Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, Characterization, and Remediation of Larval Oyster Mortalities

Abstract

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortality rates in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family, and two were members of the Siphoviridae In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% 6 days after challenge with lethal doses of V. coralliilyticus Larval survival depended on the oyster species, the quantities of phages and vibrios applied, and the species and strain of Vibrio A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phages vB_VcorM-GR7B, vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A, and 28A, respectively). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by >90% (P < 0.0001) over 6 days with phage treatment compared to those of untreated controls. Genomic sequencing of phages 7B, 11A, and 28A revealed 207,758-, 194,800-, and 154,046-bp linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries.IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of hatcheries and the commercial shellfish industry. Shellfish pathogens like Vibrio coralliilyticus and Vibrio tubiashii have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and from both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach for stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.

Keywords: Vibrio coralliilyticus; Vibrio tubiashii; bacteriophage therapy; characterization; coral; genomic sequencing; isolation; larvae; mortalities; oyster; phage.

FIG 1

Transmission electron micrographs of 16 vibriophage isolates. Bars, 100 nm.

FIG 2

Proof-of-principle testing in a 24-well-plate assay showing the percent survival of Eastern oyster (Crassostrea virginica) larvae (A) and Pacific oyster (Crassostrea gigas) larvae (B) after the addition of V. coralliilyticus strain RE98 or ATCC 19105 or V. tubiashii strain ATCC 19106 with or without treatment with phage cocktail VTP100. The negative control is the larvae only, while the positive controls are the larvae plus individual Vibrio strains (without phages). (A) For Eastern oysters, the titer of phage treatment is 3.45 × 10⁷ PFU/ml, titers of V. coralliilyticus strains RE98 and 19105 are both 1.0 × 10⁷ CFU/ml, and the titer of V. tubiashii ATCC 19106 is 6.5 × 10⁴ CFU/ml. (B) For Pacific oyster larvae, the titer of the phage treatment was 1.1 × 10⁷ PFU/ml, and the titer of V. coralliilyticus RE98 was 8.0 × 10⁷ CFU/ml, that of V. coralliilyticus ATCC 19105 was 7.0 × 10⁶ CFU/ml, and that of V. tubiashii ATCC 19106 was 1.4 × 10⁶ CFU/ml (N = 3; n = 18). Bars represent standard errors of the means (SEM).

FIG 3

Survival of larval oysters after challenge with Vibrio coralliilyticus with and without phage treatment in 24-well-plate assays. Seven-day-old postfertilized Pacific oyster larvae were treated with phage cocktail VCP300 at 3.45 × 10⁶ PFU/ml of seawater followed by inoculation 15 min later with a lethal dose of V. coralliilyticus strain RE98 at 9.5 × 10⁴ CFU/ml seawater or V. coralliilyticus OCN008 at 1.1 × 10⁵ CFU/ml of seawater. Negative controls consisted of larvae only (NC) and larvae plus the phage cocktail (Phage Cont). Mortalities were recorded 6 days later. Results are from three experiments performed in quadruplicate (N = 3; n = 12). Significance levels of paired data are shown. Bars represent standard deviations (SD).

FIG 4

Pairwise alignment of phage sequences. Nucleotide sequences of phages 7B (GenBank accession number MT366760), 11A (accession number MT366761), 28A (accession number MT366762), and YC (accession number MH375644.1) were aligned using BLAST and visualized with Easyfig v. 2.2.5 (57). Sequence identities with Expect values lower than 0.001 are represented by a color scale ranging from orange (64%) to blue (100%). Coding sequence (CDS) regions are represented by the small gray arrows.