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摘要:
目的 对3株分离自内蒙古自治区的布鲁氏菌噬菌体A1、NMY-1和NMY-2生物学特性展开分析，获取生物学信息。 方法 采用双层平板法纯化、增殖噬菌体，观察噬斑特征及测定效价；使用透射电镜观察噬菌体形态；用双层平板法和点滴法测定噬菌体宿主谱、最佳感染复数（MOI）、一步生长曲线和理化稳定性。 结果 经电镜观察，3株噬菌体均为二十面体结构，头部直径61.0 nm～65.6 nm，具有一个长为16.5 nm～19.2 nm短尾；在常规检测浓度（RTD）和104 × RTD下，噬菌体A1和NMY-1可以裂解光滑型牛种、羊种、猪种和沙林鼠种布鲁氏菌，NMY-2能够裂解粗糙型犬种、牛种和羊种布鲁氏菌，且在高浓度（104 × RTD）时可裂解的菌株数量增加；A1、NMY-1和NMY-2的MOI分别为 0.1、0.001、0.01；一步生长曲线结果显示A1和NMY-2潜伏期约为30～60 min，暴发期20～40 min，暴发量分别为2 210 蚀斑形成单位（PFU）/cell和6 000 PFU/cell，NMY-1的潜伏期和暴发期较长，分别为600 min和420 min，暴发量为1.90×105 PFU/cell；3株噬菌体在40～60 ℃，pH为2～13，紫外线照射30 min时保持良好活性。 结论 本研究明确了噬菌体A1、NMY-1和NMY-2的生物学特征和属性，丰富了布鲁氏菌的噬菌体分型系统，为噬菌体疗法治疗耐药布鲁氏菌提供了更多选择。 Abstract:Objective To understand the biological characteristics of three Brucella phages A1, NMY-1 and NMY-2 isolated from Inner Mongolia autonomous region. Methods The phages were purified and proliferated using double layer plate method, the characteristics of the plaques were observed, and the titers were determined, and transmission electron microscopy was used to observe the morphology of the phages. The phage host-range, optimal multiplicity of infection (MOI), one-step growth curve and physicochemical stability were determined using double layer plate method and drop method. Results All three phages in this study had icosahedral structures with a head diameter of 61.0 nm–65.6 nm and a short tail of 16.5 nm–19.2 nm, which were observed by electron microscopy; At the routine test dilution (RTD) and 104×RTD, bacteriophage A1 and NMY-1 could lyse smooth B. abortus, B. melitensis, B. suis and B. neotomae, NMY-2 could lyse rough B. abortus, B. melitensis and B. canis, and at high concentration (104×RTD), the number of strains that could be lysed by three phages increased; the MOIs of A1, NMY-1, and NMY-2 were 0.1, 0.001, and 0.01, respectively; one-step growth curve results showed that A1 and NMY-2 had a latency of about 30–60 min, an outbreak period of 20–40 min, and an outbreak amount of 2210 PFU/cell and 6000 PFU/cell, respectively, and the latency and outbreak period of NMY-1 were longer, with 600 min and 420 min, respectively, with an outbreak amount of 1.90×105 PFU/cell. The three phages maintained good activity at 40– 60 ℃, pH of 2–13 and ultraviolet irradiation for 30 min. Conclusion The biological characteristics and attributes of phages A1, NMY-1 and NMY-2 were clarified, and the phage typing system of Brucella was enriched, which can provide more choice for phage therapy in the treatment of drug-resistant Brucella infection. -
Key words:
- Brucella phage /
- Brucella /
- Biological characteristic
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图 1 噬菌体噬斑
注：Tb宿主菌为B.abortus 544A，A1宿主菌为B.abortus S19，NMY-1宿主菌为B.melitensis Isfahan，NMY-2宿主菌为B.abortus 45/20
Figure 1. Plaques of phages
图 3 噬菌体最佳感染复数及一步生长曲线
注：A. Tb、A1、NMY-1和NMY-2最佳感染复数的测定结果；B. Tb、A1、NMY-2的生长曲线；C. NMY-1的生长曲线；MOI. 最佳感染复数
Figure 3. OMIs and one-step growth curves of phages
图 4 噬菌体的理化稳定性
注：A. 噬菌体的热稳定性；B. 噬菌体对紫外线的敏感性；C. 噬菌体对pH的敏感性
Figure 4. Physicochemical stability of phages.
表 1 噬菌体宿主谱测定结果
Table 1. Results of phage host profiling
菌 种 菌株数量 Tb裂解菌株数量 A1裂解菌株数量 NMY-1裂解菌株数量 NMY-2裂解菌株数量 RTD 104×RTD RTD 104×RTD RTD 104×RTD RTD 104×RTD 牛种布鲁氏菌（S） 7 7 7 7 7 7 7 0 0 牛种布鲁氏菌（R） 2 0 0 0 0 0 0 1 2 羊种布鲁氏菌（S） 11 2 6 5 7 7 11 0 0 羊种布鲁氏菌（R） 6 0 0 0 0 0 0 3 6 猪种布鲁氏菌（S） 8 0 8 7 8 5 8 0 0 犬种布鲁氏菌（R） 11 0 0 0 0 0 0 11 11 沙林鼠种布鲁氏菌（S） 1 1 1 1 1 1 1 0 0 注：S. 光滑型；R. 粗糙型；RTD. 常规检测浓度 
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