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

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.

Keywords

• v. parahaemolyticus phage
• separation and purification
• optimal infection complex (moi)
• one-step growth curve
• genome
• bacteriostatic test