一株副溶血弧菌噬菌体生物学特性、全基因组特征及其在食品中的应用

张俊鹏; 刘文婷; 石甜; 王华娟; 王宏勋; 周敏

doi:10.13386/j.issn1002-0306.2023020018

一株副溶血弧菌噬菌体生物学特性、全基因组特征及其在食品中的应用

Biological Characteristics, Genome-wide Characteristics of A Bacteriophage of Vibrio parahaemolyticus and Its Application in Food

摘要

摘要: 目的：分析1株副溶血性弧菌噬菌体474x1的生物学特性、全基因组及在食品中的抑菌效果。方法：以副溶血性弧菌474菌株为宿主菌，从海鲜市场基围虾分离噬菌体474x1，利用透射电镜观察其形态，并绘制一步生长曲线，分析474x1对温度及pH的敏感性。分析474x1全基因组序列，根据474x1末端酶大亚基构建系统进化树。通过测定菌落总数评价噬菌体对虾肉中副溶血弧菌的抑制效果。结果：分离到１株新型的副溶血弧菌噬菌体，命名为474x1，该噬菌体能够裂解23株副溶血弧菌中的19株（19/23=82.61%）。电镜观察474x1具有典型的短尾病毒科病毒形态特征。最佳感染复数（MOI）为0.01，一步生长曲线显示474x1的潜伏期为10 min，裂解量为115 PFU/cell。该噬菌体能够在较大的温度范围（30~60 ℃）和pH（4~11）范围内维持活性。474x1全基因组长47830 bp，包含69个开放阅读框（open reading frames，ORFs），其中14个具有特定功能的基因。比较基因组学分析474x1与短尾噬菌体科弧菌属噬菌体Vp41s3基因组具有较高同源性，进化分析表明474x1与副溶血弧菌噬菌体Vp41s3亲缘性最高。在应用实验中，在4 ℃下MOI=1000的实验组在第3 h相较对照组菌量下降了0.39 lgCFU/mL。MOI=10000的实验组在第12 h相较对照组菌量下降了0.92 lgCFU/mL，在25 ℃下MOI=1000，MOI=10000的实验组相较对照组在第6 h时菌量分别下降1.04、1.82 lgCFU/mL，表明噬菌体474x1能够显著抑制虾肉中宿主菌的生长。结论：从基围虾中分离鉴定１株新的副溶血性弧菌噬菌体，该噬菌体裂解量大，潜伏期短，在60 ℃以下表现出较好的稳定性，pH耐受范围宽，在食品中也有良好的抑菌效果，为防控水产品副溶血性弧菌的致病株奠定了基础。

Abstract: Objective: The purpose of this study was to investigate the biological characteristics, whole genome, and antibacterial effect of V. parahaemolyticus phage 474x1 in food. Methods: The phage 474x1 was isolated from shrimp purchased from seafood market with V. parahaemolyticus strain 474 as host strain. The morphology of phage 474x1 was observed by transmission electron microscopy. One-step growth curve was determined, and its sensitivity to temperature and pH was assessed. The whole genome sequence of 474x1 was analyzed, and a phylogenetic tree was constructed based on the sequence of large subunit of phage terminase. The inhibitory effect of the phage on V. parahaemolyticus in shrimp meat was evaluated using the total colony count method. Results: A lytic phage against V. parahaemolyticus was isolated which named 474x1, and it could lyse 19 out of 23 strains of V. parahaemolyticus (82.61%). The phage had typical characteristics of a tailed bacteriophage in Myoviridae. The optimal multiplicity of infection (MOI) was 0.01, and the one-step growth curve showed that the latent period of 474x1 was 10 min, and the burst size was 115 PFU/cell. The phage was active over a wide range of temperature (30~60 ℃) and pH (4~11). The whole genome of 474x1 was 47830 bp long, and 69 open reading frames were predicted and annotated, including 14 genes with specific functions. Comparative genomics analysis showed that 474x1 had high homology with Vibrio phage Vp41s3, and phylogenetic analysis also indicated that 474x1 was most closely related to the phage Vp41s3. In the application experiment, the number of bacteria in the experimental group with MOI=1000 at 4 ℃ decreased by 0.39 lgCFU/mL compared to the control group at 3 h, while the experimental group with MOI=10000 at 4 ℃ decreased by 0.92 lgCFU/mL compared to the control group at 12 h. At 25 ℃, the number of bacteria in the MOI=1000 and MOI=10000 groups decreased by 1.04 lgCFU/mL and 1.82 lgCFU/mL at 6 h, respectively. The results indicated that phage 474x1 could significantly inhibit the growth of host bacteria in shrimp meat. Conclusion: A new V. parahaemolyticus phage was isolated from shrimp, which had large burst size, short latent period, and showed good stability below 60 ℃ and tolerated a wide range of pH. In addition, the phage had good bacteriostatic effect in food, laying a foundation for the prevention and control of pathogenic strains of V. parahaemolyticus.