一株金黄色葡萄球菌噬菌体的分离鉴定与生物学特性

Isolation and biological characterization of a bacteriophage against Staphylococcus aureus

摘要:

【背景】金黄色葡萄球菌作为一种条件致病菌，在临床感染中扮演着重要的角色，需要研究更多更有效的防治手段。【目的】分离金黄色葡萄球菌噬菌体，研究其生物学特性，从而为金黄色葡萄球菌的替代防治提供理论借鉴。【方法】以金黄色葡萄球菌D085为宿主从污水中分离得到一株噬菌体，命名为vB_SauS_SAP3，用PEG8000浓缩、氯化铯密度梯度离心获得高纯度的噬菌体，1%乙酸双氧铀染色噬菌体进行电镜观察。测定噬菌体宿主谱、最佳感染复数(Multiplicity of infection，MOI)、一步生长曲线、热稳定性、pH值稳定性及有机溶剂对噬菌体活性的影响。平衡酚法获取噬菌体基因组，illumina测序技术和SPAdes软件进行基因组测序分析。【结果】电镜下，噬菌体SAP3头部直径为60±5 nm，尾部长170±5 nm。噬菌体vB_SauS_SAP3还能裂解5株金黄色葡萄球菌和1株产色葡萄球菌，其最佳感染复数为0.1。噬菌体vB_SauS_SAP3潜伏期为20 min，60 min后进入平台期，裂解量约为210 PFU/cell；对45 °C以下温度有一定耐受性，超过45 °C开始急剧失活；能在中性pH值时保持稳定活性，酸性环境会使其急剧失活，对碱性环境有一定的抵抗力；对氯仿和异戊醇不耐受。其基因组分析结果表明：噬菌体基因组大小为41 950 bp，GC含量为35.42%，预测有65个开放阅读框(Open Reading Frame，ORF)。进化分析表明，噬菌体vB_SauS_SAP3属于长尾科噬菌体，是一种新型的噬菌体。【结论】噬菌体vB_SauS_SAP3是一株窄谱的能够耐受一定温度和碱性环境的长尾科新型噬菌体，对其研究可为金黄色葡萄球菌噬菌体的研究提供理论材料。

Abstract:

[Background] As a conditioned pathogen, Staphylococcus aureus plays an important role in clinical infection. Therefore, the effective prevention and treatment methods should be studied more. [Objective] To isolate and analyze the biological characteristics of staphylococcus aureus phage, and to provide theoretical reference for the alternative prevention and treatment of Staphylococcus aureus. [Methods] A phage was isolated from sewage using pathogenic Staphylococcus aureus D085 as host and called vB_SauS_SAP3. The phage was obtained by PEG8000 concentration and cesium chloride density gradient centrifugation. It was stained with uranium acetate and observed via transmission electron microscopy. The phage host spectrum, optimal multiplicity of infection (MOI), one-step growth curve, temperature sensitivity, pH sensitivity and the effects of organic reagents on the activity of phage were determined. The phage’s genome was obtained by the equilibrium phenol method and illumina sequencing technology for genome sequencing analyzed. [Results] via transmission electron microscopy, phage vB_SauS_SAP3 has a head with diameter of 60±5 nm and a tail with length of 170±5 nm. It was also able to lyse five strains of Staphylococcus aureus and one strain of Staphylococcus chromogenes. The optimal multiplicity of infection was 0.1. One step growth curve showed that the latent period and the burst period was 20 min and 60 min. The burst size was about 210 PFU per infected cell. The phage vB_SauS_SAP3 was resistanted to temperatures below 45 °C and sharply inactivated beyond 45 °C. It can maintain stable activity in neutral pH and has a certain resistance in alkaline environment, but acid environment will make it sharply inactivated. The phage vB_SauS_SAP3 were completely inactivated when treatment with chloroform and isoamyl alcohol. The size of phage vB_SauS_SAP3 genome was 41 950 base pairs (bp) and ecoding 65 ORFs by predicted. The genome include GC content was 35.42%. Phylogenic analysis indicated that the phage vB_SauS_SAP3 was a member of siphoviridae and a new staphylococcus aureus phage. [Conclusion] The phage vB_SauS_SAP3 has a narrow host spectrum and can withstand certain temperature and alkaline environment, which was a new Staphylococcus aureus phage. The study of vB_SauS_SAP3 can provide theoretical materials for the study of Staphylococcus aureus phage.