Characterization of the Novel Phage vB_VpaP_FE11 and Its Potential Role in Controlling Vibrio parahaemolyticus Biofilms

Abstract

Vibrio parahaemolyticus causes aquatic vibriosis. Its biofilm protects it from antibiotics; therefore, a new different method is needed to control V. parahaemolyticus for food safety. Phage therapy represents an alternative strategy to control biofilms. In this study, the lytic Vibrio phage vB_VpaP_FE11 (FE11) was isolated from the sewers of Guangzhou Huangsha Aquatic Market. Electron microscopy analysis revealed that FE11 has a typical podovirus morphology. Its optimal stability temperature and pH range were found to be 20-50 °C and 5-10 °C, respectively. It was completely inactivated following ultraviolet irradiation for 20 min. Its latent period is 10 min and burst size is 37 plaque forming units/cell. Its double-stranded DNA genome is 43,397 bp long, with a G + C content of 49.24% and 50 predicted protein-coding genes. As a lytic phage, FE11 not only prevented the formation of biofilms but also could destroy the formed biofilms effectively. Overall, phage vB_VpaP_FE11 is a potential biological control agent against V. parahaemolyticus and the biofilm it produces.

Keywords: Vibrio parahaemolyticus; aquaculture; biofilm; biological control; phage.

Figure 1

TEM images of vB_VpaP_FE11 (FE11), scale bar = 50 nm.

Figure 2

Average nucleotide identity heatmap. The percentage identity values range from 0 (0%, yellow) to 1 (100%, orange).

Figure 3

Comparison of the genomes of FE11, and vB_VpaP_KF1, and vB_VpaP_KF2 using Easyfig. Different colored arrows represent 50 predicted open reading frames with different functions.

Figure 4

Phylogenetic tree of Vibrio phages in the subfamily Autographiviridae, conducted using the neighbor-joining method in MEGAX by RNA polymerases.

Figure 5

Protein-sharing network of FE11 benchmarked against ICTV-accepted viral taxonomy. (A) The network consists of 3445 phages (nodes) and 80,374 relationships (edges). Each node represents a viral genome, and each distinct color represents the viral family. (B) The protein cluster where FE11 was located. Different shapes represent phages with different hosts and each distinct color represents the viral genus.

Figure 6

General characterizations of phage FE11. (A) One-step growth curve. (B) Effects of ultraviolet irradiation on phage activity. (C) Temperature tolerance. (D) pH tolerance.

Figure 7

Effects of phage on biofilm. (A) Effects of FE11 of different titers on the preformed biofilm. Different treatments are indicated by different bar colors. ****, p < 0.0001. (B) Effects of phage co-incubation on biofilm formation. Shown are the results on the effects of FE11 at different MOIs on the biofilm formation after 6, 9, and 12 h of co-incubation. Control: Vibrio parahaemolyticus strain O5–15 (VP O5–15) without any phages. ****, p < 0.0001.

Figure 8

Scanning electron microscope imagery of V. parahaemolyticus O5–15 treated with phage FE11: (A,C) V. parahaemolyticus O5–15, and (B,D) V. parahaemolyticus O5–15 + FE11.