绒山羊源大肠杆菌噬菌体φPTK的分子生物学特性及对小鼠大肠杆菌感染的防治效果

Molecular biological characteristics of cashmere goat-derived Escherichia coli phage φPTK and the effect on mice infected with E. coli

摘要:

【背景】抗生素耐药问题是影响人类及养殖业健康的重要因素，噬菌体能特异性裂解细菌，成为抗生素替代品研究热点，是解决抗生素耐药难题、促进养殖业健康发展的新途径。【目的】通过研究绒山羊源大肠杆菌烈性噬菌体φPTK (phage target of K1)的分子生物学特性，同时利用小鼠感染模型研究φPTK对小鼠大肠杆菌感染的防治效果，为绒山羊大肠杆菌病的防控提供新策略。【方法】用聚乙二醇-氯化钠(PEG 8000-NaCl)浓缩φPTK后，采用透射电子显微镜观察其超微形态结构；运用苯酚-氯仿法提取φPTK核酸后通过Illumina HiSeq高通量测序分析其全基因组结构，使用Mauve比较基因组学分析，通过MEGA绘制噬菌体进化树；通过构建小鼠感染模型分析φPTK对小鼠感染大肠杆菌的防治效果。【结果】透射电镜显示φPTK头部为正多面体形，直径90 nm，有长约112 nm、直径约18 nm的可收缩长尾；φPTK基因组全长169 688 bp，GC含量37.72%，有264个开放阅读框，含穿孔素-裂解酶(holin-lysin)裂解系统，有抗穿孔素蛋白和裂解抑制辅助蛋白，未发现抗生素耐药基因和毒力基因；比较基因组分析表明，φPTK为一株新的绒山羊源大肠杆菌烈性噬菌体；小鼠大肠杆菌感染前和感染后分别使用φPTK进行预防和治疗的试验表明，未使用φPTK的阳性对照组小鼠全部死亡，预防组和治疗组小鼠存活率分别为80%和60%。【结论】噬菌体φPTK是一株能够在小鼠大肠杆菌感染中具有较好预防效果的有尾噬菌体目(Caudovirales)肌尾噬菌体科(Myoviridae)绒山羊源大肠杆菌烈性噬菌体，本研究为绒山羊噬菌体生物制剂的创制奠定了基础。

Abstract:

[Background] Antibiotic resistance is arguably the biggest current threat to global health and animal husbandry development. As an emerging solution, bacteriophage can specifically infect and kill bacteria, thus becoming a candidate substitute of antibiotics to enable healthy development of the animal husbandry. [Objective] To study the molecular biological characteristics of the cashmere goat-associated Escherichia coli virulent bacteriophage φPTK, and use mouse model to examine the effect of φPTK in the prevention and treatment of E. coli infections, so as to provide a new strategy for effective control of E. coli infections in cashmere goats.[Methods] The φPTK was concentrated with polyethylene glycol (PEG) 8000-sodium chloride (NaCl) and then the morphological structure was observed by transmission electron microscopy. The nucleic acid of φPTK was extracted with phenol-chloroform method, and the whole genome was sequenced by Illumina HiSeq. Mauve was employed for comparative genomics analysis, and the polygenetic tree was plotted by MEGA. Finally, the effect of φPTK in the prevention and treatment of E. coli infection in mice was analyzed.[Results] The φPTK had isometric head (90 nm diameter) and long contractile tail (about 112 nm in length and 18 nm in diameter). The genome was 169 688 bp with GC content of 37.72% and 264 open reading frames (ORFs). It had the holin-lysin lysis system, anti-holin and lysis inhibition accessory protein, and no virulence-associated and antibiotic resistance genes. The comparative genome analysis indicated it was a novel lytic cashmere goat-associated E. coli bacteriophage. φPTK was applied before and after E. coli infection in mice for prevention and treatment. The result showed that all mice in the positive control group without φPTK died, and the survival rate of mice in the prevention and treatment groups reached 80% and 60%, respectively. [Conclusion] The virulent cashmere goat-associated E. coli phage φPTK (Myoviridae, Caudovirale) is effective in the prevention and treatment of E. coli infections. This study lays a basis for the development of bacteriophage agents for biological control of colibacillosis in cashmere goats.