Isolation and Characterization of Two Bacteriophages and Their Preventive Effects against Pathogenic Vibrio coralliilyticus Causing Mortality of Pacific Oyster (Crassostrea gigas) Larvae

Abstract

Vibrio coralliilyticus is one of the major pathogens causing mass mortality in marine bivalve larvae aquaculture. To prevent and control Vibrio spp. infections in marine bivalve hatcheries, various antibiotics are overused, resulting in environmental pollution and the creation of multi-drug-resistant strains. Therefore, research on the development of antibiotic substitutes is required. In this study, we isolated two bacteriophages (phages) that specifically infected pathogenic V. coralliilyticus from an oyster hatchery and designated them as pVco-5 and pVco-7. Both phages were classified as Podoviridae and were stable over a wide range of temperatures (4–37 °C) and at pH 7.0–9.0. Thus, both phages were suitable for application under the environmental conditions of an oyster hatchery. The two phages showed confirmed significant bactericidal efficacy against pathogenic V. coralliilyticus in an in vitro test. In the in vivo experiment, the phage pre-treated groups of Pacific oyster larvae showed significantly lower mortality against V. coralliilyticus infection than untreated control larvae. The results of the present study suggest that both phages could be used in the artificial marine bivalve seedling industry; not only to prevent pathogenic V. coralliilyticus infection, but also to reduce antibiotic overuse.

Bacteriophage Cocktail for the Prevention of Multiple-Antibiotic-Resistant and Mono-Phage-Resistant Vibrio coralliilyticus Infection in Pacific Oyster (Crassostrea gigas) Larvae

“…Vaccination is an ideal method to prevent diseases caused by pathogen infections, but it cannot be applied to invertebrates because of the absence of an acquired immune system. Therefore, the phage application is actively studied to prevent bacterial infection in invertebrates and has shown a substantial prophylactic efficacy [ 16 , 17 ].…”

“…Therefore, a phage cocktail could be utilized to prevent the emergence of phage-resistant bacteria that may occur when a mono-phage is applied. pVco-5, pVco-7, and pVco-14 have been reported to inhibit Vco58 and other V. coralliilyticus strains at 27 °C [ 16 , 17 ], indicating that the three phages are suitable for bivalve hatchery environments.…”

“…Three previously isolated V. coralliilyticus specific phages, pVco-5 and pVco-7 [ 17 ] and pVco-14 [ 16 ], were used to prepare the phage cocktail (designated pVco-C) ( Figure 4 ). All phages were confirmed as lytic phages through the whole-genome sequence analysis [ 37 ], unpublished data.…”

“…To evaluate the bactericidal effect of each phage and pVco-C, the phage-resistant variant induced in this study was used as the host. Bacterial cell lysis tests were performed as previously described by Kim et al [ 17 ]. The multiplicity of infection (MOIs) of each phage and pVco-C was regulated to 0, 0.1, 1, and 10.…”

“…Among these alternative approaches is the potential prophylactic and therapeutic use of bacteriophages (phages) for the treatment of bacterial infections. In the aquaculture field, the problem of antibiotic abuse has been continuously emphasized, and alternative studies on the application of bacteriophages have been actively performed in invertebrate [ 16 , 17 , 18 , 19 , 20 , 21 ] and fish [ 22 , 23 , 24 , 25 ]. However, if the phage used also has low infectivity, it can induce phage resistance variants [ 26 , 27 , 28 ].…”