A novel approach to reducing multiple-drug resistance in foodborne bacteria: application of CRISPR technology

降低食源性细菌多重耐药性的新方法：CRISPR技术的应用

• 批准号: 2443565
• 金额: --
• 依托单位: University of Reading
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2443565
• 关键词: novel; approach; reducing; multiple; drug

The testable hypothesis is that the CRISPR-Cas systems may be exploited to reduce the burden of drug resistance in bacterial populations in animal production such that (a) the risk of resistance passing into the human food chain is dramatically reduced and (b) that targeted removal of specific resistance will permit use of the antibiotic, thus expanding the potential utility of all antibiotics. The system consists of a sequence-specific nuclease (Cas9) and aguide RNA (gRNA) that provides sequence specificity to its DNA cleavage activity. CRISPR-Cas9 is a versatile and highly specific next-generation antimicrobial that allows targeting of specific pathogenic bacteria while leaving the remaining microbiome intact. Preliminary data generated by the commercial partner, Folium Science, has demonstrated the first in vivo application of CRISPR technology to specifically reduce the burden of Salmonella in poultry (Cogan et al, in press ). Conjugative delivery of CRISPR-Cas9 and target-specific gRNA by a mobilisable plasmid from a donor bacterium (a 'probiotic') to Salmonella caused degradation of the DNA of the targeted bacteriumand subsequent bacterial cell death.Alternative delivery systems such as modified bacteriophages (bacterial viruses) can also be utilised. resistant (MDR) bacteriain animal production. Recent studies demonstrated very high prevalence of MDR bacteria in commercial poultry production. Avian Pathogenic E. coli (APEC) have been identified as a risk factor in Urinary Tract Infection (UTI) in humans and resistance means treatment failure. The overall goal is to devise a CRISPR-Cas system that effectively reduces the incidence of selected test antibiotic resistance genes (e.g. CTX-M 15) for use in poultry in the first instance. Four objectives will be delivered, each a specialism of the two partner Universities. (1) In silico analysis of WGS data from commercial broiler poultry microbiomes will be undertaken to identify antibiotic resistance genes, their prevalence and probable vectors (e.g. plasmid incompatibility (Inc) groups). Targets (guides with relevant PAM motif for Cas activity) will be selected and tested by BLAST searches for specificity to the target gene. This process will also identify suitable gene positive/negative strains for in vitro and in vivo testing and information on the distribution of CRISPR-Cas systems within those strains. (2) Ta r ge t sequences will be synthesised and assembled by Golden Gate cloning into Folium vectors constitutively expressing Cas9 or Type I CRISPR-Cas genes. The testing of the utility of the guide sequences is empirical using gene positive/negative recipients from [1] above. The constructs will be mobilised into test strains and loss of the targeted resistance monitored both phenotypically and genotypically. (3) Prior to in vivo studies, in vitro gut poultry models will be exploited to determine the dynamics of delivery of the CRISPR-Cas system to the poultry gut microbial community and assess reduction of the target resistance gene and its associated vector. Mathematical modelling of rates of transfer and diversity of recipient microbes will be developed to provide information related to enhancing and/or specifying range of conjugation recipients. (4) The CRISPR-Cas probiotic strain developed in 1-3 above will then be used in commercial broiler chicks un-primed and primed with a target strain using appropriate third-party chicken containment facilities e.g. APHA (Weybridge). Detailed microbial molecular profiling will be undertaken to examine dissemination of the CRISPR-Cas system, the loss of the targeted AMR gene and any population shifts in the gut microbiome.

可检验的假设是，CRISPR-Cas系统可用于减少动物生产中细菌群体的耐药性负担，使得（a）抗性传递到人类食物链中的风险显著降低，以及（B）特异性抗性的靶向去除将允许使用抗生素，从而扩大所有抗生素的潜在效用。该系统由序列特异性核酸酶（Cas9）和向导RNA（gRNA）组成，向导RNA为其DNA切割活性提供序列特异性。CRISPR-Cas9是一种多功能和高度特异性的下一代抗菌剂，可以靶向特定的病原菌，同时保持剩余的微生物组完整。由商业合作伙伴Cogan Science生成的初步数据已经证明了CRISPR技术的首次体内应用，以特异性地减少家禽中沙门氏菌的负担（Cogan等人，出版中）。CRISPR-Cas9和靶标特异性gRNA通过来自供体细菌（“益生菌”）的可移动质粒缀合递送至沙门氏菌引起靶细菌的DNA降解和随后的细菌细胞死亡。也可以使用替代递送系统，例如修饰的噬菌体（细菌病毒）。耐药（MDR）菌在动物生产中的作用。最近的研究表明，MDR细菌在商业家禽生产中的流行率非常高。禽致病性大肠杆菌大肠杆菌（APEC）已被确定为人类尿路感染（UTI）的危险因素，耐药意味着治疗失败。总体目标是设计一种CRISPR-Cas系统，该系统首先有效地降低用于家禽的选定测试抗生素抗性基因（例如CTX-M15）的发生率。将实现四个目标，每个目标都是两所合作大学的专长。(1)将对来自商业肉鸡家禽微生物组的WGS数据进行计算机模拟分析，以鉴定抗生素耐药基因、其流行率和可能的载体（例如质粒不相容性（Inc）组）。将选择靶标（具有Cas活性相关PAM基序的指导物），并通过BLAST搜索检测对靶基因的特异性。该过程还将鉴定用于体外和体内测试的合适的基因阳性/阴性菌株以及关于CRISPR-Cas系统在这些菌株中的分布的信息。(2)将合成标记序列并通过Golden Gate克隆组装到组成型表达Cas9或I型CRISPR-Cas基因的cDNA载体中。使用来自上述[1]的基因阳性/阴性接受者对指导序列的效用进行测试是经验性的。将构建体动员到试验菌株中，并在表型和基因型上监测靶向耐药性的丧失。(3)在体内研究之前，将利用体外肠道家禽模型来确定CRISPR-Cas系统向家禽肠道微生物群落递送的动力学，并评估靶抗性基因及其相关载体的减少。将开发受体微生物的转移速率和多样性的数学建模，以提供与增强和/或指定缀合受体范围相关的信息。(4)然后将在上述1-3中开发的CRISPR-Cas益生菌菌株用于未引发的商业肉鸡和使用适当的第三方鸡遏制设施例如APHA（Weybridge）用靶菌株引发的商业肉鸡。将进行详细的微生物分子分析，以检查CRISPR-Cas系统的传播，靶向AMR基因的丢失以及肠道微生物组中的任何群体变化。