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.

Unusual Surge of Acute Hepatitis A Cases in 2016 and 2017 in Malaga, Southern Spain: Characterization and Relationship with Other Concurrent European Outbreaks.

We aimed to describe the Hepatitis A virus (HAV) cases that arose in Malaga (Spain) in 2016 and 2017 when the European Centre for Disease Prevention and Control (ECDC) reported several outbreaks among men who have sex with men (MSM). Therefore, we conducted a retrospective study gathering demographic, clinical, and immunological data from the acute HAV patients attending our hospital between March 2016 and December 2017. Additionally, VP1/P2A region was amplified from serum samples, sequenced, and genotyped. We finally performed a phylogenetic analysis, including the HAV strains from the other European outbreaks. A total of 184 HAV cases were reported, with the highest number in March 2017. The cohort mostly comprised Spaniards (81.0%), males (84.8%), and MSM (72.3%), with a median age of 33.0 years (interquartile range (IQR) = 25.0-43.0). Most patients exhibited symptoms. In addition, a successful amplification and sequencing of the VP1/P2A region was performed in 25 out of 106 serum samples (23.6%). All the sequences belonged to the genotype IA, and 20 were phylogenetically related to VRD_521_2016, first described in the United Kingdom (UK). In conclusion, HAV cases emerged in Malaga in 2016 and 2017, showing an epidemic character phylogenetically related to the predominant strain first detected in the UK. Characteristics of the cohort were similar to those from the European outbreaks.

SpPdp11 Administration in Diet Modified the Transcriptomic Response and Its Microbiota Associated in Mechanically Induced Wound Sparus aurata Skin.

Skin lesions are a frequent fact associated with intensive conditions affecting farmed fish. Knowing that the use of probiotics can improve fish skin health, SpPdp11 dietary administration has demonstrated beneficial effects for farmed fish, so its potential on the skin needs to be studied more deeply. The wounded specimens that received the diet with SpPdp11 showed a decrease in the abundance of Enterobacteriaceae, Photobacterium and Achromobacter related to bacterial biofilm formation, as well as the overexpression of genes involved in signaling mechanisms (itpr3), cell migration and differentiation (panxa, ttbk1a, smpd3, vamp5); and repression of genes related to cell proliferation (vstm4a, areg), consistent with a more efficient skin healing processes than that observed in the wounded control group. In addition, among the groups of damaged skin with different diets, Achromobacter, f_Ruminococcaceae, p_Bacteroidetes, Fluviicola and Flavobacterium genera with significant differences showed positive correlations with genes related to cell migration and negative correlations with inflammation and cell proliferation and may be the target of future studies.

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.

Estudio mediante técnicas moleculares del tanatomicrobioma intestinal en la estimación del intervalo post mortem temprano empleando un modelo de ratón / Early post mortem interval estimation in a mouse model using molecular analyses of the gut thanatomicrobiome

Introducción: en la actualidad, la falta de métodos cuantitativos fiables ha llevado a distintas líneas de investigación a buscar un modelo que prediga el intervalo post mortem (IPM). El tanatomicrobioma, presente desde el momento de la muerte, parece sufrir cambios predecibles y que siguen una correlación con el IPM.Materiales y métodosse ha analizado experimentalmente el comportamiento del tanatomicrobioma en la región del intestino delgado posterior y del colon ascendente durante las primeras 24 h de descomposición en Mus musculus. Para ello, se ha llevado a cabo una aproximación molecular basada en el análisis del gen ribosomal 16S (ARNr 16S) mediante electroforesis en gel con gradiente desnaturalizante (DGGE) y, seguidamente un análisis de la alfa y beta diversidad.Resultadoslos resultados basados en el análisis de los índices de diversidad ecológica reflejaron cambios estadísticamente significativos antes de las 12 h, y un descenso de la diversidad a partir de esas 12 h postmortem, siendo este estadísticamente significativo en las 2 regiones intestinales analizadas. Por otro lado, el estudio comparativo de las comunidades microbianas mostró que cambian estructurada y diferenciablemente desde el momento de la muerte, alejándose en similitud de las mostradas en vida (IPM 0 h).Discusiónestos resultados coinciden con el descenso de la diversidad sugerido a largo plazo por distintos autores. Sin embargo, en las condiciones del estudio, se ha visto que este descenso no se inicia hasta las 12 h. Conclusión como conclusión, se han podido establecer, según los cambios en la diversidad bacteriana, fases de la dinámica bacteriana durante la descomposición que podrían ayudar a mejorar los modelos de correlación microbiana para la estimación del IPM. (AU)

Introduction: Currently, the lack of reliable quantitative methods has led different research lines to find a model that predicts the postmortem interval (PMI). The thanatomicrobiome, present from the moment of death, has been shown to change in predictable ways, allowing a correlation with PMI.Materials and methodsIn this study, the shifts of the thanatomicrobiome in the region of the posterior small intestine and the ascending colon in Mus musculus during the first 24 hrs of decomposition have been analyzed experimentally. For this purpose, a molecular approach based on the analysis of the 16S ribosomal gene (16S rRNA) and a denaturing gradient gel electrophoresis (DGGE) was adopted, followed by analyses of the ecological diversity indices Alpha and beta diversity.ResultsThe results based on the analysis of the ecological diversity indices reflected statistically significant changes before 12 hrs, and a decrease in diversity after 12 hrs postmortem, this being statistically significant in the two intestinal regions analyzed. Moreover, the comparative study of microbial communities indicated distinct and structured changes from the moment of death, with shifts in the degree of similarity from the composition detected in life (PMI 0 hrs).DiscussionThese results agree with other studies demonstrating a decrease in microbial diversity. However, under the conditions of the study, this decrease does not begin until 12 hrs after death. Conclusions: In conclusion, by examining the dynamics of bacterial diversity our study has identified phases during decomposition that could help to improve microbial correlation models for PMI estimation. (AU)

Revisión sobre las nuevas perspectivas de datación cadavérica desde el necrobioma / Review of cadaveric dating methods and new perspectives from the necrobiome

Hoy en día existen numerosas estrategias desde un punto de vista científico que ayudan a esclarecer los casos forenses, entre ellas la datación cadavérica. La ausencia de métodos fiables cuantitativos para estimar el intervalo post mortem explica el incremento de nuevas líneas de investigación prometedoras con dicha finalidad. Tras la aparición de las nuevas técnicas de secuenciación masiva y bioinformáticas, surge también el estudio del necrobioma como un área novedosa y poco estudiada dentro de las ciencias forenses, que se ha llegado a denominar «microbiología forense». En esta revisión se realiza un breve recorrido por las técnicas y procedimientos existentes de datación cadavérica, centrándose en la utilidad del tanatomicrobioma, o conjunto de microorganismos presentes en el momento de la muerte, que podría ser un método prometedor para la estimación del intervalo post mortem en el futuro. (AU)

Nowadays there are numerous scientific strategies that helping to clarify forensic cases, including time since death. The absence of reliable quantitative methods to estimate the post-mortem interval explains the increase in promising new lines of research for this purpose. After the appearance of the new techniques of massive sequencing and bioinformatics, also arises the study of the necrobiome through a new and little studied area within the forensic sciences, Forensic Microbiology. In this review, a tour of the existing techniques and procedures of cadaveric dating is made, which includes new cutting-edge techniques in different areas of knowledge and also mentions the utilities of Forensic Microbiology, where the thanatomicrobiome, present from the moment of death, according to recent studies, points to be a promising method for estimating the post-mortem interval in the future. (AU)

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.

Solea senegalensis Bacterial Intestinal Microbiota Is Affected by Low Dietary Inclusion of Ulva ohnoi.

The inclusion of macroalgae in the diets of farmed fish offers the opportunity for an added-value dietary ingredient to the nutraceutical feed. The composition of algae varies greatly among species. Several Ulva species have been considered in aquafeed formulations for different farmed fish, and Ulva ohnoi is being applied recently. However, the effects of seaweed dietary inclusion on the host must be evaluated. Considering the important role of the host intestinal microbiota, the potential effects of U. ohnoi dietary inclusion need to be studied. In this study, the characterization of the intestinal microbiome of Solea senegalensis, a flatfish with high potential for aquaculture in South Europe, receiving U. ohnoi (5%)-supplemented diet for 90 days has been carried out. In addition, the functional profiles of bacterial communities have been determined by using PICRUSt, a computational approach to predict the functional composition of a metagenome by using marker gene data and a database of reference genomes. The results show that long-term dietary administration of U. ohnoi (5%)-supplemented feed modulates S. senegalensis intestinal microbiota, especially in the posterior intestinal section. Increased relative abundance of Vibrio jointly with decreased Stenotrophomonas genus has been detected in fish receiving Ulva diet compared to control-fed fish. The influence of the diet on the intestinal functionality of S. senegalensis has been studied for the first time. Changes in bacterial composition were accompanied by differences in predicted microbiota functionality. Increased abundance of predicted genes involved in xenobiotic biodegradation and metabolism were observed in the microbiota when U. ohnoi diet was used. On the contrary, predicted percentages of genes associated to penicillin and cephalosporin biosynthesis as well as beta-lactam resistance were reduced after feeding with Ulva diet.

TarSynFlow, a workflow for bacterial genome comparisons that revealed genes putatively involved in the probiotic character of Shewanella putrefaciens strain Pdp11.

Probiotic microorganisms are of great interest in clinical, livestock and aquaculture. Knowledge of the genomic basis of probiotic characteristics can be a useful tool to understand why some strains can be pathogenic while others are probiotic in the same species. An automatized workflow called TarSynFlow (Targeted Synteny Workflow) has been then developed to compare finished or draft bacterial genomes based on a set of proteins. When used to analyze the finished genome of the probiotic strain Pdp11 of Shewanella putrefaciens and genome drafts from seven known non-probiotic strains of the same species obtained in this work, 15 genes were found exclusive of Pdp11. Their presence was confirmed by PCR using Pdp11-specific primers. Functional inspection of the 15 genes allowed us to hypothesize that Pdp11 underwent genome rearrangements spurred by plasmids and mobile elements. As a result, Pdp11 presents specific proteins for gut colonization, bile salt resistance and gut pathogen adhesion inhibition, which can explain some probiotic features of Pdp11.

Modulation of Intestinal Microbiota in Solea senegalensis Fed Low Dietary Level of Ulva ohnoi.

Gastrointestinal (GI) microbiota has a relevant role in animal nutrition, modulation of the immune system and protection against pathogen invasion. Interest in algae as source of nutrients and functional ingredients for aquafeeds is increasing in order to substitute conventional feedstuffs by more sustainable resources. The diet is an important factor in the modulation of the microbiota composition, and functional ingredients have been proposed to shape the microbiota and contribute benefits to the host. However, fish microbiome research is still limited compared to other hosts. Solea senegalensis is a flat fish with high potential for aquaculture in South Europe. In this study, a characterization of the microbiome of S. senegalensis (GI) tract and the effects of feeding Ulva ohnoi supplemented diet has been carried out. Differences in the composition of the microbiota of anterior and posterior sections of S. senegalensis GI tract have been observed, Pseudomonas being more abundant in the anterior sections and Mycoplasmataceae the dominant taxa in the posterior GI tract sections. In addition, modulation of the GI microbiota of juvenile Senegalese sole fed for 45 days a diet containing low percentage of U. ohnoi has been observed in the present study. Microbiota of the anterior regions of the intestinal tract was mainly modulated, with higher abundance of Vibrio spp. in the GI tract of fish fed dietary U. ohnoi.

Dietary administration of the probiotic Shewanella putrefaciens Pdp11 promotes transcriptional changes of genes involved in growth and immunity in Solea senegalensis larvae.

Senegalese sole (Solea senegalensis) has been proposed as a high-potential species for aquaculture diversification in Southern Europe. It has been demonstrated that a proper feeding regimen during the first life stages influences larval growth and survival, as well as fry and juvenile quality. The bacterial strain Shewanella putrefaciens Pdp11 (SpPdp11) has shown very good probiotic properties in Senegalese sole, but information is scarce about its effect in the earliest stages of sole development. Thus, the aim of this study was to investigate the effect of SpPdp11, bioencapsulated in live diet, administered during metamorphosis (10-21 dph) or from the first exogenous feeding of Senegalese sole (2-21 dph). To evaluate the persistence of the probiotic effect, we sampled sole specimens from metamorphosis until the end of weaning (from 23 to 73 dph). This study demonstrated that probiotic administration from the first exogenous feeding produced beneficial effects on Senegalese sole larval development, given that specimens fed this diet exhibited higher and less dispersed weight, as well as increases in both total protein concentration and alkaline phosphatase activity, and in non-specific immune response. Moreover, real-time PCR documented changes in the expression of a set of genes involved in central metabolic functions including genes related to growth, genes coding for proteases (including several digestive enzymes), and genes implicated in the response to stress and in immunity. Overall, these results support the application of SpPdp11 in the first life stages of S. senegalensis as an effective tool with the clear potential to benefit sole aquaculture.

Mucus glycosylation, immunity and bacterial microbiota associated to the skin of experimentally ulcered gilthead seabream (Sparus aurata).

Interest in fish skin immunity and its associated microbiota has greatly increased among immunologists. The objective of this study is to know if skin ulcers may be associated with changes in the mucus composition and microbial diversity. The abundance of terminal carbohydrates, several enzymes (protease, antiprotease, peroxidase, lysozyme) and total immunoglobulin M levels were evaluated in skin mucus of experimentally ulcered gilthead seabream (Sparus aurata L.). Furthermore, the composition of the microbiota of ulcered and non-ulcered skin has been determined using Illumina Miseq technology. Significant decreases of terminal abundance of α-D-mannose, α-D-glucose and N-acetyl-galactosamine in skin mucus of ulcered fish, compared to control fish were detected. The levels of IgM and all the tested enzymes in mucus were decreased in ulcered fish (compared to control fish) although the observed decreases were only statistically significant for proteases and antiproteases. Concomitantly, the analysis of the composition of the skin microbiota showed clear differences between ulcered and non-ulcered areas. The genus taxonomic analysis showed that Staphylococcus and Lactobacillus were more abundant in non-ulcered skin whereas in ulcered area were Streptococcus and Granulicatella. Important decreases of the number of sequences related to Alteromonas, Thalassabius and Winogradskyella were detected in ulcered skin whilst slight increases of sequences related to Flavobacterium, Chryseobacterium and Tenacibaculum genera were observed. Overall these results demonstrated that the presence of skin ulcers provide microenvironments that perturb both the mucus composition and microbial biodiversity of this important external surface which seem to be more vulnerable to diseases.

Effects on intestinal microbiota and immune genes of Solea senegalensis after suspension of the administration of Shewanella putrefaciens Pdp11.

The interaction host-intestinal microbiota is essential for the immunological homeostasis of the host. Probiotics, prebiotics and synbiotics are promising tools for the manipulation of the intestinal microbiota towards beneficial effects to the host. The objective of this study was to evaluate the modulation effect on the intestinal microbiota and the transcription of genes involved in the immune response in head kidney of Solea senegalensis after administration of diet supplemented with the prebiotic alginate and the probiotic Shewanella putrefaciens Pdp11 CECT 7627 (SpPdp11). The results showed higher adaptability to dietary changes in the intestinal microbiota of fish fed diet with alginate and SpPdp11 together compared to those fish that received an alginate-supplemented diet. The alginate-supplemented diet produced up-regulation of genes encoding proteins involved in immunological responses, such as complement, lysozyme G and transferrin, and oxidative stress, such as NADPH oxidase and glutation peroxidase. On the other hand, the administration of alginate combined with SpPdp11 resulted in a significant increase of the transcription of genes encoding for glutation peroxidase and HSP70, indicating a potential protective effect of SpPdp11 against oxidative stress. In addition, these effects were maintained after the suspension of the probiotic treatment. The relationship between the modulation of the intestinal microbiota and the expression of genes with protective effect against the oxidative stress was demonstrated by the Principal Components Analysis.

Modulation of immunity and gut microbiota after dietary administration of alginate encapsulated Shewanella putrefaciens Pdp11 to gilthead seabream (Sparus aurata L.).

The potential benefits of probiotics when administering to fish could improve aquaculture production. The objective of this study was to examine the modulation of immune status and gut microbiota of gilthead seabream (Sparus aurata L.) specimens by a probiotic when administered encapsulated. Commercial diet was enriched with Shewanella putrefaciens Pdp11 (SpPdp11, at a concentration of 10(8) cfu g(-1)) before being encapsulated in calcium alginate beads. Fish were fed non-supplemented (control) or supplemented diet for 4 weeks. After 1, 2 and 4 weeks the main humoral and cellular immune parameters were determined. Furthermore, gene expression profile of five immune relevant genes (il1ß, bd, mhcIIα, ighm and tcrß) was studied by qPCR in head kidney. On the other hand, intestinal microbiota of fish was analysed at 7 and 30 days by DGGE. Results demonstrated that administration of alginate encapsulated SpPdp11 has immunostimulant properties on humoral parameters (IgM level and serum peroxidase activity). Although no immunostimulant effects were detected on leucocyte activities, significant increases were detected in the level of mRNA of head-kidney leucocytes for mhcIIα and tcrß after 4 weeks of feeding the encapsulated-probiotic diet. The administration of SpPdp11 encapsulated in alginate beads produced important changes in the DGGE patterns corresponding to the intestinal microbiota. Predominant bands related to lactic acid bacteria, such as Lactococcus and Lactobacillus strains, were sequenced from the DGGE patterns of fish fed the probiotic diet, whereas they were not sequenced from fish receiving the control diet. The convenience or not of probiotic encapsulation is discussed.

Probiotic supplementation influences the diversity of the intestinal microbiota during early stages of farmed senegalese sole (Solea Senegalensis, Kaup 1858).

Ingestion of bacteria at early stages results in establishment of a primary intestinal microbiota which likely undergoes several stages along fish life. The role of this intestinal microbiota regulating body functions is crucial for larval development. Probiotics have been proved to modulate this microbiota and exert antagonistic effects against fish pathogens. In the present study, we aimed to determine bacterial diversity along different developmental stages of farmed Senegalese sole (Solea senegalensis) after feeding probiotic (Shewanella putrefaciens Pdp11) supplemented diet for a short period (10-30 days after hatching, DAH). Intestinal lumen contents of sole larvae fed control and probiotic diets were collected at 23, 56, 87, and 119 DAH and DNA was amplified using 16S rDNA bacterial domain-specific primers. Amplicons obtained were separated by denaturing gradient gel electrophoresis (DGGE), cloned, and resulting sequences compared to sequences in GenBank. Results suggest that Shewanella putrefaciens Pdp11 induces a modulation of the dominant bacterial taxa of the intestinal microbiota from 23 DAH. DGGE patterns of larvae fed the probiotic diet showed a core of bands related to Lactobacillus helveticus, Pseudomonas acephalitica, Vibrio parahaemolyticus, and Shewanella genus, together with increased Vibrio genus presence. In addition, decreased number of clones related to Photobacterium damselae subsp piscicida at 23 and 56 DAH was observed in probiotic-fed larvae. A band corresponding to Shewanella putrefaciens Pdp11 was sequenced as predominant from 23 to 119 DAH samples, confirming the colonization by the probiotics. Microbiota modulation obtained via probiotics addition emerges as an effective tool to improve Solea senegalensis larviculture.

Dietary probiotic supplementation (Shewanella putrefaciens Pdp11) modulates gut microbiota and promotes growth and condition in Senegalese sole larviculture.

Probiotic supplementation in fish aquaculture has significantly increased in the last decade due to its beneficial effect on fish performance. Probiotic use at early stages of fish development may contribute to better face metamorphosis and weaning stress. In the present work, we studied the influence of Shewanella putrefaciens Pdp11 supplementation on growth, body composition and gut microbiota in Senegalese sole (Solea senegalensis) during larval and weaning development. S. putrefaciens Pdp11 was incorporated using Artemia as live vector (2.5 × 107 cfu mL⁻¹) and supplied to sole specimens in a co-feeding regime (10-86 DAH) by triplicate. Probiotic addition promoted early metamorphosis and a significantly higher growth in length at 24 DAH larvae. S. putrefaciens Pdp11 also modulated gut microbiota and significantly increased protein content and DHA/EPA ratios in sole fry (90 DAH). This nutritional enhancement is considered especially important after weaning, where significantly higher growth in length and weight was observed in probiotic fish. Moreover, a less heterogeneous fish size in length was detected since metamorphosis till the end of weaning, being of interest for sole aquaculture production. After weaning, fish showed significantly higher growth (length and weight) and less variable lengths in fish when supplemented with probiotics. Both the enhancement of nutritional condition and the decrease in size variability associated with probiotic addition are highly interesting for sole aquaculture production.

Changes in intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis in gilthead sea bream (Sparus aurata L.) specimens.

Changes produced in gilthead sea bream (Sparus aurata L.) intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis have been studied. Gilthead sea bream specimens were fed diets containing 0 (control), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)), or B. subtilis + inulin (10(7) cfu g(-1) + 10 g kg(-1)) for four weeks. Curiously, fish fed the experimental diets (inulin, B. subtilis, or B. subtilis + inulin) showed the same morphological alterations when studied by light and electron microscopy, while significant differences in the signs of intestinal damage were detected by the morphometric study. All of the observed alterations were present only in the gut mucosa, and intestinal morphometric study revealed no effect of inulin or B. subtilis on the intestinal absorptive area. Furthermore, experimental diets cause important alterations in the intestinal microbiota by significantly decreasing bacterial diversity, as demonstrated by the specific richness, Shannon, and range-weighted richness indices. The observed alterations demonstrate that fish fed experimental diets had different signs of gut oedema and inflammation that could compromise their body homeostasis, which is mainly maintained by the epithelial lining of the gastrointestinal tract. To our knowledge, this is the first in vivo study regarding the implications of the use of synbiotics (conjunction of probiotics and prebiotics) on fish gut morphology and microbiota.

Histological alterations and microbial ecology of the intestine in gilthead seabream (Sparus aurata L.) fed dietary probiotics and microalgae.

The effects on histology and microbial ecology in gilthead seabream (Sparus aurata) intestine caused by dietary probiotic and microalgae were studied. Fish were fed non-supplemented (C, control) or supplemented diets with Tetraselmis chuii, Phaeodactylum tricornutum and Bacillus subtilis single or combined (diets T, P, B, BT and BP) for 4 weeks. Curiously, fish fed the experimental diets showed similar morphological alterations when studied by light and electron microscopy and significant signs of intestinal damage were detected. No effect of microalgae or B. subtilis on the intestinal absorptive area was observed, whereas the number of goblet cells and IELs were significantly lower in fish fed the T, P, B and BT diets and T, BT and BP diets, respectively. Interestingly, only the diets containing B. subtilis resulted in a significant reduction of microvilli height. Alterations such as wide intercellular spaces and large vacuoles in enterocytes were observed in fish fed T, B, BT, BT and P in lesser degrees. These observations demonstrate that fish fed experimental diets presented different signs of oedema and inflammation that could compromise their body homeostasis. Moreover, the experimental diets cause important alterations in the intestinal microbiota by a significant decrease in bacterial diversity, as demonstrated by the fall in specific richness, Shannon and range-weighted richness indices. To our knowledge, this is the first in vivo study regarding the implications of the use of probiotics in combination with immunostimulants on fish intestinal morphology and microbiota. More morphofunctional studies are needed in order to correlate the nutritional and immune aspects of fish gut.