The Fate of Bacteriophages in Recirculating Aquaculture Systems (RAS)-Towards Developing Phage Therapy for RAS

Abstract

Aquaculture production has increased tremendously during the last decades, and new techniques have been developed, e.g., recirculating aquaculture systems (RAS). In RAS, the majority of water volume is circulated via mechanical and biological filters and reused in the tanks. However, the prevention and treatment of diseases in these systems are challenging, as the pathogens spread throughout the system, and the addition of chemicals and antibiotics disrupts the microbiome of the biofilters. The increasing antibiotic resistance has made phage therapy a relevant alternative for antibiotics in food production. Indeed, as host-specific and self-replicating agent they might be optimal for targeted pathogen eradication in RAS. We tested the survival and spread of Flavobacterium columnare -infecting phage FCL-2 in recirculating aquaculture fish farm with rainbow trout (Oncorhynchus mykiss) in a fully controlled study. After a single addition, phage persisted in water samples collected from tank, fixed bed, moving bed, and aeration unit up to 14 days, and in the water of rearing tanks, rainbow trout mucus, and bioreactor carrier media from the fixed and moving bed biofilters for 21 days. Furthermore, phage adsorbed preferentially to moving bed carrier media, which contained biofilm attached and from which higher phage numbers were recovered. This study shows phages as a potent strategy for maintaining biosecurity in RAS systems.

Keywords: RAS; aquaculture; bacteriophage; biofilter; disease; phage therapy; recirculating aquaculture systems.

Figure 1

Phage persistence over time in the recirculating aquaculture system (RAS) tanks and fish. Phage FCL-2 titers (pfu/mL) on (A) the fish tank water, (B) the moving bed water, (C) the fixed-bed water, (D) the aeration unit water, (E) fish mucus, (F) the moving bed plastic carrier media, (G) the fixed-bed plastic carrier media, (H) on fish gills (seven days after phage exposure). In A, B, C, and D, each tank was sampled once until day seven and in triplicates from day nine onwards. In E, three fish per tank were sampled in the first and fifth day, then three fish per control tank, and ten fish per phage-treated tank were measured from day seven onwards. In F and G, five carrier media pieces per tank were sampled in all time points. In H, three fish from each control tank and ten fish from each phage treated tank were measured.

Figure 2

Adsorption of phage to plastic carrier media. Each condition was tested in triplicates. Unpaired t-tests were used for comparing the controls and tested conditions. * Statistical difference found (exact p values are mentioned in the text).

Figure 3

RAS farm location and an example of the fish tanks used. (A) Fish farm location in Laukaa, Finland. (B) Fish tank (in green) and solids removal units. (C) Fixed and moving bed biofilters.