鸡源大肠杆菌噬菌体vB_EcoM-E33的分离鉴定和基因组特征及其治疗效果评价

Chicken-derived coliphage vB_EcoM-E33: isolation, identification, genomic characterization, and evaluation of its therapeutic effect on colibacillosis

摘要:

【背景】大肠杆菌是导致规模化养殖家禽死亡的重要病原体，噬菌体防治耐药性细菌感染具有广阔的应用前景。【目的】从鸡场环境样本中分离出噬菌体，研究其生物学活性特征及其对白羽肉鸡大肠杆菌病的治疗效果。【方法】采用双层平板法分离纯化噬菌体vB_EcoM-E33(E33)；利用透射电镜观察其形态特征；克隆噬菌体E33基因组序列并分析其基因组特征；通过裂解谱、最佳感染复数、理化因子耐受性和一步生长曲线确定噬菌体E33的生物学活性；构建白羽肉鸡大肠杆菌感染模型，检测噬菌体E33的治疗效果。【结果】从鸡场粪便样本中分离到一株Straboviridae科噬菌体E33，其宿主谱为35.4%(34/96)，基因组全长为170625bp，包含271个开放阅读框和2个tRNA，无毒力基因和耐药基因。以Escherichia coli E32为宿主菌增殖，噬菌体E33的潜伏期为10min，暴发量为60PFU/cell；当感染复数为0.001时，噬菌体E33效价最高，达到1.93×10⁹PFU/mL；在50℃以下和pH3.0–11.0范围内活性稳定；对紫外线敏感。口服10⁸CFU/mL的致病性E.coliO78构建白羽肉鸡感染模型，肌肉注射10⁸PFU/mL噬菌体E33具有良好的治疗效果。【结论】噬菌体E33具有宿主谱宽、裂解性能高、理化因子耐受性强、不携带有害基因等优点，具有较好的开发价值，有望作为抗生素替代品用于鸡场致病性大肠杆菌病的防控。

Abstract:

[Background] Drug-resistant Escherichia coli is a major pathogen that causes the death of poultry in large-scale breeding. Virulent phages have broad application prospects in preventing and treating drug-resistant bacterial infections. [Objective] To isolate a phage from the environmental samples of chicken farms, identify the biological characteristics, and evaluate its therapeutic effect on colibacillosis in white feather broilers.[Methods] Phage vB_EcoM-E33 (E33) was isolated via double-layer plate method. Its morphological characteristics were observed under a transmission electron microscope. After cloning of the genome sequence, the genomic features of the phage were analyzed. The host range, multiplicity of infection (MOI), stability, and one-step growth curve of phage E33 were determined by double-layer plate method. The colibacillosis model was established with white feather broilers to test the therapeutic effect of phage E33. [Results] A bacteriophage E33 of the family Straboviridae was isolated from a chicken feces sample, with a host range of 35.4% (34/96). The full-length genome of phage E33 was 170 625 bp, including 271 open reading frames (ORFs), two tRNAs, and no virulence or resistance gene. E.coli E32 was used as the host strain for the proliferation of E33, which showed an incubation period of 10 min and a burst size of 60 PFU/cell. When the MOI was 0.001, phage E33 had the highest titer of 1.93×10⁹ PFU/mL. Phage E33 kept stable below 50 ℃ and within the range of pH 3.0-11.0, while it was sensitive to ultraviolet light. The colibacillosis model of white feather broilers was established by oral administration with 10⁸ CFU/mL E.coli O78, and intramuscular injection of 10⁸ PFU/mL phage E33 demonstrated good therapeutic effect on the model. [Conclusion] Phage E33 has a broad host range, high lysis ability, and good tolerance to physical and chemical factors and does not carry harmful genes. It demonstrates a good development value and is expected to be used as an antibiotic alternative for the prevention and control of colibacillosis in chicken farms.