人体肠道菌群中耐药基因分布和增殖机制研究

The fecal samples etc. are collected from healthy population and some disease populations following questionnaire on each people. The distribution characteristic of antibiotic resistance genes in human intestinal microbiota are studied to reveal the characteristic antibiotic resistance genes of disease population through detection and analysis on bacterial isolates, antibiotic resistant plasmids and antibiotic resistance genes by genome sequencing, high throughput sequencing and PCR, and determination on antibiotic residues in each sample by LC-MS. By building simulator of human intestinal microbiota ecosystem (SHIME), the proliferation mechanisms of antibiotic resistance genes undergoing antibiotics stresses are investigated in vitro with known fluorescent tags intestinal antibiotic resistant plasmids as the indicators to detect the proliferation of plasmids and combining with the knowledge of antibiotic resistant plasmids in each sample. The isolates of opportunistic pathogens from intestinal microbiota are used to investigate the resistant mechanism against antibiotics, and to reveal the rapid evolution of antibiotic resistance under antibiotic stresses. The pathogenic mechanism and health effects of pathogens are investigated by the sequence analysis on plasmids and bacterial genome, the tests on biofilms, bacterial capsule and serology, and galleiamellonella inoculation experiments etc. The potential health effects of antibiotic resistance genes on human are involved in the study. This study will provide basic data for understanding the distribution and proliferation mechanism and potential health effects of antibiotic resistance genes in human intestinal microbiota.

采集各类人群样本并问卷调查抗生素使用和病史等相关信息，通过分菌测序、高通量测序、高通量PCR等方法检测耐药细菌、耐药质粒和耐药基因，开展各类人群肠道耐药基因分布研究，揭示疾病人群特征耐药基因；构建人体外胃肠道模拟系统(SHIME)，通过跟踪荧光标记耐药质粒并模拟实验分析耐药质粒的增殖扩散，阐明抗生素胁迫肠道耐药基因增殖机制；利用分离获得的肠道条件致病菌，通过细菌基因组测序、质粒全测序注释、生物膜和荚膜等检测，开展条件致病菌耐药机制研究，揭示抗生素胁迫肠道条件致病菌快速进化的增强耐药机制；针对耐药性和丰度显著增加的条件致病菌，通过模式生物接种和血清学检测等，开展致病机制和健康效应研究，揭示耐药基因的潜在健康效应。本研究为认识耐药基因在各类人群肠道菌群中的分布特征和增殖机制及潜在健康效应提供基础数据。

抗生素被广泛应用于临床治疗，同时大量抗生素被排放进入环境，通过饮食进入人体。但在抗生素胁迫下，人体肠道菌群演替、耐药基因增殖、细菌（包括条件/致病菌）耐药性和致病力变化、及其引起的潜在健康效应，亟待被揭示。本研究中针对肠道耐药基因分布、增殖机制、和肠道细菌演替规律展开研究，揭示了抗生素胁迫下细菌耐药、传播和致病性的进化机制。具体包括揭示了中国健康人群和疾病人群（使用抗生素的病人）肠道中抗生素抗性基因的差异分布和抗性组的差异特征；揭示出健康人群与疾病人群肠道中的代表性差异基因；揭示了肠道菌群改变是耐药基因增殖的重要机制之一；揭示了抗生素胁迫是驱动肠道耐药基因增殖的主要因素；揭示了抗生素胁迫下肠道菌群演替及其潜在健康效应；揭示了抗生素胁迫下细菌和耐药基因丰度的协同变化规律；揭示了抗生素胁迫促进质粒在肠道细菌间的定殖和传播；揭示了抗生素胁迫细菌增殖、传播和耐药等进化机制。研究成果将为认识耐药细菌和耐药基因在人体肠道中的进化和演变以及潜在健康效应奠定基础，为科学防控病原菌感染提供理论依据。