An ex vivo Approach in European Seabass Leucocytes Supports the in vitro Regulation by Postbiotics of Aip56 Gene Expression of Photobacterium damselae subsp. piscicida.

Shewanella putrefaciens Pdp11 (SpPdp11) is a probiotic strain assayed in aquaculture; however, its postbiotic potential is unknown. Postbiotics are bacterial metabolites, including extracellular products (ECPs) that improve host physiology and immunity. Their production and composition can be affected by different factors such as the growing conditions of the probiotics. Photobacterium damselae subsp. piscicida strain Lg 41/01 (Phdp) is one of the most important pathogens in marine aquaculture. The major virulent factor of this bacterium is the exotoxin aip56, responsible for inducing apoptosis of fish leucocytes. Viable SpPdp11 cells have been reported to increase resistance to challenges with Phdp. This work aimed to evaluate the effect of two ECPs, T2348-ECP and FM1548-ECP, obtained from SpPdp11 grown under different culture conditions that previously demonstrated to exert different degradative and non-cytotoxic activities, as well as the effect on pathogens biofilm formation. These SpPdp11-ECPs were then analyzed by their effect on the viability, phagocytosis, respiratory burst and apoptogenic activity against European sea bass leucocytes infected or not with Phdp supernatant. Both ECPs, T2348-ECP and FM1548-ECP, were not cytotoxic against leucocytes and significantly reduced their apoptosis. Phagocytosis and respiratory burst of leucocytes were significantly reduced by incubation with Phdp supernatant, and not influenced by incubation with T2348-ECP or FM1548-ECP. However, both activities were significantly increased after leucocyte incubation with combined T2348-ECP and FM1548-ECP with Phdp supernatant, compared to those incubated only with Phdp supernatant. Finally, both T2348-ECP and FM1548-ECP significantly reduced the relative in vitro expression of the Phdp aip56 encoding gene.

Evaluation of the Differential Postbiotic Potential of Shewanella putrefaciens Pdp11 Cultured in Several Growing Conditions.

The increased knowledge of functional foods has led to the development of a new generation of health products, including those containing probiotics and products derived from them. Shewanella putrefaciens Pdp11 (SpPdp11) is a strain described as a probiotic that exerts important beneficial effects on several farmed fish. However, the use of live probiotic cells in aquaculture has limitations such as uncertain survival and shelf life, which can limit their efficacy. In addition, its efficacy can vary across species and hosts. When probiotics are administered orally, their activity can be affected by the environment present in the host and by interactions with the intestinal microbiota. Furthermore, live cells can also produce undesired substances that may negatively impact the host as well as the risk of potential virulence reversion acquired such as antibiotic resistance. Therefore, new alternatives emerged such as postbiotics. Currently, there is no knowledge about the postbiotic potential of SpPdp11 in the aquaculture industry. Postbiotic refers to the use of bacterial metabolites, including extracellular products (ECPs), to improve host physiology. However, the production of postbiotic metabolites can be affected by various factors such as cultivation conditions, which can affect bacterial metabolism. Thus, the objective of this study was to evaluate the postbiotic potential of ECPs from SpPdp11 under different cultivation conditions, including culture media, temperature, growth phase, and salinity. We analyzed their hydrolytic, antibacterial, antiviral, and cytotoxic capacity on several fish cell lines. The results obtained have demonstrated how each ECP condition can exert a different hydrolytic profile, reduce the biofilm formation by bacterial pathogens relevant to fish, lower the titer of nervous necrosis virus (NNV), and exert a cytotoxic effect on different fish cell lines. In conclusion, the ECPs obtained from SpPdp11 have different capacities depending on the cultivation conditions used. These conditions must be considered in order to recover the maximum number of beneficial capacities or to choose the appropriate conditions for specific activities.

Microalgal and Cyanobacterial Biomasses Modified the Activity of Extracellular Products from Bacillus pumilus: An In Vitro and In Vivo Assessment.

This study investigates the postbiotic potential of extracellular products (ECPs) from Bacillus pumilus strains cultivated on microalgae-supplemented media. We assessed enzymatic and antimicrobial activities to select ECPs that enhance the digestive processes in gilthead seabream. Additionally, we explored the in vitro enzymatic capacity of the chosen postbiotics to hydrolyze macromolecules in microalgae. Finally, a feeding trial was conducted to determine the in vivo effects of the ECPs on Sparus aurata. In vitro enzymatic assays demonstrated diverse hydrolytic capacities among ECPs. All conditions exhibited antimicrobial activity against Photobacterium damselae subsp. piscicida, with variation in inhibitory effects against Vibrio harveyi and Tenacibaculum maritimum. Furthermore, in vitro assays revealed differences in protein hydrolysis and soluble protein concentration, influencing amino acid and reducing sugar release from microalgal biomass. These analyses facilitated a selection to test ECPs in vivo. Lastly, the in vivo experiment revealed no differences in the growth performance, nutrient utilization, and general metabolism of S. aurata fed the experimental diets. Dietary inclusion of postbiotics increased the activity of key digestive enzymes in fish compared to the control group, and particularly, values increased significantly when the fish were fed with the ECP-nanoparticulate-supplemented diet. In conclusion, the inclusion of microalgae in the culture media significantly influences the activity of extracellular products from B. pumilus strains, as evidenced in both in vitro and in vivo assays.

Combined Dietary Administration of Chlorella fusca and Ethanol-Inactivated Vibrio proteolyticus Modulates Intestinal Microbiota and Gene Expression in Chelon labrosus.

The use of functional feeds in aquaculture is currently increasing. This study aimed to assess the combined impact of dietary green microalgae Chlorella fusca and ethanol-inactivated Vibrio proteolyticus DCF12.2 (CVP diet) on thick-lipped grey mullet (Chelon labrosus) juvenile fish. The effects on intestinal microbiota and the transcription of genes related to metabolism, stress, and the immune system were investigated after 90 days of feeding. Additionally, the fish were challenged with Aeromonas hydrophila and polyinosinic-polycytidylic acid (poly I:C) to evaluate the immune response. Microbiota analysis revealed no significant differences in alpha and beta diversity between the anterior and posterior intestinal sections of fish fed the control (CT) and CVP diets. The dominant genera varied between the groups; Pseudomonas and Brevinema were most abundant in the CVP group, whereas Brevinema, Cetobacterium, and Pseudomonas were predominant in the CT group. However, microbial functionality remained unaltered. Gene expression analysis indicated notable changes in hif3α, mhcII, abcb1, mx, and tnfα genes in different fish organs on the CVP diet. In the head kidney, gene expression variations were observed following challenges with A. hydrophila or poly I:C, with higher peak values seen in fish injected with poly I:C. Moreover, c3 mRNA levels were significantly up-regulated in the CVP group 72 h post-A. hydrophila challenge. To conclude, incorporating C. fusca with V. proteolyticus in C. labrosus diet affected the microbial species composition in the intestine while preserving its functionality. In terms of gene expression, the combined diet effectively regulated the transcription of stress and immune-related genes, suggesting potential enhancement of fish resistance against stress and infections.

Pathogenic strains of Shewanella putrefaciens contain plasmids that are absent in the probiotic strain Pdp11.

Shewanella putrefaciens Pdp11 is a strain described as a probiotic for use in aquaculture. However, S. putrefaciens includes strains reported to be pathogenic or saprophytic to fish. Although the probiotic trait has been related to the presence of a group of genes in its genome, the existence of plasmids that could determine the probiotic or pathogenic character of this bacterium is unknown. In the present work, we searched for plasmids in several strains of S. putrefaciens that differ in their pathogenic and probiotic character. Under the different conditions tested, plasmids were only found in two of the five pathogenic strains, but not in the probiotic strain nor in the two saprophytic strains tested. Using a workflow integrating Sanger and Illumina reads, the complete consensus sequences of the plasmids were obtained. Plasmids differed in one ORF and encoded a putative replication initiator protein of the repB family, as well as proteins related to plasmid stability and a toxin-antitoxin system. Phylogenetic analysis showed some similarity to functional repB proteins of other Shewanella species. The implication of these plasmids in the probiotic or pathogenic nature of S. putrefaciens is discussed.

A Tentative Study of the Effects of Heat-Inactivation of the Probiotic Strain Shewanella putrefaciens Ppd11 on Senegalese Sole (Solea senegalensis) Intestinal Microbiota and Immune Response.

Concerns about safety, applicability and functionality associated with live probiotic cells have led to consideration of the use of non-viable microorganisms, known as paraprobiotics. The present study evaluated the effects of dietary administration of heat-inactivated cells of the probiotic strain Shewanella putrefaciens Ppd11 on the intestinal microbiota and immune gene transcription in Solea senegalensis. Results obtained were evaluated and compared to those described after feeding with viable Pdp11 cells. S. senegalensis specimens were fed with basal (control) diet or supplemented with live or heat inactivated (60 °C, 1 h) probiotics diets for 45 days. Growth improvement was observed in the group receiving live probiotics compared to the control group, but not after feeding with a probiotic heat-inactivated diet. Regarding immune gene transcription, no changes were observed for tnfα, il-6, lys-c1, c7, hsp70, and hsp90aa in the intestinal samples based on the diet. On the contrary, hsp90ab, gp96, cd4, cd8, il-1ß, and c3 transcription were modulated after probiotic supplementation, though no differences between viable and heat-inactivated probiotic supplemented diets were observed. Modulation of intestinal microbiota showed remarkable differences based on the viability of the probiotics. Thus, higher diversity in fish fed with live probiotic cells, jointly with increased Mycoplasmataceae and Spirochaetaceae to the detriment of Brevinemataceae, was detected. However, microbiota of fish receiving heat-inactivated probiotic cells showed decreased Mycoplasmataceae and increased Brevinemataceae and Vibrio genus abundance. In short, the results obtained indicate that the viable state of Pdp11 probiotic cells affects growth performance and modulation of S. senegalensis intestinal microbiota. On the contrary, minor changes were detected in the intestinal immune response, being similar for fish receiving both, viable and inactivated probiotic cell supplemented diets, when compared to the control diet.