大肠杆菌K99噬菌体的分离鉴定及在小鼠上的防治效果

Isolation and identification of a phage against Enterotoxigenic Escherichia coli K99 and its function verification in mice

摘要:

【背景】产肠毒素大肠杆菌K99是引起仔猪腹泻的主要致病菌之一，目前对该病的治疗主要依赖于抗生素，但大量抗生素的长期使用造成了病原菌耐药性的增强，亟待寻求一种新的产品来解决这一问题。【目的】以一株产肠毒素大肠杆菌K99为宿主菌，从畜禽粪水中分离噬菌体并对其基本生物学特性进行研究，同时在小鼠上检验噬菌体对小鼠大肠杆菌感染的防治效果。【方法】双层平板法分离筛选烈性噬菌体并观察噬菌斑形态；纯化后进行电镜观察、核酸类型鉴定；测定噬菌体热稳定性、pH稳定性、最佳感染复数及一步生长曲线；通过小鼠体内实验检测噬菌体对小鼠大肠杆菌感染的防治效果。【结果】从畜禽粪水中分离出1株产肠毒素大肠杆菌K99强裂解性噬菌体，命名为ФK99-1，经电镜观察及核酸类型鉴定，应属于肌尾噬菌体科(Myoviridae)。噬菌体能耐受50 °C左右的高温，在pH 3.0?10.0范围内效价稳定。最佳感染复数为0.000 01，暴发量为108 333 pfu/cell；运用噬菌体ФK99-1预防和治疗小鼠产肠毒素大肠杆菌K99感染，结果显示小鼠发病症状较阳性对照组明显减轻，小鼠血液内大肠杆菌K99含量也显著低于阳性对照组，通过病理切片观察显示脏器发病情况也较阳性对照组减轻。【结论】ФK99-1是一株在不同温度、不同酸碱性环境中有较强适应能力，且在小鼠大肠杆菌感染上表现出良好防治效果的裂解性肌尾科大肠杆菌噬菌体。

Abstract:

[Background] Enterotoxigenic Escherichia coli K99 is one of the main pathogens causing diarrhea in piglets. So far the treatment of this disease mainly depends on antibiotics, but the long-term use of antibiotics has caused the increase of drug resistance, therefore, new product is urgently needed to solve this problem. [Objective] A lytic phage against Enterotoxigenic Escherichia coli K99 was isolated from the manure of livestock and identified, and its biological properties were assayed as well. The preventive and therapeutic effects of the bacteriophage on Enterotoxigenic Escherichia coli K99 infection were examined in mice. [Methods] The lytic phage was isolated by the double-layer agar culture method and the phage plaque was checked. Morphology of the phage was observed by transmission electron microscopy and the type of nucleic acids was also identified after the phage purification. The biological parameters including the thermal stability, pH stability and optimal multiplicity of infection (MOI) were assayed separately. The inhibitory effects of the bacteriophage on Escherichia coli infection in mice was tested by in vivo experiments with mice. [Results] A lytic phage (designated as ФK99-1) against Enterotoxigenic Escherichia coli K99 was successfully isolated from livestock manure. ФK99-1 belongs to family Myoviridae according to the electron microscopy and type identification of nucleic acid. The phage could withstand the temperature up to 50 °C and maintain stable titer under pH 3.0–10.0. The optimal MOI of ФK99-1 was 0.000 01, and the burst size was 108 333 PFU/cell. Application of ФK99-1 in mice which were infected with Enterotoxigenic Escherichia coli K99 could alleviate the related symptoms and viscera lesions. [Conclusion] The isolated phage, ФK99-1, belongs to lytic phage of Myoviridae, which shows strong adapting capacity to various temperature and acid-base environments. At the same time the isolated phage shows inhibitory effect on E. coli infection in mice.