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Aquaculture is a growing industry worldwide, but it faces challenges related to animal health. These challenges include infections by parasites, bacteria, and viral pathogens. These harmful pathogens have devastating effects on the industry, despite efforts to control them through vaccination and antimicrobial treatments. Unfortunately, these measures have proven insufficient to address the sanitary problems, resulting in greater environmental impact due to the excessive use of antimicrobials. In recent years, probiotics have emerged as a promising solution to enhance the performance of the immune system against parasitic, bacterial, and viral pathogens in various species, including mammals, birds, and fish. Some probiotics have been genetically engineered to express and deliver immunomodulatory molecules. These promote selective therapeutic effects and specific immunization against specific pathogens. This review aims to summarize recent research on the use of probiotics in fish aquaculture, with a particular emphasis on genetically modified probiotics. In particular, we focus on the advantages of using these microorganisms and highlight the main barriers hindering their widespread application in the aquaculture industry.

Bifidobacterium longum is considered a microorganism with probiotic potential, which has been extensively studied, but these probiotic effects are strain dependent. This work aims to characterize the probiotic potential, based on the biochemical and genomic functionality, of B. longum LBUX23, isolated from neonates' feces. B. longum LBUX23 contains one circular genome of 2,287,838 bp with a G+C content of 60.05%, no plasmids, no CRISPR-Cas operon, possesses 56 tRNAs, 9 rRNAs, 1 tmRNA and 1776 coding sequences (CDSs). It has chromosomally encoded resistance genes to ampicillin and dicloxacillin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and to some emergent pathogen's clinical strains. B. longum LBUX23 was able to utilize lactose, sucrose, fructooligosaccharides (FOS), and lactulose. The maximum peak of bacterial growth was observed in sucrose and FOS at 6 h; in lactose and lactulose, it was shown at 8 h. B. longum LBUX23 can survive in gastrointestinal conditions (pH 4 to 7). A decrease in survival (96.5 and 93.8%) was observed at pH 3 and 3.5 during 120 min. argC , argH , and dapA genes could be involved in this tolerance. B. longum LBUX23 can also survive under primary and secondary glyco- or tauro-conjugated bile salts, and a mixture of bile salts due to the high extracellular bile salt hydrolase (BSH) activity (67.3 %), in taurocholic acid followed by taurodeoxycholic acid (48.5%), glycocholic acid (47.1%), oxgall (44.3%), and glycodeoxycholic acid (29.7%) probably due to the presence of the cbh and gnlE genes which form an operon (start: 119573 and end: 123812). Low BSH activity was determined intracellularly (<7%), particularly in glycocholic acid; no intracellular activity was shown. B. longum LBUX23 showed antioxidant effects in DPPH radical, mainly in intact cells (27.4%). In the case of hydroxyl radical scavenging capacity, cell debris showed the highest reduction (72.5%). In the cell-free extract, superoxide anion radical scavenging capacity was higher (90.5%). The genome of B. longum LBUX23 contains PNPOx , AhpC , Bcp , trxA, and trxB genes, which could be involved in this activity. Regarding adherence, it showed adherence up to 5% to Caco-2 cells. B. longum LBUX23 showed in vitro potential probiotic properties, mainly in BSH activity and antioxidant capacity, which indicates that it could be a good candidate for antioxidant or anti-cholesterol tests using in vivo models.

Butyrate is a microbiota-produced metabolite, sensed by host short-chain fatty acid receptors FFAR2 (Gpr43), FFAR3 (Gpr41), HCAR2 (Gpr109A), and Histone deacetylase (HDAC) that promotes microbiota-host crosstalk. Butyrate influences energy uptake, developmental and immune response in mammals. This microbial metabolite is produced by around 79 anaerobic genera present in the mammalian gut, yet little is known about the role of butyrate in the host-microbiota interaction in salmonid fish. To further our knowledge of this interaction, we analyzed the intestinal microbiota and genome of Atlantic salmon ( Salmo salar ), searching for butyrate-producing genera and host butyrate receptors. We identified Firmicutes, Proteobacteria, and Actinobacteria as the main butyrate-producing bacteria in the salmon gut microbiota. In the Atlantic salmon genome, we identified an expansion of genes orthologous to FFAR2 and HCAR2 receptors, and class I and IIa HDACs that are sensitive to butyrate. In addition, we determined the expression levels of orthologous of HCAR2 in the gut, spleen, and head-kidney, and FFAR2 in RTgutGC cells. The effect of butyrate on the Atlantic salmon immune response was evaluated by analyzing the pro and anti-inflammatory cytokines response in vitro in SHK-1 cells by RT-qPCR. Butyrate decreased the expression of the pro-inflammatory cytokine IL-1β and increased anti-inflammatory IL-10 and TGF-β cytokines. Butyrate also reduced the expression of interferon-alpha, Mx, and PKR, and decreased the viral load at a higher concentration (4 mM) in cells treated with this molecule before the infection with Infectious Pancreatic Necrosis Virus (IPNV) by mechanisms independent of FFAR2, FFAR3 and HCAR2 expression that probably inhibit HDAC. Moreover, butyrate modified phosphorylation of cytoplasmic proteins in RTgutGC cells. Our data allow us to infer that Atlantic salmon have the ability to sense butyrate produced by their gut microbiota via different specific targets, through which butyrate modulates the immune response of pro and anti-inflammatory cytokines and the antiviral response., Competing Interests: “The authors declare no conflict of interest.” “The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results”., (© 2023 The Authors.)

Experimental and clinical evidence has demonstrated the potential of probiotic strains in the prevention or treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, there is little data on what the methodology leading to the identification of such strains should be. In this work, we propose a new flowchart to identify strains with probiotic potential for the management of IBS and IBD, which we tested on a collection of 39 lactic acid bacteria and Bifidobacteria strains. This flowchart included in vitro tests of immunomodulatory properties on intestinal and peripheral blood mononuclear cells (PBMCs), assessment of the barrier-strengthening effect by measuring transepithelial electric resistance (TEER) and quantification of short-chain fatty acids (SCFAs) and aryl hydrocarbon receptor (AhR) agonists produced by the strains. The in vitro results were then combined in a principal component analysis (PCA) to identify strains associated with an anti-inflammatory profile. To validate our flowchart, we tested the two most promising strains identified in the PCA in mouse models of post-infectious IBS or chemically induced colitis to mimic IBD. Our results show that this screening strategy allows the identification of strains with potential beneficial effects on colonic inflammation and colonic hypersensitivity.

Probiotic supplementation can help to mitigate the pathogenesis of irritable bowel syndrome (IBS) by reinforcing the intestinal barrier, and reducing both inflammation and proteolytic activity. Here, a combination of in vitro tests was performed on 33 Bifidobacterium strains as probiotic candidates for IBS. In addition to the classical tests performed, the detection of the serine protease inhibitor (serpin) enzyme capable of decreasing the high proteolytic activity found in IBS patients was included. Three serpin-positive strains were selected: Bifidobacterium breve CNCM I-5644, Bifidobacterium longum subsp. infantis CNCM I-5645 and B. longum CNCM I-5646 for their immunomodulation properties and protection of intestinal epithelial integrity in vitro. Furthermore, we found that B. breve CNCM I-5644 strain prevented intestinal hyperpermeability by upregulating Cingulin and Tight Junction Protein 1 mRNA levels and reducing pro-inflammatory markers. The ability of CNCM I-5644 strain to restore intestinal hyperpermeability (FITC-dextran) was shown in the murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS). This effect of this strain was corroborated in a second model of IBS, the neonatal maternal separation model in mice. Altogether, these data suggest that serpin-positive B. breve CNCM I-5644 may partially prevent disorders associated with increased barrier permeability such as IBS., (© 2022. The Author(s).)

Bifidobacteria have been investigated due to their mutualistic microbe-host interaction with humans throughout their life. This work aims to make a biochemical and genomic characterization of Bifidobacterium pseudocatenulatum JCLA3. By multilocus analysis, the species of B. pseudocatenulatum JCLA3 was established as pseudocatenulatum . It contains one circular genome of 2,369,863 bp with G + C content of 56.6%, no plasmids, 1937 CDSs, 54 tRNAs, 16 rRNAs, 1 tmRNA, 1 CRISPR region, and 401 operons predicted, including a CRISPR-Cas operon; it encodes an extensive number of enzymes, which allows it to utilize different carbohydrates. The ack gene was found as part of an operon formed by xfp and pta genes. Two genes of ldh were found at different positions. Chromosomally encoded resistance to ampicillin and cephalothin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 were demonstrated by B. pseudocatenulatum JCLA3; it can survive 100% in simulated saliva, can tolerate primary and secondary glyco- or tauro-conjugated bile salts but not in a mix of bile; the strain did not survive at pH 1.5-5. The cbh gene coding to choloylglycine hydrolase was identified in its genome, which could be related to the ability to deconjugate secondary bile salts. Intact cells showed twice as much antioxidant activity than debris. B. pseudocatenulatum JCLA3 showed 49% of adhesion to Caco-2 cells. The genome and biochemical analysis help to elucidate further possible biotechnological applications of B. pseudocatenulatum JCLA3.

Early in the 1900s, it was proposed that health could be improved and senility delayed by manipulating gut microbiota with the host-friendly bacteria found in yogurt. Later, in 1990, the medical community reconsidered this idea and today probiotics represent a developed area of research with a billion-dollar global industry. As a result, in recent decades, increased attention has been paid to the isolation and characterization of novel probiotic bacteria from fermented foods and dairy products. Most of the identified probiotic strains belong to the lactic acid bacteria group and the genus Bifidobacterium . However, current molecular-based knowledge has allowed the identification and culture of obligatory anaerobic commensal bacteria from the human gut, such as Akkermansia spp. and Faecalibacterium spp., among other human symbionts. We are aware that the identification of new strains of these species does not guarantee their probiotic effects and that each effect must be proved through in vitro and in vivo preclinical studies before clinical trials (before even considering it as a probiotic strain). In most cases, the identification and characterization of new probiotic strain candidates may lack the appropriate set of in vitro experiments allowing the next assessment steps. Here, we address some innovative strategies reported in the literature as alternatives to classical characterization: (i) identification of alternatives using whole-metagenome shotgun sequencing, metabolomics, and multi-omics analysis; and (ii) probiotic characterization based on molecular effectors and/or traits to target specific diseases (i.e., inflammatory bowel diseases, colorectal cancer, allergies, among others).

The possibility of exploiting the human immune response to glycan α-Gal for the control of multiple infectious diseases has been the objective of recent investigations. In this field of research, the strain of Escherichia coli O86:B7 has been at the forefront, but this Gram-negative microorganism presents a safety concern and therefore cannot be considered as a probiotic. To address this challenge, this study explored the identification of novel lactic acid bacteria with a safe history of use, producing α-Gal and having probiotic potential. The lactic acid bacteria were isolated from different traditionally fermented foods (kununn-zaki, kindirmo, and pulque) and were screened for the production of α-Gal and some specific probiotic potential indicators. The results showed that Ten (10) out of forty (40) [25%] of the tested lactic acid bacteria (LAB) produced α-Gal and were identified as Limosilactobacillus fermentum, Levilactobacillus brevis, Agrilactobacillus composti, Lacticaseibacillus paracasei, Leuconostoc mesenteroides and Weissella confusa. Four (4) LAB strains with highest levels of α-Gal were further selected for in vivo study using a mouse model (α1,3GT KO mice) to elucidate the immunological response to α-Gal. The level of anti-α-Gal IgG observed were not significant while the level of anti-α-Gal IgM was lower in comparison to the level elicited by E. coli O86:B7. We concluded that the lactic acid bacteria in this study producing α-Gal have potential probiotic capacity and can be further explored in α-Gal-focused research for both the prevention and treatment of various infectious diseases and probiotic development., (© 2022. The Author(s).)