The human gut microbiome as a reservoir of antibiotic resistance

目的：A.将人类肠道微生物组中的抗生素耐药基因(ARGs)与其细菌宿主连接起来。为了评估人类肠道微生物群中ARGs传播到机会性病原体的能力。背景人类肠道中含有一个复杂的微生物群落(肠道微生物群)，对人类健康和疾病有贡献。Van Schaik教授和其他人之前的工作揭示了肠道微生物组由大量的抗生素耐药基因(ARGs)组成。1、2目前研究肠道微生物组的方法，如元基因组鸟枪测序或高通量定量PCR，不能识别携带ARG的细菌和可移动的遗传元件。3 ARGs可以在肠道微生物组的成员之间传播的程度，包括肠道栖息的条件致病菌，如大肠杆菌和肠球菌，也尚不清楚。假设肠道微生物群中的大部分ARG将被厌氧肠道共生体包裹。这些ARG可以转移给革兰氏阳性和革兰氏阴性条件致病菌。实验方法和研究计划1。肠道微生物群中ARGs的细菌储存库在这个项目中，我们将在人类粪便样本中确定ARGs与其细菌宿主之间的联系。我们将使用来自儿科和成人患者和健康人的粪便(n=5)，总共15个样本。这些凳子可以通过合作者获得，并可根据当前的研究方案和伦理法规共享用于研究目的。首先将通过定量聚合酶链式反应筛选粪便样本中是否存在重要的耐药基因blaNDM、blaKPC、mcr-1、vanA和vanB，如果发现阳性，则将通过一种新开发的方法来确定抗药性基因与其细菌宿主之间的联系，该方法称为用于乳化、配对分离和串联PCR.4.抗药性基因对机会致病菌的可转移性一旦确定携带args的细菌的身份，将使用各种增菌方法从相关样本中培养这些细菌。在分离后，分离的细菌的基因组将被测序，使用短读和长读测序相结合的方式，导致染色体和质粒的完整组装。这些注解的基因组序列将用于确定抗药性基因是与可移动的遗传元件，特别是质粒和接合元件相关，还是携带在染色体上，这将使其水平转移的可能性降至最低。5在完成携带抗药性基因的细菌的基因组特征后，我们将进行接合分析，以评估这些细菌作为抗药性基因供体的能力，以对抗条件致病菌大肠杆菌和粪肠球菌。ARGs的转移将通过实验室介质和人类粪便悬浮液中的分析来量化。预期结果和影响该项目将导致对肠道微生物组中ARGs动态的重要洞察。由于微生物群在各种复杂疾病和条件(包括炎症性肠病、结肠癌和肥胖症)中的作用，以及抗生素的广泛使用，这是导致抗生素耐药性出现的主要原因，因此研究肠道微生物群中Args的动态变化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来靶向肠道微生物组中的特定细菌组的治疗方法，同时将ARG的获得降至最低，从而导致耐药性。这位博士生将在需氧和厌氧微生物学和分子生物学方面获得最先进的方法方面的专业知识，特别是在人体肠道微生物组快速移动的领域。此外，这名学生将被

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