在抗微生物耐药性(AMR)正在以前所未有的速度出现和传播的世界中,噬菌体(噬菌体)是传统抗微生物剂的理想替代品。此外,由于它们的宿主范围很窄,噬菌体也是调节肠道微生物群的理想工具,其中特定细菌菌株的改变是人类疾病的基础,而广谱抗生素引起的生态失调可能是有害的。这里,我们设计了一个λ噬菌体(Eλ)来靶向肠出血性大肠杆菌(EHEC),有时致命的人类肠道感染。我们通过将CRISPR-Cas3系统掺入野生型λ(wtλ)来增强Eλ噬菌体的杀伤能力,并通过引入多个靶向EHEC的CRISPR间隔区同时敲除裂解基因cro来增强特异性。体外实验表明,Eλ抑制EHEC的生长长达18小时,而wtλ仅为6小时;在感染复数(MOI)为10时,Eλ以约100%的效率杀死EHEC细胞,并且不影响其他实验室和人肠分离的大肠杆菌菌株的生长。此外,EHEC细胞没有对Eλ产生抗性。小鼠实验进一步证实了Eλ对EHEC的增强和菌株特异性杀伤,而整个小鼠肠道微生物群没有受到干扰。我们的方法可用于靶向负责抗生素抗性基因和/或人类毒素的其他基因,工程师其他噬菌体,并支持工程噬菌体的体内应用。重要性大肠杆菌的致病性菌株每年造成80万人死亡,在所有致病性物种中排名第一。这里,我们得到了,第一次,一个工程噬菌体,Eλ,可以特别有效地消除EHEC,可以在人与人之间传播的最常见且通常致命的病原体之一。我们通过体外和体内实验验证了Eλ优于野生型噬菌体的性能,并表明Eλ可以将EHEC生长抑制到不可检测的水平,完全拯救感染EHEC的小鼠,并重新研究受干扰的小鼠肠道微生物群。我们的结果还表明,EHEC没有对Eλ产生抗性,这是噬菌体治疗中最大的挑战。我们相信我们的方法可用于靶向大肠杆菌的其他致病菌株并支持工程化噬菌体的体内应用。
Bacteriophages (phages) are ideal alternatives to traditional antimicrobial agents in a world where antimicrobial resistance (AMR) is emerging and spreading at an unprecedented speed. In addition, due to their narrow host ranges, phages are also ideal tools to modulate the gut microbiota in which alterations of specific bacterial strains underlie human diseases, while dysbiosis caused by broad-spectrum antibiotics can be harmful. Here, we engineered a lambda phage (Eλ) to target enterohemorrhagic Escherichia coli (EHEC) that causes a severe, sometimes lethal intestinal infection in humans. We enhanced the killing ability of the Eλ phage by incorporating a CRISPR-Cas3 system into the wild-type λ (wtλ) and the specificity by introducing multiple EHEC-targeting CRISPR spacers while knocking out the lytic gene cro. In vitro experiments showed that the Eλ suppressed the growth of EHEC up to 18 h compared with only 6 h with the wtλ; at the multiplicity of infection (MOI) of 10, the Eλ killed the EHEC cells with ~100% efficiency and did not affect the growth of other laboratory- and human-gut isolated E. coli strains. In addition, the EHEC cells did not develop resistance to the Eλ. Mouse experiments further confirmed the enhanced and strain-specific killing of the Eλ to EHEC, while the overall mouse gut microbiota was not disturbed. Our methods can be used to target other genes that are responsible for antibiotic resistance genes and/or human toxins, engineer other phages, and support in vivo application of the engineered phages. IMPORTANCE Pathogenic strains of Escherichia coli are responsible for 0.8 million deaths per year and together ranked the first among all pathogenic species. Here, we obtained, for the first time, an engineered phage, Eλ, that could specifically and efficiently eliminate EHEC, one of the most common and often lethal pathogens that can spread from person to person. We verified the superior performance of the Eλ over the wild-type phage with in vitro and in vivo experiments and showed that the Eλ could suppress EHEC growth to nondetectable levels, fully rescue the EHEC-infected mice, and rescore disturbed mouse gut microbiota. Our results also indicated that the EHEC did not develop resistance to the Eλ, which has been the biggest challenge in phage therapy. We believe our methods can be used to target other pathogenic strains of E. coli and support in vivo application of the engineered phages.