Potential of Marine Strains of Pseudoalteromonas to Improve Resistance of Juvenile Sea Bass to Pathogens and Limit Biofilm Development
Authors: Rahmani Alexandra , Parizadeh Leila , Baud Marine , Francois Yoannah , Bazire Alexis , Rodrigues Sophie , Fleury Yannick , Cuny Helena , Debosse Eric , Cabon Joelle , Louboutin Lenaïg , Bellec Laure , Danion Morgane , Morin Thierry 
Published: March 2023

Abstract
The European sea bass (Dicentrarchus labrax) is one of the most produced marine fish species in Europe. Its larval stage is particularly sensitive and is thus characterized by relatively low survival rates, making it acutely vulnerable to multiple infectious hazards that can occur during its intensive production cycle in hatcheries and at sea. In this study, we investigated the potential probiotic effect of marine Pseudoalteromonas bacterial strains against two major pathogens of this species, Vibrio harveyi (a bacterial pathogen) and the nervous necrosis virus (NNV); we also investigated antibiofilm effect of these Pseudoalteromonas strains. Over an 8 to 12-week impregnation phase, seabass juveniles were immersed fortnightly for 4 hours in static hyperoxygenated seawater containing the probiotic candidates of Pseudoalteromonas strains at a concentration of 106 CFU/mL. We tested four candidates: (1) a combination of two strains producing antimicrobial compounds, hCg-42 and hOe-125; (2) strain 3J6, with known antibiofilm properties and (3) strain RA15, from the same genus, but with no identified probiotic effect. At the end of the impregnation phase, fish underwent an infection challenge with an intraperitoneal injection at a dose of 4.6 × 108 CFU/mL of V. harveyi or a 4-hour bath with a pathogenic strain of NNV. Thereafter, mortality was monitored for 2 and 6 weeks, respectively, at temperatures allowing the development of clinical signs. Immunological analyses were carried out during impregnation and after infection. The probiotic candidates were detected at different sampling times after the 4-hour immersion session in the gills and mucus, but there was no evidence of long-term persistence. For the V. harveyi challenge, no statistical difference in mortality was observed between the non-impregnated control (63%) and the 3J6-impregnated (68.7%) group, but improved survival rates of 10 and 25% were obtained for the RA15- and the double strain (hCg-42+hOe-125)-impregnated groups, respectively. For the NNV challenge, no significant benefic effect of the probiotics on infection kinetics or cumulative mortality was observed. Leucocyte mortality and phagocytosis activity revealed only slight significant differences between the treatment groups, either during impregnation or after infection challenges. Regarding biofilm development during impregnation with probiotic candidates, the maximal thickness of biofilm was significantly lower in the 3J6, double-strain and RA15 groups, compared with the non-impregnated control group. This study highlights the interesting probiotic potential of marine bacteria to limit mortalities induced by bacterial pathogens as well as biofilm development. Further investigations are in progress to investigate the mechanisms of action of these probiotics and to improve their formulation for larger-scale tests.