Isolation and Characterization of a Novel Phage against Vibrio alginolyticus Belonging to a New Genus

Abstract

Vibrio alginolyticus causes substantial economic losses in the aquaculture industry. With the rise of multidrug-resistant Vibrio strains, phages present a promising solution. Here, a novel lytic Vibrio phage, vB_ValC_RH2G (RH2G), that efficiently infects the pathogenic strain V. alginolyticus ATCC 17749^T, was isolated from mixed wastewater from an aquatic market in Xiamen, China. Transmission electron microscopy revealed that RH2G has the morphology of Siphoviruses, featuring an icosahedral head (73 ± 2 nm diameter) and long noncontractile tail (142 ± 4 nm). A one-step growth experiment showed that RH2G had a short latent period (10 min) and a burst size of 48 phage particles per infected cell. Additionally, RH2G was highly species-specific and was relatively stable at 4-55 °C and pH 4-10. A genomic analysis showed that RH2G has a 116,749 bp double-stranded DNA genome with 43.76% GC content. The intergenomic similarity between the genome sequence of RH2G and other phages recorded in the GenBank database was below 38.8%, suggesting that RH2G represents a new genus. RH2G did not exhibit any virulence or resistance genes. Its rapid lysis capacity, lytic activity, environmental resilience, and genetic safety suggested that RH2G may be a safe candidate for phage therapy in combatting vibriosis in aquaculture settings.

Keywords: Vibrio alginolyticus; biological characterization; genomic analysis; phage; phage therapy.

Figure 1

Isolation and growth curve of vB_ValC_RH2G. (A) Transmission electron micrograph of vB_ValC_RH2G. (B) One-step growth curve of vB_ValC_RH2G. Error bars indicate standard deviations among triplicate samples.

Figure 2

Biological features of phage vB_ValC_RH2G. (A) Growth curve for V. alginolyticus ATCC 17749T infected by vB_ValC_RH2G. (B) Absorbance (OD600) of the host at 24 h. (C) Thermal stability profile. (D) pH stability profile. Error bars indicate standard deviations among triplicate samples. Letters on the columns indicate statistical significance at p < 0.05.

Figure 3

Genome map of the V. alginolyticus phage vB_ValC_RH2G. Protein-coding sequences are presented by arrows. Gene features classified into different functional modules are color-coded according to the legend below the figure.

Figure 4

Taxonomy and phylogeny of vB_ValC_RH2G. (A) Phylogenetic tree of vB_ValC_RH2G and other closely related phages, constructed using the Virus Classification and Tree Building Online Resource (VICTOR) web service. Pairwise comparisons of the amino acid sequences were conducted using the Genome-BLAST Distance Phylogeny (GBDP) method with settings recommended for prokaryotic viruses. (B) Pairwise intergenomic distances/similarities among viral genomes according to the Virus Intergenomic Distance Calculator. Intergenomic similarity values are in right half, alignment indicators are in left half and top annotation.

Figure 5

Maximum-likelihood phylogenetic tree (1000 bootstraps) based on amino acid sequences of (A) major capsid protein, (B) portal protein, and (C) terminase large subunit.