的补救治疗研究提供了新的思路。