The human gut microbiome as a reservoir of antibiotic resistance

人类肠道微生物组是抗生素耐药性的储存库

• 批准号: 2084366
• 金额: --
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2084366
• 关键词: human; gut; microbiome; reservoir; antibiotic

Aims:A. To link antibiotic resistance genes (ARGs) in the human gut microbiome to their bacterial hostsB. To assess the ability of ARGs in the human gut microbiome to spread to opportunistic pathogens.BackgroundThe human gut harbours a complex microbial community ('the gut microbiome'), that contributes to human health and disease. Previous work by the group of Prof. Van Schaik and others have revealed that the gut microbiome comprises a large number of antibiotic resistance genes (ARGs).1,2 Current methodologies to study the gut microbiome, like metagenomic shotgun sequencing or high-throughput quantitative PCRs, do not allow for the identification of the bacteria and mobile genetic elements that carry ARGs.3 The extent by which ARGs can spread between members of the gut microbiome, including to gut-dwelling opportunistic pathogens like Escherichia coli and the enterococci, also remains unclear. HypothesisMost of the ARGs in the gut microbiome will be harboured by anaerobic gut commensals. These ARGs may be transferred to both Gram-positive and Gram-negative opportunistic pathogens. Experimental Methods and Research Plan1. Bacterial reservoirs of ARGs in the gut microbiome In this project, we will determine the linkage of ARGs with their bacterial hosts in human stool samples. We will use faeces from paediatric and adult patients and healthy individuals (n = 5 for each, 15 samples in total). The stools are available through collaborators and can be shared for research purpose in compliance with current study protocols and ethical regulations. The faecal samples will first be screened for the presence of the important resistance genes blaNDM, blaKPC, mcr-1, vanA and vanB by quantitative PCR, and, if found to be positive, linkage between resistance genes and their bacterial hosts will be determined by a recently developed method, termed epicPCR for Emulsion, Paired Isolation and Concatenation PCR.4 2. Transferability of resistance genes to opportunistic pathogensOnce the identity of the bacteria carrying ARGs has been established, these bacteria will be cultured from the relevant samples using a variety of enrichment methods. After their isolation, the genomes of the isolated bacteria will be sequenced, using a combination of short- and long-read sequencing, resulting in complete assemblies of the chromosome and plasmids. The annotated genome sequences will be used to determine whether resistance genes are associated with mobile genetic elements, specifically plasmids and conjugative elements, or are carried on the chromosome, which would minimize their potential for horizontal transfer.5 Upon completion of the genomic characterization of the resistance-gene-carrying bacteria, we will perform conjugation assays to assess the ability of these bacteria to serve as donors of resistance genes to the opportunistic pathogens Escherichia coli and Enterococcus faecium. Transfer of ARGs will be quantified by assays on laboratory media, and in human faecal suspensions. Expected Outcomes and ImpactThis project will lead to important insights into the dynamics of ARGs in the gut microbiome. Due to the role of the microbiome in a variety of complex diseases and conditions (including inflammatory bowel disease, colon cancer, and obesity) and the widespread use of antibiotics, which is the main driver of the emergence of antibiotic resistance, it is of considerable importance to study the dynamics of the reservoir of ARGs in the gut microbiome. This information can be used for the development of therapies that use antibiotics or other antimicrobials to target specific groups of bacteria in the gut microbiome, while minimizing the acquisition of ARG, leading to drug resistance. The PhD student will acquire expertise in state-of-the-art methods in aerobic and anaerobic microbiology and molecular biology, specifically in the fast-moving area of the human gut microbiome. In addition, the student will be

目的:A。将人类肠道微生物组中的抗生素耐药基因（ARGs）与其细菌宿主联系起来。评估人类肠道微生物群中ARGs向条件致病菌传播的能力。人体肠道内有一个复杂的微生物群落（“肠道微生物组”），对人类健康和疾病有贡献。Van Schaik教授和其他人之前的研究表明，肠道微生物组包含大量抗生素耐药基因（ARGs） 1,2目前研究肠道微生物组的方法，如宏基因组霰弹枪测序或高通量定量pcr，不允许鉴定携带args的细菌和可移动遗传元件ARGs在肠道微生物群成员之间传播的程度，包括肠道栖息的机会性病原体，如大肠杆菌和肠球菌，也尚不清楚。假设肠道微生物组中的大多数ARGs将被厌氧肠道共生体所庇护。这些ARGs可能转移到革兰氏阳性和革兰氏阴性条件致病菌。实验方法及研究计划在这个项目中，我们将确定人类粪便样本中ARGs与其细菌宿主的联系。我们将使用儿童、成人患者和健康个体的粪便（n = 5，共15个样本）。这些粪便可通过合作者获得，并可在符合当前研究方案和伦理法规的情况下用于研究目的共享。首先通过定量PCR筛选粪便样本中是否存在重要的抗性基因blaNDM、blaKPC、mcr-1、vanA和vanB，如果抗性基因呈阳性，将通过一种新开发的方法来确定抗性基因与其细菌宿主之间的联系，这种方法被称为乳状PCR、配对分离和串联PCR。一旦确定了携带ARGs的细菌的身份，将使用各种富集方法从相关样品中培养这些细菌。分离后，将对分离细菌的基因组进行测序，使用短读和长读测序相结合，从而得到染色体和质粒的完整组装。带注释的基因组序列将用于确定抗性基因是否与可移动的遗传元件（特别是质粒和共轭元件）相关，或者是否携带在染色体上，这将最大限度地减少其水平转移的可能性在完成耐药基因携带细菌的基因组表征后，我们将进行偶联试验，以评估这些细菌作为条件致病菌大肠杆菌和屎肠球菌耐药基因供体的能力。将通过对实验室培养基和人粪便悬浮液的测定来定量ARGs的转移。预期结果和影响该项目将有助于深入了解肠道微生物组中ARGs的动态。由于微生物组在多种复杂疾病和病症（包括炎症性肠病、结肠癌和肥胖）中的作用，以及抗生素的广泛使用，这是抗生素耐药性出现的主要驱动因素，因此研究肠道微生物组中ARGs储存库的动力学具有相当重要的意义。这些信息可用于开发使用抗生素或其他抗菌剂靶向肠道微生物组中特定细菌群的治疗方法，同时最大限度地减少ARG的获得，从而导致耐药性。博士生将获得有氧和厌氧微生物学和分子生物学最先进方法的专业知识，特别是在人类肠道微生物组的快速发展领域。此外，学生将