Targeting antibiotic resistance genes in Vibrio cholerae using a phage-encoded CRISPR-Cas system to improve efficacy of phage prophylaxis

项目摘要 细菌病原体中多药耐药性的不断上升造成了一种可怕的局面， 发展预防和治疗传染病的新模式。噬菌体预防和噬菌体 治疗代表了这样一种方法，因为抗生素抗性表型不影响细菌。的 霍乱的病原体，霍乱弧菌，在过去已经成为广泛耐药（XDR 由于低收入和中等收入社区滥用和广泛使用抗生素， 国家我们最近报道了一种噬菌体产物，由毒力很强的噬菌体ICP 1、ICP 2和ICP 3组成， 在动物模型中有效预防霍乱，可用于降低高危人群的感染率。 然而，这项工作使用了1980年以前的霍乱弧菌菌株。在这里，我们试图测试两个 假设，如果得到证实，可以大大提高霍乱的噬菌体预防， 它本身可以改善噬菌体产品，用于由多重耐药细菌引起的其他疾病。第一 有一种假设是，将CRISPR间隔区整合到ICP 1中，其特异性靶向抗生素耐药性， XDR霍乱弧菌中的基因，可以提高ICP 1在体外和动物中杀死这些菌株的能力 从而更好地保护动物免受感染。第二个假设是， 通过CRISPR-Cas切割抗生素抗性基因，我们可以大大降低它们的频率， 水平基因转移

PROJECT SUMMARY The incessant rise of multidrug resistance in bacterial pathogens has created a dire situation that necessitates development of new modalities for preventing and treating infectious diseases. Phage prophylaxis and phage therapy represent one such approach, since phages are not affected by antibiotic resistance phenotypes. The causative agent of cholera, Vibrio cholerae, has become extensively drug resistant (XDR) in just the past decade due to indiscriminate and widespread antibiotic use in the community in low- and middle-income countries. We recently reported a phage product, comprised of virulent phages ICP1, ICP2 and ICP3, that effectively prevents cholera in animal models, which could be used to reduce infection rates those at-risk. However, that work used an antibiotic-sensitive, pre-1980 strain of V. cholerae. Here, we seek to test two hypotheses that, if substantiated, could dramatically improve phage prophylaxis for cholera and would lend itself to improving phage products for other diseases caused by multidrug-resistant bacteria. The first hypothesis is that incorporating CRISPR spacers into ICP1, which specifically target antibiotic resistance genes in XDR V. cholerae, can improve the ability of ICP1 to kill these strains both in vitro and in animal models, thereby better protecting the animals from infection. The second hypothesis is that by targeting antibiotic resistance genes for cleavage by CRISPR-Cas, we can dramatically reduce their frequencies of horizontal gene transfer.