肠道菌群耐药基因组与肝硬化并发自发性腹膜炎的致病菌耐药性之间的关联研究

China has a highest incidence of cirrhosis, usually resulting from chronic infection of HBV virus. The cirrhosis is often complicated by bacterial infection leading to a high percentage of death. Bacterial antibiotic resistance (AR) is one of the most important threats in bacterial infection therapy. The human gut microbiota is a great reservoir of antibiotic resistance genes, and intestinal bacterial translocation (BT) probably play an important role in the progress of bacterial infection in cirrhosis, but little is known about AR gene diversity and richness within the gut，and none has done in the comparison in spontaneous bacteria peritonitis (SBP) ascitic bacteria and their AR genes with gut microbial AR genes. Taking advantages in quick advances in the next-generation sequencing (NGS) and metagenomic technologies, our lab has successfully found gut microbial markers for cirrhosis development (N Qin, et al. 2014, Nature), and it also provided basic database for this study. In this project, we aim to collect 20 ascites and feces samples from SBP-complicated liver cirrhosis patients, and use Illumina-based metagenomic sequencing and most updated bioinformatics tools to achieve the following goals: reveal and compare the bacterial communcity structure and AR genes in the gut and ascites microbiota; assembly, gene annotation and homologous analysis of AR genes in both sample types, to reveal ascitic AR gene (bacteria) origin, evolution and migration; functional studies of AR genes by construction of BAC library and drug-screen for new AR genes, and knock-out experiment of target gene-containing ascites-isolated bacterial strain, for confirmation of the gene's AR function and AR mechanism; Compare metagenomic and gene function data with clinical examination results and prognosis for potential methods to improve diagnosis and therapy.

细菌感染并发症是导致肝硬化患者病死率增加的主要原因之一，细菌耐药是抗感染治疗中的重要问题。肠道细菌移位在肝硬化并发自发性腹膜炎（SBP）的发生发展中起重要作用，但是人们对肠道菌群这一“抗生素耐药基因的巨大贮存库”的了解甚少，对人体腹水菌群及耐药基因也需要通过最新的宏基因学进行全面的再认识。本项目在已找到肝硬化患者肠道菌群失衡规律的基础上，计划通过采集肝硬化并发SBP患者的大便和腹水样品，采用宏基因组生物信息和多元分析技术，揭示其菌群组成和耐药基因组特征；并首次建立肝硬化SBP腹水菌群数据库，通过与肠道菌群耐药基因组的同源、聚类、差异和进化关系的关联分析，结合临床表型及对腹水分离菌耐药性和耐药基因功能的研究，阐明腹水致病菌耐药基因肠道产生、进化和体内转移途径及致病机理，为临床抗生素使用指导及肠道微生态干预改善预后奠定研究基础。

细菌感染并发症是导致肝硬化患者病死率增加的主要原因之一，细菌耐药是抗感染治疗中的重要问题。肠道细菌移位在肝硬化并发自发性腹膜炎（SBP）的发生发展中起重要作用，但是人们对肠道菌群这一“抗生素耐药基因的巨大贮存库”的了解甚少，对人体腹水菌群及耐药基因也需要通过最新的宏基因学进行全面的再认识。本项目在已找到肝硬化患者肠道菌群失衡规律的基础上，通过采集肝硬化并发SBP患者的大便和腹水样品，进行鸟枪法高通量测序，首次建立肝硬化SBP腹水菌群数据库，并采用宏基因组生物信息和多元分析技术，揭示了肠道和腹水菌群组成和耐药基因组特征。物种差异分析发现肝硬化并发自发性腹膜炎患者腹水及肠道两个部位的物种组成具有显著性差异。在门水平，腹水和肠道中丰度最高的物种分别为Protobacteria和Bacteroidetes；属水平，腹水和肠道中丰度最高的物种分别为Alcanivorax和Bacteroides。随后分别构建了腹水及肠道中菌群的基因集，腹水基因集中包含20956个非冗余基因，肠道基因集中包含222034个非冗余基因，共有基因有1115个。通过对肝硬化并发自发性腹膜炎患者肠道和腹水耐药基因与健康人肠道耐药基因的比较，发现肝硬化并发自发性腹膜炎患者耐药基因的丰度显著高于健康人，而且耐药基因型并不完全一致。36%的腹水耐药基因能够比对到肠道菌群宏基因组数据上，包括对四环素类，β-内酰胺类，红霉素等多种药物耐药类型及外排泵、内酰胺酶、核糖体保护或甲基化转移酶修饰等多种耐药机制。分析耐药基因的起源，发现肝硬化并发自发性腹膜炎患者的肠道菌群耐药基因更多起源于变形菌门（Proteobacteria），与健康人表现不同。本研究揭示了肝硬化并发自发性腹膜炎患者肠道和腹水的菌群结构和耐药基因组特征，为阐明腹水致病菌耐药基因肠道产生、进化和体内转移途径及致病机理提供实验支撑，为临床抗生素使用指导及肠道微生态干预改善预后奠定研究基础。