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

可验证的假设是，CRISPR-Cas系统可能被用来减轻动物生产中细菌种群的耐药性负担，这样(a)耐药性进入人类食物链的风险大大降低，(b)有针对性地去除特定耐药性将允许使用抗生素，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）和为其DNA切割活性提供序列特异性的指导RNA （gRNA）组成。CRISPR-Cas9是一种多功能和高度特异性的下一代抗菌剂，可以靶向特定的致病菌，同时保持剩余的微生物组完整。商业合作伙伴Folium Science产生的初步数据已经证明了CRISPR技术首次在体内应用，专门减少了家禽中沙门氏菌的负担（Cogan等人，已出版）。通过来自供体细菌（益生菌）的可移动质粒将CRISPR-Cas9和目标特异性gRNA结合递送到沙门氏菌，导致目标细菌的DNA降解和随后的细菌细胞死亡。也可以利用诸如修饰噬菌体（细菌病毒）之类的替代递送系统。动物生产中的耐药（MDR）细菌。最近的研究表明，商业家禽生产中耐多药细菌的流行率非常高。禽致病性大肠杆菌（APEC）已被确定为人类尿路感染（UTI）的危险因素，耐药性意味着治疗失败。总体目标是设计一种CRISPR-Cas系统，该系统可以有效地降低选定的测试抗生素耐药基因（例如ctx - m15）的发生率，以便首次在家禽中使用。将实现四个目标，每个目标都是两所合作大学的专业。(1)将对来自商品肉鸡微生物组的WGS数据进行计算机分析，以确定抗生素耐药基因、其流行程度和可能的载体（例如质粒不相容（Inc）群）。目标（与Cas活性相关的PAM基序的指南）将被选择并通过BLAST搜索对目标基因的特异性进行测试。该过程还将确定合适的基因阳性/阴性菌株进行体外和体内测试，并获得CRISPR-Cas系统在这些菌株中的分布信息。(2)通过金门克隆技术合成并组装到组成表达Cas9或I型CRISPR-Cas基因的Folium载体中。利用上述[1]的基因阳性/阴性受体对引导序列的效用进行实证检验。这些构建物将被动员到测试菌株中，并对目标抗性的丧失进行表型和基因表型监测。(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的传递动力学，并评估目标耐药基因及其相关载体的减少。将开发转移率和受体微生物多样性的数学模型，以提供有关增强和/或指定偶联受体范围的信息。(4)上述1-3中开发的CRISPR-Cas益生菌菌株将在未引物的商业肉鸡中使用，并使用适当的第三方鸡围护设施（如APHA (Weybridge)）用目标菌株引物。将进行详细的微生物分子谱分析，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种群变化。

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.