一株福氏志贺氏菌噬菌体的分离鉴定及其生物学特性

Isolation and biological characterization of a bacteriophage infecting Shigella flexneri

摘要:

[背景] 志贺氏菌是一类能引起人和动物腹泻的致病菌，由于抗生素滥用导致其耐药问题日益严重，寻找新的抗菌药物和治疗方法成为目前亟待解决的问题。[目的] 检测志贺氏菌对肉鸡的致病性，分离纯化出一株可裂解强致病性志贺氏菌的噬菌体，并对其生物学特性进行研究。[方法] 从病鸡肠道黏膜分离志贺氏菌；以健康肉鸡为动物模型进行攻毒，测定强致病性菌株的耐药性；并以此为宿主菌分离噬菌体，聚乙二醇（Polyethylene Glycol，PEG）沉淀法纯化浓缩噬菌体后，用透射电子显微镜观察其形态特征。利用双层平板法测定噬菌体的宿主谱、最佳感染复数、一步生长曲线、pH和热稳定性对噬菌体活性的影响。[结果] 分离得到26株志贺氏菌，分别命名为BDS1-BDS26，其中BDS8致病性最强，经鉴定属于福氏志贺氏菌，而且存在多重耐药性，灌喂后的肉鸡出现严重腹泻和血便；解剖病症主要表现为心脏肥大、肠系膜出血明显等。以BDS8为宿主菌，分离得到噬菌体ΦDS8。透射电镜结果显示噬菌体ΦDS8的头部呈二十面体形状，直径61±2 nm，尾长165±2 nm，属长尾噬菌体科。噬菌体ΦDS8在pH 4.0-10.0、50℃以下范围内能保持较高活性，感染周期约为120 min，其中包括75 min的潜伏期和45 min的暴发期，暴发量为52 PFU/cell。[结论] 噬菌体ΦDS8具有宿主特异性，在常规环境下具有较好的耐受能力，为噬菌体治疗福氏志贺氏菌病提供理论依据。

关键词:福氏志贺氏菌;噬菌体;分离鉴定;生物学特性

Abstract:

[Background] Shigella is a class of pathogenic bacteria that can cause diarrhea in humans and animals. As the problem of drug resistance is becoming more and more serious due to the abuse of antibiotics, alternative antimicrobial agents and treatment methods have become an urgent need. [Objective] To detect the pathogenicity of Shigella to broiler chickens, isolate and purify a bacteriophage (phage) that can lyse the strongly pathogenic Shigella, and study its biological characteristics. [Methods] Shigella was isolated from intestinal mucosa of diseased chickens. Healthy broiler chickens were used as animal models to test the drug resistance of highly pathogenic strains. The phage was isolated by using the host bacterium, and concentrated phage was purified by polyethylene glycol (PEG) precipitation. The morphological characteristics of the phage were observed by transmission electron microscopy (TEM). The double-layer plate method was used to determine the host spectrum, multiplicity of infection, one-step growth curve, pH, and thermal stability of the phage. [Results] This study has isolated 26 Shigella species, designated BDS1-BDS26. Among, BDS8 was the most pathogenic with multiple drug resistance and was identified as Shigella flexneri. Severe diarrhea and bloody stools were observed in the broiler chickens after feeding BDS8. Anatomical symptoms were mainly manifested as cardiac hypertrophy and obvious mesenteric haemorrhage, etc. Furthermore, the phage ΦDS8 was isolated from BDS8. TEM results showed that the head of ΦDS8 was icosahedral, with a diameter of 61±2 nm and a tail length of 165±2 nm. ΦDS8 belongs to the family of Siphoviridae. ΦDS8 could maintain high activity in the range of pH 4.0-10.0 and below 50℃. The infection cycle of ΦDS8 was about 120 min, including an incubation period of 75 min and an outbreak period of 45 min. The outbreak volume was 52 PFU/cell. [Conclusion] The phage ΦDS8 has host specificity and a better tolerance to general environments. This study provides a theoretical basis for phage therapy of shigellosis flexneri.

Key words:Shigella flexneri;bacteriophage;isolation and identification;biological characterization