Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

Dietary ARA, DHA, and Carbohydrate Ratios Affect the Immune Status of Gilthead Sea Bream Juveniles upon Bacterial Challenge.

This study aims to assess the effects of different dietary n-6/n-3 long-chain polyunsaturated fatty acid ratios and CHO content in the immune response of gilthead seabream. For that purpose, gilthead sea bream juveniles (initial body weight = 47.5 g) were fed for 84 days with four isoproteic (47% crude protein) and isolipidic (18% crude lipids) diets with high (20%) or low (5%) level of gelatinized starch (HS or LS diets, respectively) and included approximately 2.4% ARA or DHA. At the end of the trial, the DHA-enriched groups presented increased red blood cell (RBC) count, hemoglobin, plasmatic nitric oxide (NO) content, and antiprotease and alternative complement activities. The ARA groups had increased thrombocyte count, and plasmatic bactericidal activity against Vibrio anguillarum was lower in the fish fed the ARA/LS diet. After the feeding trial, the fish were challenged with an intraperitoneal injection (i.p.) of killed Photobacterium damselae subsp. piscicida (Phdp) and sampled at 4 and 24 h after the challenge. At 4 h after i.p., the ARA groups presented increased plasma total immunoglobulins (Ig) and bactericidal activity against V. anguillarum. In addition, the fish fed the ARA/LS diet presented lower white blood cell (WBC) and alternative complement activity. At 24 h after i.p., the ARA groups presented increased RBC, WBC, and thrombocyte numbers, total IG, plasma peroxidase activity, and casp3 expression in the distal intestine. The HS groups presented increased plasma NO content and bactericidal activity against Phdp and decreased protease, antiprotease activity, and bactericidal activity against V. anguillarum. In conclusion, high dietary DHA levels seemed to improve the immune status of unchallenged gilthead sea bream juveniles, while high dietary ARA levels improved the fish immune response to a bacterial challenge. The energy provided by dietary starch seems to be important to promote a fast response by the fish immune system after a challenge.

LPS-Induced Mortality in Zebrafish: Preliminary Characterisation of Common Fish Pathogens.

Disease outbreaks are a common problem in aquaculture, with serious economic consequences to the sector. Some of the most important bacterial diseases affecting aquaculture are caused by Gram-negative bacteria including Vibrio spp. (vibriosis), Photobacterium damselae (photobacteriosis), Aeromonas spp. (furunculosis; haemorrhagic septicaemia) or Tenacibaculum maritimum (tenacibaculosis). Lipopolysaccharides (LPS) are important components of the outer membrane of Gram-negative bacteria and have been linked to strong immunogenic responses in terrestrial vertebrates, playing a role in disease development. To evaluate LPS effects in fish, we used a hot-phenol procedure to extract LPS from common fish pathogens. A. hydrophila, V. harveyi, T. maritimum and P. damselae purified LPS were tested at different concentrations (50, 100, 250 and 500 µg mL-1) at 3 days post-fertilisation (dpf) Danio rerio larvae, for 5 days. While P. damselae LPS did not cause any mortality under all concentrations tested, A. hydrophila LPS induced 15.5% and V. harveyi LPS induced 58.3% of zebrafish larvae mortality at 500 µg mL-1. LPS from T. maritimum was revealed to be the deadliest, with a zebrafish larvae mortality percentage of 80.6%. Analysis of LPS separated by gel electrophoresis revealed differences in the overall LPS structure between the bacterial species analysed that might be the basis for the different mortalities observed.

Lung ultrasound-guided PEEP titration in COVID-19 patients treated with CPAP.

OBJECTIVES: An increasing number of COVID-19 patients were treated with continuous positive airways pressure (CPAP). To evaluate the clinical effects of personalized positive end-expiratory pressure (PEEP) compared to standard fixed PEEP in COVID-19 patients requiring CPAP. METHODS: This is a single center, prospective, randomized clinical study. Sixty-three COVID-19 patients with hypoxemic respiratory failure and bilateral pneumonia were randomized in two Groups: Group A received CPAP with fixed PEEP of 10 cm H2O, Group B performed the "PEEP trial", that consists in the evaluation of best PEEP defined as the PEEP value that precedes the echographic appearance of "lung pulse" determining a PaO2/FiO2 increase. Primary outcome was composite in-hospital mortality + intubation, secondary outcome was the percentage increase of PaO2/FiO2. As safety indicator, the incidence of pneumothorax was collected. RESULTS: Thirty-two patients were enrolled in Group A and 31 in Group B. The two groups were comparable for clinical characteristics and laboratory parameters. The primary outcome occurred in 36 (57.1 %) patients: 23 (71.8 %) in Group A and 13 (41.9 %) in Group B (p<0.01). Mortality was higher in Group A (53.1 vs. 19.3 %, p<0.01), while intubation rate was comparable between groups. Group B showed a higher PaO2/FiO2 increase than Group A (34.9 vs. 13.1 %, p<0.01). Five cases of pneumothorax were reported in Group A, none in Group B. CONCLUSIONS: Lung ultrasound-guided PEEP trial is associated with lower mortality in COVID-19 patients treated with CPAP. Identifying the best PEEP is useful to increase oxygenation and reduce the incidence of complications.

Feeding Yellow Worms to Meagre: Effects on Whole-Body Fatty Acid Profile and Hepatic and Intestine Oxidative Status.

This study aimed to determine the effects of dietary inclusion of Tenebrio molitor larvae (yellow worms) meal (TM) on meagre fish (Argyrosomus regius) whole-body fatty acids (FA) profile and hepatic and intestine oxidative status. For that purpose, fish were fed for 9 weeks a fishmeal-based diet (control) or diets including 10%, 20%, or 30% TM. With the increase in dietary TM level, whole-body oleic acid, linoleic acid, monounsaturated FA, and n-6 polyunsaturated FA (PUFA) increased while saturated FA (SFA), n-3 PUFA, n-3 long chain-PUFA, SFA:PUFA ratio, n3:n6 ratio, and FA retention decreased. Hepatic superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G6PDH), and glutathione reductase (GR) activities increased and catalase (CAT) and glutathione peroxidase (GPX) activities decreased with dietary TM inclusion. Hepatic total and reduced glutathione were lower in fish fed 20% TM. Intestinal CAT activity and oxidized glutathione increased and GPX activity decreased with dietary TM inclusion. Intestine SOD, G6PDH, and GR activities increased and malondialdehyde concentration decreased in fish fed the diets with lower TM inclusion levels. Liver and intestine oxidative stress index and liver malondialdehyde concentration were unaffected by dietary TM. In conclusion, to avoid major whole-body FA changes or antioxidant status imbalances, it is recommended to limit TM to 10% inclusion in meagre diets.

The animal's microbiome and cancer: A translational perspective.

Cancer is a substantial global health problem both in humans and animals with a consistent increase in mortality and incidence rate. The commensal microbiota has been involved in the regulation of several physiological and pathological processes, both within the gastrointestinal system and at distant tissue locations. Cancer is not an exception, and different aspects of the microbiome have been described to have anti- or pro-tumour effects. Using new techniques, for example high-throughput DNA sequencing, microbial populations of the human body have been largely described and, in the last years, studies more focused on companions' animals have emerged. In general, the recent investigations of faecal microbial phylogeny and functional capacity of the canine and feline gut have shown similarities with human gut. In this translational study we will review and summarize the relation between the microbiota and cancer, in humans and companion animals, and compare their resemblance in the type of neoplasms already studied in veterinary medicine: multicentric and intestinal lymphoma, colorectal tumours, nasal neoplasia and mast cell tumours. In the context of One Health, microbiota and microbiome integrative studies may contribute to the understanding of the tumourigenesis process, besides offering an opportunity to develop new diagnostics and therapeutic biomarkers both for veterinary and human oncology.

Oxidative Stress Response of Meagre to Dietary Black Soldier Fly Meal.

This study aimed to assess the effect of Hermetia illucens meal (HM) dietary inclusion on meagre oxidative status. Thus, fish were fed a fishmeal-based diet (CTR diet) and three other diets with increasing levels of HM inclusion, namely 10%, 20%, and 30% (diets HM10, HM20, and HM30, respectively). At the end of the trial, hepatic and intestine superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities and malondialdehyde concentration were unaffected by the diet composition. Liver glutathione peroxidase activity was higher in the fish fed the HM20 diet than in the fish fed the CTR and HM30 diets, and glutathione reductase activity linearly increased with the dietary HM level. The hepatic total glutathione and reduced glutathione contents were significantly lower in fish fed the HM20 diet than in fish fed the CTR and HM10 diets. In the intestine, the oxidized glutathione (GSSG) content and oxidative stress index linearly increased with the increase in dietary HM level, with the GSSG content of fish fed the HM20 diet being significantly higher than of fish fed the CTR diet. In conclusion, 30% HM might be included in meagre diets without negatively affecting hepatic and intestine oxidative status.

Oral vaccination of fish against vibriosis using spore-display technology.

Oral vaccines are highly demanded by the aquaculture sector, to allow mass delivery of antigens without using the expensive and labor-intensive injectable vaccines. These later require individual handling of fish, provoking stress-related mortalities. One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores, extremely resistant structures with wide biotechnological applications, including as probiotics, display systems, or adjuvants. Bacterial spores, in particular those of Bacillus subtilis, have been shown to behave as mucosal vaccine adjuvants in mice models. However, such technology has not been extensively explored against fish bacterial disease. In this study, we used a laboratory strain of B. subtilis, for which a variety of genetic manipulation tools are available, to display at its spores surface either a Vibrio antigenic protein, OmpK, or the green fluorescence protein, GFP. When previously vaccinated by immersion with the OmpK- carrying spores, zebrafish survival upon a bacterial challenge with V. anguillarum and V. parahaemolyticus, increased up to 50 - 90% depending on the pathogen targeted. Further, we were able to detect anti-GFP-antibodies in the serum of European seabass juveniles fed diets containing the GFP-carrying spores and anti-V. anguillarum antibodies in the serum of European seabass juveniles fed the OmpK-carrying spores containing diet. More important, seabass survival was increased from 60 to 86% when previously orally vaccinated with in-feed OmpK- carrying spores. Our results indicate that B. subtilis spores can effectively be used as antigen-carriers for oral vaccine delivery in fish.

Isolation of Chitinolytic Bacteria from European Sea Bass Gut Microbiota Fed Diets with Distinct Insect Meals.

Insect meal (IM), recently authorized for use in aquafeeds, positions itself as a promising commodity for aquafeed inclusion. However, insects are also rich in chitin, a structural polysaccharide present in the exoskeleton, which is not digested by fish, resulting in lower fish performance. Through the application of a dietary pressure, this study aimed to modulate European sea bass gut microbiota towards the enrichment of chitinolytic bacteria to allow the isolation of novel probiotics capable of improving the use of IM-containing diets, overcoming chitin drawbacks. Five isoproteic (44%) and isolipidic (18%) diets were used: a fish meal (FM)-based diet (diet CTR), a chitin-supplemented diet (diet CHIT5), and three diets with either 25% of Hermetia illucens and Tenebrio molitor larvae meals (HM25 and TM25, respectively) or H. illucens exuviae meal (diet HEM25) as partial FM substitutes. After an 8-week feeding trial, the results showed a clear modulatory effect towards spore-forming bacteria by HM25 and HEM25 diets, with the latter being responsible for the majority of the chitinolytic fish isolates (FIs) obtained. Sequential evaluation of the FI hemolytic activity, antibiotic resistance, total chitinolytic activity, sporulation, and survival in gastrointestinal-like conditions identified FI645 and FI658 as the most promising chitinolytic probiotics for in vivo application.

Immunostimulant properties of full-length and truncated Marinobacter algicola flagellins, and their effects against viral hemorrhagic septicemia virus (VHSV) in trout.

Adjuvants that would help optimize fish vaccines against bacterial and viral pathogens are highly demanded by the aquaculture sector. Flagellin has been proposed as an immunostimulant and an adjuvant for more than a decade. However, the adjuvant ability of flagellins with hypervariable region deleted is still unclear in fish. In this study, we evaluated the immune-stimulating capacity of two recombinant flagellins, the wild-type flagellin F from Marinobacter algicola and a version with the hypervariable region deleted (FredV2), to induce the transcription of a wide range of immune genes using two rainbow trout cell lines: a monocyte/macrophage-cell line (RTS-11) and an epithelial cell line from intestine (RTgutGC). Additionally, we studied the capacity of both flagellins to limit the replication of viral hemorrhagic septicemia virus (VHSV) on the RTgutGC cell line. Our results demonstrated that both recombinant flagellins can significantly increase the transcription of IL-1ß1, IL-6, and IL-8 in both cell lines. However, other cytokines such as IFNγ1, and TNFα or antimicrobial peptides such as hepcidin were induced by both flagellins in RTgutGC but not in RTS-11 cells. Furthermore, both flagellins were capable of reducing the replication of VHSV in RTgutGC cells. Although the immunostimulatory and the antiviral capacities exerted by F were slightly more potent than those obtained with FredV2, the effects were retained after losing the hypervariable region. Our results provide new information on the immunostimulating and antiviral capacities of flagellins that point to their potential as suitable adjuvants for the future optimization of vaccines in aquaculture.

Bacillus subtilis Expressing the Infectious Pancreatic Necrosis Virus VP2 Protein Retains Its Immunostimulatory Properties and Induces a Specific Antibody Response.

Bacillus subtilis has been documented in the past years as an effective probiotic for different aquacultured species, with recognized beneficial effects on water quality, fish growth and immune status. Furthermore, its potential as a vaccine adjuvant has also been explored in different species. In the current work, we have used B. subtilis spores as delivery vehicles for the presentation of the VP2 protein from infectious pancreatic necrosis virus (IPNV). For this, the VP2 gene was amplified and translationally fused to the crust protein CotY. The successful expression of VP2 on the spores was confirmed by Western blot. We then compared the immunostimulatory potential of this VP2-expressing strain (CRS208) to that of the original B. subtilis strain (168) on rainbow trout (Oncorhynchus mykiss) leukocytes obtained from spleen, head kidney and the peritoneal cavity. Our results demonstrated that both strains significantly increased the percentage of IgM+ B cells and the number of IgM-secreting cells in all leukocyte cultures. Both strains also induced the transcription of a wide range of immune genes in these cultures, with small differences between them. Importantly, specific anti-IPNV antibodies were detected in fish intraperitoneally or orally vaccinated with the CRS208 strain. Altogether, our results demonstrate B. subtilis spores expressing foreign viral proteins retain their immunomodulatory potential while inducing a significant antibody response, thus constituting a promising vaccination strategy.

Differential Modulation of the European Sea Bass Gut Microbiota by Distinct Insect Meals.

The aquaculture industry is one of the fastest-growing sectors in animal food production. However, farming of carnivorous fish strongly relies on the use of wild fish-based meals, a practice that is environmentally and economically unsustainable. Insect-based diets constitute a strong candidate for fishmeal substitution, due to their high nutritional value and low environmental footprint. Nevertheless, data on the impact of insect meal (IM) on the gut microbiome of farmed fish are so far inconclusive, and very scarce in what concerns modulation of microbial-mediated functions. Here we use high-throughput 16S rRNA gene amplicon sequencing and quantitative PCR to evaluate the impact of different IMs on the composition and chitinolytic potential of the European sea bass gut digesta- and mucosa-associated communities. Our results show that insect-based diets of distinct origins differently impact the gut microbiota of the European sea bass (Dicentrarchus labrax). We detected clear modulatory effects of IM on the gut microbiota, which were more pronounced in the digesta, where communities differed considerably among the diets tested. Major community shifts were associated with the use of black soldier fly larvae (Hermetia illucens, HM) and pupal exuviae (HEM) feeds and were characterized by an increase in the relative abundance of the Firmicutes families Bacillaceae, Enterococcaceae, and Lachnospiraceae and the Actinobacteria family Actinomycetaceae, which all include taxa considered beneficial for fish health. Modulation of the digesta community by HEM was characterized by a sharp increase in Paenibacillus and a decrease of several Gammaproteobacteria and Bacteroidota members. In turn, a mealworm larvae-based diet (Tenebrio molitor, TM) had only a modest impact on microbiota composition. Further, using quantitative PCR, we demonstrate that shifts induced by HEM were accompanied by an increase in copy number of chitinase ChiA-encoding genes, predominantly originating from Paenibacillus species with effective chitinolytic activity. Our study reveals an HEM-driven increase in chitin-degrading taxa and associated chitinolytic activity, uncovering potential benefits of adopting exuviae-supplemented diets, a waste product of insect rearing, as a functional ingredient.

Novel protein carrier system based on cyanobacterial nano-sized extracellular vesicles for application in fish.

Aquaculture has been one of the fastest-growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP-loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

In vitro modulation of gilthead seabream (Sparus aurata L.) leukocytes by Bacillus spp. extracellular molecules upon bacterial challenge.

Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1ß, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1ß, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.

Bacillus spp. Inhibit Edwardsiella tarda Quorum-Sensing and Fish Infection.

The disruption of pathogen communication or quorum-sensing (QS) via quorum-quenching (QQ) molecules has been proposed as a promising strategy to fight bacterial infections. Bacillus spp. have recognizable biotechnology applications, namely as probiotic health-promoting agents or as a source of natural antimicrobial molecules, including QQ molecules. This study characterized the QQ potential of 200 Bacillus spp., isolated from the gut of different aquaculture fish species, to suppress fish pathogens QS. Approximately 12% of the tested Bacillus spp. fish isolates (FI). were able to interfere with synthetic QS molecules. Ten isolates were further selected as producers of extracellular QQ-molecules and their QQ capacity was evaluated against the QS of important aquaculture bacterial pathogens, namely Aeromonas spp., Vibrio spp., Photobacterium damselae, Edwardsiela tarda, and Shigella sonnei. The results revealed that A. veronii and E. tarda produce QS molecules that are detectable by the Chr. violaceum biosensor, and which were degraded when exposed to the extracellular extracts of three FI isolates. Moreover, the same isolates, identified as B. subtilis, B. vezelensis, and B. pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae, increasing its survival by 50%. Taken together, these results identified three Bacillus spp. capable of extracellularly quenching aquaculture pathogen communication, and thus become a promising source of bioactive molecules for use in the biocontrol of aquaculture bacterial diseases.

Methionine and Tryptophan Play Different Modulatory Roles in the European Seabass (Dicentrarchus labrax) Innate Immune Response and Apoptosis Signaling-An In Vitro Study.

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes' immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1ß and nf-κb expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-κb and p65. This study showed the ability of methionine surplus to improve leucocytes' response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.

Isolation and Characterization of Fish-Gut Bacillus spp. as Source of Natural Antimicrobial Compounds to Fight Aquaculture Bacterial Diseases.

Aquaculture is responsible for more than 50% of global seafood consumption. Bacterial diseases are a major constraint to this sector and associated with misuse of antibiotics, pose serious threats to public health. Fish-symbionts, co-inhabitants of fish pathogens, might be a promising source of natural antimicrobial compounds (NACs) alternative to antibiotics, limiting bacterial diseases occurrence in aquafarms. In particular, sporeforming Bacillus spp. are known for their probiotic potential and production of NACs antagonistic of bacterial pathogens and are abundant in aquaculture fish guts. Harnessing the fish-gut microbial community potential, 172 sporeforming strains producing NACs were isolated from economically important aquaculture fish species, namely European seabass, gilthead seabream, and white seabream. We demonstrated that they possess anti-growth, anti-biofilm, or anti-quorum-sensing activities, to control bacterial infections and 52% of these isolates effectively antagonized important fish pathogens, including Aeromonas hydrophila, A. salmonicida, A. bivalvium, A. veronii, Vibrio anguillarum, V. harveyi, V. parahaemolyticus, V. vulnificus, Photobacterium damselae, Tenacibaculum maritimum, Edwardsiela tarda, and Shigella sonnei. By in vitro quantification of sporeformers' capacity to suppress growth and biofilm formation of fish pathogens, and by assessing their potential to interfere with pathogens communication, we identified three promising candidates to become probiotics or source of bioactive molecules to be used in aquaculture against bacterial aquaculture diseases.

Gut microbiota dynamics in carnivorous European seabass (Dicentrarchus labrax) fed plant-based diets.

A healthy gastrointestinal microbiota is essential for host fitness, and strongly modulated by host diet. In aquaculture, a current challenge is to feed carnivorous fish with plant-feedstuffs in substitution of fish meal, an unsustainable commodity. Plants have a limited nutritive value due to the presence of non-starch polysaccharides (NSP) which are not metabolized by fish. In this work we assessed the effects of NSP-enriched diets on European seabass gut microbiota and evaluate the selective pressure of plant feedstuffs towards gut microbes with NSP-hydrolytic potential, i.e. capable to convert indigestible dietary constituents in fish metabolites. Triplicate groups of European seabass juveniles were fed a fish meal-based diet (control) or three plant-based diets (SBM, soybean meal; RSM, rapeseed meal; SFM, sunflower meal) for 6 weeks, before recovering intestinal samples for microbiota analysis, using the Illumina's MiSeq platform. Plant-based diets impacted differently digesta and mucosal microbiota. A decrease (p = 0.020) on species richness, accompanied by a decline on the relative abundance of specific phyla such as Acidobacteria (p = 0.030), was observed in digesta samples of SBM and RSM experimental fish, but no effects were seen in mucosa-associated microbiota. Plant-based diets favored the Firmicutes (p = 0.01), in particular the Bacillaceae (p = 0.017) and Clostridiaceae (p = 0.007), two bacterial families known to harbor carbohydrate active enzymes and thus putatively more prone to grow in high NSP environments. Overall, bacterial gut communities of European seabass respond to plant-feedstuffs with adjustments in the presence of transient microorganisms (allochthonous) with carbohydrolytic potential, while maintaining a balanced core (autochthonous) microbiota.

Time-weighted lactate as a predictor of adverse outcome in acute heart failure.

AIMS: The role of dynamic changes in lactate concentrations on prognosis in acute heart failure has been poorly investigated. The aim of this study was to explore the predictive value of 24 h time-weighted lactate (LACTW ) in patients with acute heart failure. METHODS AND RESULTS: Ninety-six consecutive acute heart failure patients presenting to the Emergency Department of San Paolo Hospital, Naples, Italy, were prospectively enrolled. Arterial blood lactate was measured at admission and during the following 24 h at random time intervals. LACTW was obtained by the sum of the average lactate values among consecutive time points multiplied by the intervals between consecutive time points and dividing the sum by the total time (24 h). The outcome was a composite of need of admission to the intensive care unit, hospitalization duration >7 days, or intra-hospital death. Admission lactate, maximum measured lactate, and LACTW were collected. Univariate and multivariate Cox regression analysis was applied to determine the hazard ratio (HR) of developing the outcome. Forty-three patients experienced the pre-specified outcome. In sex-adjusted and age-adjusted multivariable analysis, LACTW predicted the outcome occurrence (HR: 1.51, 95% confidence interval: 1.24, 1.84, P < 0.001). Risk stratification analysis based on LACTW tertiles demonstrated a gradual increase in risk of developing the outcome (HR: 17.32, 95% confidence interval: 2.30, 130.23, P = 0.006) for the highest LACTW tertile. CONCLUSIONS: In acute heart failure patients, 24 h LACTW had a significant independent predictive value for adverse intra-hospital outcome. LACTW could be a useful index at identifying high-risk patients who may require a more aggressive treatment during hospitalization.