Characterization of a Vibriophage Infecting Pathogenic Vibrio harveyi

Abstract

Bacterial diseases caused by Vibrio spp. are prevalent in aquaculture and can lead to high mortality rates among aquatic species and significant economic losses. With the increasing emergence of multidrug-resistant Vibrio strains, phage therapy is being explored as a potential alternative to antibiotics for biocontrol of infectious diseases. Here, a new lytic phage named vB_VhaS_R21Y (R21Y) was isolated against Vibrio harveyi BVH1 obtained from seawater from a scallop-farming area in Rongcheng, China. Its morphology, infection cycle, lytic profile, phage stability, and genetic features were characterized. Transmission electronic microscopy indicated that R21Y is siphovirus-like, comprising an icosahedral head (diameter 73.31 ± 2.09 nm) and long noncontractile tail (205.55 ± 0.75 nm). In a one-step growth experiment, R21Y had a 40-min latent period and a burst size of 35 phage particles per infected cell. R21Y was highly species-specific in the host range test and was relatively stable at pH 4-10 and 4-55 °C. Genomic analysis showed that R21Y is a double-stranded DNA virus with a genome size of 82,795 bp and GC content of 47.48%. Its high tolerance and lytic activity indicated that R21Y may be a candidate for phage therapy in controlling vibriosis in aquacultural systems.

Keywords: Vibrio phage; biological characteristics; genomic analysis; phage therapy; siphovirus.

Figure 1

Isolation and biological features of Vibrio phage vB_VhaS_R21Y. (A) Plaques of vB_VhaS_R21Y formed on a lawn of Vibrio harveyi BVH1. (B) Transmission electron micrograph of vB_VhaS_R21Y. (C) One-step growth curve of vB_VhaS_R21Y. Error bars indicate standard deviations among triplicate samples.

Figure 2

Full genome of phage vB_VhaS_R21Y. Putative open reading frames were assigned functional categories and are depicted as arrows.

Figure 3

Evidence supporting the taxonomy and phylogeny of vB_VhaS_R21Y. (A) Protein-sharing network indicating evolutionary affinity among vB_VhaS_R21Y and its related phages sharing pairwise similarity scores of >1. Each node represents a phage genome and is colored according to its host taxonomy. Edges connecting pairwise phages from the same viral cluster determined by vConTACT2 are displayed. Thicker edges indicate a strong connection between the two phages. The valid names of existing phage genera are displayed. (B) Pairwise intergenomic distances/similarities among viral genomes for 20 phages as per the Virus Intergenomic Distance Calculator. (C) GBDP tree based on complete or partial genomes of compared phages using the web tool, VICTOR. (D) Neighbor-joining tree (1000 bootstraps) of vB_VhaS_R21Y and similar phages based on the amino acid sequences of major capsid protein as per MEGA 7. The asterisk indicates that the protein sequence has been manually annotated as the major capsid protein. (E) Neighbor-joining tree (1000 bootstraps) of vB_VhaS_R21Y and similar phages based on amino acid sequences of the terminase large subunit as per MEGA 7.

Figure 4

In vitro lysis of vB_VhaS_R21Y at different multiplicities of infection. (A) Growth curve for V. harveyi BVH1 infected by vB_VhaS_R21Y. (B) Absorbance (OD₆₀₀) of the host at 24 h. Error bars indicate standard deviation among triplicate samples. Statistical significance is indicated by ** and *** at p < 0.01 and p < 0.001, respectively, compared with the control.

Figure 5

Stability profiles of phage vB_VhaS_R21Y over a range of temperatures and pHs. (A) Thermal stability profile. (B) pH stability profile. Error bars indicate standard deviation among triplicate samples. Letters on the columns indicate statistical significance at p < 0.05.