Introduction
Disease outbreaks in marine aquaculture continue to pose major challenges, particularly during early larval stages when fish are highly susceptible to opportunistic infections and their immune system is still immature . F ish larvae depend exclusively on live feeds such as Rotifers and Artemia , which often serve as reservoirs of pathogenic bacteria—especially members of the Vibrio genus. Given the global effort to reduce antibiotic use, bacteriophages—viruses that exclusively infect and lyse bacteria—have gained attention as a highly specific, eco-friendly alternative for bacterial control in aquaculture environments.
In this study, we have tested the efficacy of f ully characterized phages against known pathogenic vibrios but also against randomly selected vibrios from the live feed section of a research fish hatchery. The goal was to identify optimal phage candidates and cocktail combinations for further application in hatchery systems as a preventive intervention against fish infections by member of the Vibrio genus.
Materials and Methods
Water samples were collected from Artemia and Rotifer culture tanks at the hatchery of the Institute of Marine Biology, Biotechnology and Aquaculture (IMBBC) . B acterial strains were isolated on thiosulfate-citrate-bile salts-sucrose (TCBS) agar, a selective medium for Vibrio species. Seventeen distinct isolates were selected based on colony morphology and preliminary biochemical characteristics for subsequent testing . To enhance the diversity and pathogenic relevance of this bacterial panel, four previously characterized Vibrio strains from the IMBBC lab collection were also included; Vibrio harveyi VH2, V. alginolyticus V1, V. anguillarum VAK, and V. harveyi KEF80 , all of which linked to disease outbreaks in fish hatcheries.
A panel of bacteriophages from IMBBC lab collection was screened against the full bacterial set. Phages had been previously isolated from marine environments and propagated on either Vibrio alginolyticus or V. harveyi. Five phages (Figure 1) , Vibrio phage Virtus (Droubogiannis and Katharios, 2022), Vibrio phage ELK1, Vibrio phage Artemius (Droubogiannis et al., 2022), ST2 (Kalatzis et al., 2016), and Vibrio phage FG which is a novel phage , were selected based on their plaque clarity, host range, and genomic profiles. Whole genome sequencing confirmed the absence of undesirable genes (e.g., lysogeny markers, toxins).
Each phage was evaluated in in vitro killing assays against the bacterial panel. Cultures were incubated with phage suspensions in liquid media, and optical density (OD600 ) was monitored over time to generate detailed lysis curves. Phage cocktails were then formulated using two or three phages, targeting maximal bacterial coverage while aiming to reduce the likelihood of resistance emergence. The same in vitro lysis assays were applied to test cocktail performance.
Results and Discussion
The results highlighted clear variability in host susceptibility. Certain phages, such as Virtus, ST2 and FG , showed strong, rapid lysis and a relative broader host range and were selected for further downstream applications. Several cocktails demonstrated improved bacterial clearance compared to individual phages, suggesting additive or synergistic interactions (Figure 2) .
Collectively, our results demonstrate the feasibility of isolating representative bacteria from live feed systems and effectively targeting them with well-characterized phages. These in vitro findings establish a strong foundation for the development of phage-based disinfection protocols tailored to hatchery settings. Our findings provide meaningful insights into phage-host interactions, cocktail optimization, and the potential for routine phage supplementation of live feeds to reduce microbial risks during early fish development. Phage therapy could be used to selectively exclude unwanted bacteria from fish hatcheries leaving beneficial bacteria unaffected by a targeted modulation of the microbiome.
The study has been funded in part by the European Union’s Horizon Europe Research and Innovation Programme under grant agreement 101084204 Cure4Aqua.
References
Droubogiannis , S., Katharios, P., 2022. Genomic and Biological Profile of a Novel Bacteriophage, Vibrio phage Virtus, Which Improves Survival of Sparus aurata Larvae Challenged with Vibrio harveyi. Pathogens 11, 630. https://doi.org/10.3390/pathogens11060630
Droubogiannis , S., Pavlidi , L., Tsertou , M.I., Kokkari , C., Skliros , D., Flemetakis, E., Katharios, P., 2022. Vibrio phage Artemius, a novel phage infecting Vibrio alginolyticus. Pathogens 11, 848.
Kalatzis , P.G., Bastías , R., Kokkari , C., Katharios, P., 2016. Isolation and Characterization of Two Lytic Bacteriophages , φ St2 and φ Grn1 ; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds. PLoS One 1–18. https://doi.org/10.1371/journal.pone.0151101