食品科学 ›› 2021, Vol. 42 ›› Issue (1): 52-58.doi: 10.7506/spkx1002-6630-20200105-053

• 基础研究 • 上一篇    下一篇

噬菌体vB_SauM_RS对乳源金黄色葡萄球菌生物被膜的清除作用

阮红日,王宇辉,徐若洋,陈立,靳育琦,王建发,宋军,郑家三   

  1. (1.黑龙江八一农垦大学动物科技学院,黑龙江 大庆 163319;2.黑龙江省牛病防制重点实验室,黑龙江 大庆 163319)
  • 发布日期:2021-01-18
  • 基金资助:
    国家自然科学基金青年科学基金项目(31802226);黑龙江省青年科学基金项目(QC2017021); 黑龙江省政府博士后资助项目(LBH-Z17185);黑龙江省牛病防制重点实验室开放课题(PCBD201710); 黑龙江八一农垦大学三横三纵支持计划项目(TDJH201903);黑龙江八一农垦大学引进人才项目(XYB201807)

Effect of Phage vB_SauM_RS on Removal of Milk-Derived Staphylococcus aureus Biofilms

RUAN Hongri, WANG Yuhui, XU Ruoyang, CHEN Li, JIN Yuqi, WANG Jianfa, SONG Jun, ZHENG Jiasan   

  1. (1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China; 2. Key Laboratory of Prevention and Control of Bovine Diseases of Heilongjiang Province, Daqing 163319, China)
  • Published:2021-01-18

摘要: 目的:以金黄色葡萄球菌为宿主菌,分离裂解性噬菌体vB_SauM_RS,测定其生物学特性,并探究其在两种牛奶中的抑菌效果及对生物被膜的清除作用。方法:将噬菌体与金黄色葡萄球菌以1∶100的比例(菌体数量比)分别接种于脱脂牛奶和全脂牛奶中,分别在4 ℃和25 ℃环境下处理,测定不同时间脱脂牛奶和全脂牛奶中细菌浓度和噬菌体效价,分析噬菌体在两种牛奶中的抑菌效果;之后通过结晶紫染色、平板计数法和双层平板法测定经噬菌体处理后不同时间膜内细菌浓度和噬菌体效价,分析vB_SauM_RS对不同材料表面生物被膜的清除效果,并通过荧光显微镜观察生物被膜清除前后膜内细菌数量的变化。结果:vB_SauM_RS形成的噬菌斑清晰透亮、外有晕环;透射电子显微镜观察其属于肌尾噬菌体科;最佳感染复数为0.01、潜伏期为20 min、平均裂解量为39 PFU/cell,其在20~50 ℃和pH 4.0~11.0条件下稳定,具有高效的裂解效率;vB_SauM_RS在牛奶中抑菌作用明显,与对照组相比,在低温(4 ℃)条件下24 h使脱脂牛奶和全脂牛奶中细菌浓度分别下降1.079(lg(CFU/mL))和1.021(lg(CFU/mL)),在室温(25 ℃)条件下24 h使脱脂牛奶和全脂牛奶中细菌浓度分别下降5.418(lg(CFU/mL))和5.740(lg(CFU/mL));通过平板计数法和荧光显微镜观察显示vB_SauM_RS对生物被膜内细菌的清除效果明显,对不同材料表面生物被膜均有良好的清除效果。结论:vB_SauM_RS不仅对牛奶中金黄色葡萄球菌有良好的裂解效果,对生物被膜也有很好的清除效果,具有作为金黄色葡萄球菌生物抗菌剂的应用潜力。

关键词: 裂解性噬菌体;金黄色葡萄球菌;生物学特性;牛奶;生物被膜

Abstract: Objective: Staphylococcus aureus was used as the host bacterium for isolating a lytic bacteriophage vB_SauM_RS. Its biological characteristics were determined, and its antibacterial effect in milk and its efficiency in removing biofilms were studied. Methods: Bacteriophage and S. aureus were inoculated together into skim milk or whole milk at a ratio of 1:100, which was then kept at 4 or 25 ℃. At different time points, bacterial concentration and phage titer were determined and the phage’s antibacterial effect was evaluated. Then, by crystal violet staining, plate counting method and double-layer agar plate method, bacterial concentration in the biofilm of S. aureus and phage titer were determined at different time points during phage treatment, the effect of the phage on the removal of biofilms formed on the surface of different materials was analyzed, and the changes in the number of bacterial cells in the biofilm before and after being removed were observed by fluorescence microscope. Results: Phage vB_SauM_RS formed clear plaques with halo; transmission electron microscopic (TEM) observation showed that the phage belonged to the Myoviridae family. The optimal multiplicity of infection (MOI) of this phage was 0.01. The one-step growth curve showed that the latent period was 20 min, and the average lysis amount was 39 PFU/cell. The phage was stable at 20–50 ℃ or pH 4.0–11.0. It inhibited the host bacterium, reducing the bacterial concentration in skim milk and whole milk by 1.079 (lg(CFU/mL)) and 1.021 (lg(CFU/mL)) after 24 h incubation at 4 ℃, and by 5.418 (lg(CFU/mL)) and 5.740 (lg(CFU/mL)) after 24 h incubation at 25 ℃, respectively. The results of plate counting and fluorescence microscopic observation showed that this phage had a clear effect on the clearance of bacterial cells in the biofilm, and of biofilms formed on the surface of all tested materials. Conclusion: Phage vB_SauM_RS not only has a good lytic effect on S. aureus in milk, but also can effectively remove the biofilm formed by S. aureus. It is promising as an antibacterial bioagent for preventing and controlling S. aureus.

Key words: lytic bacteriophage; Staphylococcus aureus; biological characteristics; milk; biofilm

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