New insights on phage efficacy to control Aeromonas salmonicida in aquaculture systems: an in vitro preliminary study

Abstract

A major source of financial loss for the fish-farming industry is the occurrence of bacterial infections. Phage therapy can be a useful alternative tool to conventional treatments to control bacterial infections in aquaculture. The promising results obtained with phage AS-A to control the agent of furunculosis, Aeromonas salmonicida, led us to isolate two new phages and evaluate their dynamics, in cocktails and individually, to control this pathogenic bacterium. Moreover, considering that in outdoor facilities aquaculture water is exposed to the natural variability of physical and chemical parameters, the influence of pH, salinity, temperature, solar radiation and UV radiation on phage AS-D stability was also evaluated in this study, in order to develop an effective phage therapy protocol. Phages were assigned to the family Myoviridae and revealed identical morphological characteristics. Phage AS-A presents a higher burst size and a shorter latent period, decreasing the concentration of A. salmonicida sooner than phages AS-D and AS-E. However, phage AS-D presented higher rate of bacterial reduction, inducing also less bacterial resistance. Bacterial control with the cocktails was higher, namely when phage AS-A was combined with one of the two new phages or with both, but the main difference in the bacterial control was in the treatment time. Phage cocktails decrease the concentration of A. salmonicida sooner than single suspensions. The use of phage cocktails, in general, decreased phage-resistant mutants. The survival of AS-D phage was mostly affected by sunlight exposure (decrease of 3 log PFU/mL after 12 h) and high temperatures (decrease of 3 PFU/mL after 21 days and of 7 log PFU/mL after 49 days at 37 °C, but no decrease after 21 and 49 days at 25 °C and decrease of only 1 and 2 log PFU/mL after 21 and 49 days, respectively, at ambient temperature). The high bacterial control and low development of phage-resistant bacterial clones suggest that these phages can be used to control the furunculosis in aquaculture. Nonetheless, the stability of the phages was affected by solar radiation, this can be overcome by the application of phages at the end of the day or at night.