利用新型人肠类器官衍生感染模型对福氏志贺菌进行噬菌体治疗测试。
Bacteriophage Therapy Testing Against Shigella flexneri in a Novel Human Intestinal Organoid-Derived Infection Model.
机构信息
Division of Pediatric Gastroenterology and Nutrition, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital.
Department of Pediatrics, Harvard Medical School, Boston.
出版信息
J Pediatr Gastroenterol Nutr. 2019 Apr;68(4):509-516. doi: 10.1097/MPG.0000000000002203.
OBJECTIVE
Enteric bacterial pathogens cause diarrheal disease and mortality at significant rates throughout the world, particularly in children younger than 5 years. Our ability to combat bacterial pathogens has been hindered by antibiotic resistance, a lack of effective vaccines, and accurate models of infection. With the renewed interest in bacteriophage therapy, we sought to use a novel human intestinal model to investigate the efficacy of a newly isolated bacteriophage against Shigella flexneri.
METHODS
An S. flexneri 2457T-specific bacteriophage was isolated and assessed through kill curve experiments and infection assays with colorectal adenocarcinoma HT-29 cells and a novel human intestinal organoid-derived epithelial monolayer model. In our treatment protocol, organoids were generated from intestinal crypt stem cells, expanded in culture, and seeded onto transwells to establish 2-dimensional monolayers that differentiate into intestinal cells.
RESULTS
The isolated bacteriophage efficiently killed S. flexneri 2457T, other S. flexneri strains, and a strain of 2457T harboring an antibiotic resistance cassette. Analyses with laboratory and commensal Escherichia coli strains demonstrated that the bacteriophage was specific to S. flexneri, as observed under co-culture conditions. Importantly, the bacteriophage prevented both S. flexneri 2457T epithelial cell adherence and invasion in both infection models.
CONCLUSIONS
Bacteriophages offer feasible alternatives to antibiotics for eliminating enteric pathogens, confirmed here by the bacteriophage-targeted killing of S. flexneri. Furthermore, application of the organoid model has provided important insight into Shigella pathogenesis and bacteriophage-dependent intervention strategies. The screening platform described herein provides proof-of-concept analysis for the development of novel bacteriophage therapies to target antibiotic-resistant pathogens.
目的
肠细菌病原体在全球范围内以相当高的比率导致腹泻病和死亡率，尤其是在 5 岁以下的儿童中。我们对抗细菌病原体的能力受到抗生素耐药性、缺乏有效疫苗和感染准确模型的阻碍。随着对噬菌体治疗的重新关注，我们试图使用新型人体肠道模型来研究新分离的噬菌体对抗福氏志贺菌的疗效。
方法
分离并评估了一株针对福氏志贺菌 2457T 的噬菌体，通过杀菌曲线实验和感染实验，用结直肠腺癌 HT-29 细胞和新型人肠道类器官衍生的上皮单层模型进行了检测。在我们的治疗方案中，类器官由肠隐窝干细胞生成，在培养中扩增，并接种到 Transwell 上，建立 2 维单层，分化为肠细胞。
结果
分离的噬菌体能有效杀死福氏志贺菌 2457T、其他福氏志贺菌菌株和一株携带抗生素抗性盒的 2457T 菌株。对实验室和共生大肠杆菌菌株的分析表明，噬菌体特异性针对福氏志贺菌，如在共培养条件下观察到的那样。重要的是，噬菌体在两种感染模型中均能阻止福氏志贺菌 2457T 上皮细胞的黏附和侵袭。
结论
噬菌体为消除肠道病原体提供了可行的抗生素替代方案，这里通过噬菌体靶向杀死福氏志贺菌得到了证实。此外，类器官模型的应用为志贺氏菌发病机制和噬菌体依赖的干预策略提供了重要的见解。本文所述的筛选平台为开发针对抗生素耐药病原体的新型噬菌体疗法提供了概念验证分析。
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