Characterization and mechanism of anti-Aeromonas salmonicida activity of a marine probiotic strain, Bacillus velezensis V4.

The bacterium Aeromonas salmonicida is the causative agent of furunculosis, a systemic, ubiquitous disease of fish in the salmon family, characterized by high mortality and morbidity. Probiotics are a promising approach for prevention of furunculosis in aquaculture. A bacterial strain with anti-A. salmonicida properties, Bacillus velezensis V4, was isolated and the mechanisms underlying these properties were investigated. Anti-A. salmonicida compounds present in cell-free supernatant of V4 were purified and structurally identified as members of the iturin, macrolactin, and difficidin groups. The compounds contributed jointly to inhibition of A. salmonicida, and the diversity of the compounds was related to the versatility of their mode of action. Addition of the compounds to A. salmonicida cell suspensions reduced cell density. Analyses by confocal microscopy and scanning electron microscopy revealed cell membrane disruption, deletion of cellular content, and cell lysis of A. salmonicida. The V4 genome was sequenced, and gene clusters involved in synthesis of anti-Aeromonas compounds were detected and identified. A possible probiotic effect on growth performance of Oncorhynchus mykiss (rainbow trout) was investigated by addition of 0, 1, and 3 % (v/w) V4. Relative to control, mortality was reduced 27.25 % in the 1 % addition group and 81.86 % in the 3 % addition group. Feed coefficient ratio was reduced 19.49 % and weight gain ratio was increased 71.22 % in the 1 % addition group. Our findings demonstrate that V4 is an effective probiotic strain in O. mykiss and has clear potential for both control of furunculosis and growth promotion of aquaculture animals.

[Excretion of extracellular membrane nanovesicles by aeromonas].

AIM: Study of extracellular membrane nanovesicles production by Aeromonas hydrophila and Aeromonas salmonicida bacteria on a subcellular level. MATERIALS AND METHODS: 4 strains of A. hydrophila: 342-1, E 8-8, H 336 and H 1-6-05 and 1 strain of A. salmonicida A-450 as well as intact Wistar line rats were used. Methods of transmission electron microscopy: ultrathin sectioning and negative contrasting were used. RESULTS. A. hydrophila and A. salmonicida bacteria produced in pure cultures excrete into the environment extracellular membrane nanovesicles. The size of these vesicles varies from 20 to 200 nm in diameter. The process of gemmation from bacterial cell and possibility of obtaining isolated membrane nanovesicles preparations is shown. These vesicles are detected in ultrathin sections of apical surface of intact rat intestine among accumulations ofparietal microorganisms that colonize mucous membrane. Extracellular membrane nanovesicles excreted by aeromonas are analogous by size and ultrastructure to vesicles of other species of gram-negative bacteria described in the literature. CONCLUSION: During production of A. hydrophila and A. salmonicida bacteria in vitro nanovesicles are formed from the outer membranes of the cells and excreted into the environment, the nanovesicles are similar to those detected in ultrathin sections of the surface of intestine of rats among accumulations of parietal microorganisms and in glycocalix between epitheliocyte microvilli.

Histological changes in intestine of Atlantic salmon (Salmo salar L.) following in vitro exposure to pathogenic and probiotic bacterial strains.

Furunculosis and vibriosis are diseases that cause severe economic losses in the fish-farming industry. The foregut of the Atlantic salmon (Salmo salar L.) was exposed in vitro to two fish pathogens, Aeromonas salmonicida (causative agent of furunculosis) and Vibrio anguillarum (causative agent of vibriosis), and to one probiotic strain, Carnobacterium divergens, at 6 x 10(4) or 6 x 10(6) viable bacteria per milliliter. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples (foregut exposed to Ringer's solution only) and samples exposed only to C. divergens had a similar appearance to intact intestinal mucosal epithelium, with no signs of damage. However, exposure of the foregut to the pathogenic bacteria resulted in damaged epithelial cells, cell debris in the lumen, and disorganization of the microvilli. Co-incubation of the foregut with a pathogen and C. divergens did not reverse the damaging effects caused by the pathogen, although these were alleviated when probiotic bacteria were used. Based on these results, we suggest that the probiotic bacterium, C. divergens, is able to prevent, to some extent, pathogen-induced damage in the Atlantic salmon foregut.

Damaging effect of the fish pathogen Aeromonas salmonicida ssp. salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar L.).

In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.