Phage therapy in the inactivation of Aeromonas salmonicida in aquaculture systems: inactivation in water and in juvenile Senegalese sole (Solea senegalensis) Carla Pereira, Catarina Moreirinha, Adelaide Almeida* Department of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
*Corresponding author: aalmeida@ua.pt
Introduction
Results
Fish farming industry is often affected with infections by pathogenic bacteria, even multidrugresistant variants, this problem is most prominent during the early stages of fish development. It is difficult to treat these infections with traditional antibiotic treatment or vaccination. In this way, alternative environmentally-friendly biological strategies to control bacterial infections need to be implemented. Phage therapy is a potential tool for the inactivation of bacterial pathogens in aquaculture.
In in vitro the maximum of bacterial inactivation with AS-A phage was 3.9 log achieved after 8 h of phage therapy. CB – Bacteria control
A limited phage-resistant mutants was observed frequency of resistant mutants of 2.4 x 10-4
Methods Phage AS-A – Bacteria plus phage.
Semi-intensive aquaculture system Corte das Freiras located in the estuarine system Ria de Aveiro, Portugal.
Phage therapy assays in vitro Phage AS-A (107 PFU mL-1) and bacterium A. salmonicida CECT 894 (105 CFU mL-1) as host, at a multiplicity of infection (MOI) of 100 at 25 °C .
In vivo - AS-A phage inhibited A. salmonicida by ≈ 3.5 log. After 72 h of phage treatment, no fish mortality was observed contrarily to that observed for no treated fish (mortality of 36%).
Aeromonas + water Bacteria control
Aeromonas + phage
Phage therapy in vivo Fish infected with A . salmonicida (108 CFUmL-1) and treated with AS-A phage (104 PFU mL-1), at 25 °C. Water samples collected after treatment for determination of bacteria and phage concentration . Fish mortality was determined by visual inspection. Determination of phage-resistant bacteria Spontaneous A. salmonicida mutants resistant to phage were selected by plating on double agar layer with phage. The mutation frequency was determined by dividing the number of mutants (with pages) by total number of bacteria (without phages).
Phage control
Impact on bacterial community of aquaculture water
Effect of AS-A phage on the structure of bacterial community of aquaculture water and fish After incubation, water sample was filtered. For fish samples after incubation at 25 °C for 72 h, fish were homogenized, RNA was extracted, reverse transcription of mRNA was done, cDNA was amplified and DGGE was done.
This study provides evidences that the tested phage can be effective and safe against furunculosis during the production of juvenile fish.
Impact on fish bacteriome
Water samples without incubation (T0) and water incubation during 8 h (T8) R=0.148
Conclusions The addition of phages to fish culture water inactivated efficiently the agent of furunculosis in fish. No mortality of challenged fish was observed but chalenged fish not treated with phages presented a mortality of 36%.
Fish control
Water with phage in preservative solution; Water without phage (T8 –TSBCL vs T8- Phage) R=0.356
No significant impact of phage inoculation on natural bacteria of aquaculture water
Moderate separation of Fish control group and Fish+Phage (R=0.669)
Group Fish+AS similar to the group exposed to bacteria AS and phage AS-A (R=0.269).
Fish bacteria were moderately affected by the phage, however, no significant differences were observed when the phage was added in the presence of the host bacteria.