Uso del probiótico Shewanella putrefaciens Pdp11 en el cultivo de Solea senegalensis: implicaciones sobre la microbiota intestinal

Actualmente, la acuicultura es una industria en expansión. Entre las especies que se están introduciendo destaca el lenguado, Solea senegalensis, cuyo cultivo en cautividad no acaba de ser económicamente rentable debido fundamentalmente a las mortalidades producidas por infecciones bacterianas. Los antibióticos presentan una serie de limitaciones ya conocidas, siendo los probióticos una de las opciones más consideradas. Estudios previos en nuestro laboratorio han mostrado que la cepas S. putrefaciens Pdp11 (Pdp11) es capaz de inhibir el crecimiento de bacterias patógenas de peces, por lo que se planteó su uso como probiótico y el estudio de los efectos sobre la fisiología de S. senegalensis así como la potencialidad en la prevención de infecciones. La incorporación de Pdp11 a la dieta de S. senegalensis induce una modulación de la microbiota intestinal y siendo capaz de colonizar el intestino del animal y presentando mayor diversidad en la microbiota, menor susceptibilidad a infecciones y mejor composición corporal, sobre todo relativo a la cantidad de ácidos grasos beneficiosos. El estudio de la forma más adecuada de administración del probiótico mostró que las células no liofilizadas tienen un tiempo de permanencia superior en el intestino y su administración aumenta la diversidad de la microbiota intestinal al tiempo que reduce la presencia de patógenos oportunistas en la misma. Por este motivo, se consideró la forma no liofilizada como la más idónea para los estudios posteriores. La administración de Pdp11 a ejemplares de S. senegalensis sometidos a estrés por alta densidad o tratamiento con oxitetraciclina (OTC) permitió determinar la presencia de una microbiota intestinal y expresión génica diferencial en dichos ejemplares, con sobreexpresión de genes relacionados con el sistema inmune, mejoría de la histología intestinal y protección frente a infecciones por V. harveyi y V. parahaemolyticus. Por otro lado, la administración de OTC produce cambios radicales en la microbiota, pero éstos son menos acusados cuando va junto con Pdp11 apareciendo en la microbiota especies beneficiosas y desapareciendo otras patógenas. Además, el aumento de la expresión de genes relacionados con el estrés oxidativo observado tras la administración de OTC no ocurre cuando se aplica simultáneamente con Pdp11. Por último, se ha estudiado el efecto de la administración del probiótico durante la fase larvaria, fase muy susceptible a infecciones. La aplicación de forma continuada durante esta fase supone una modulación de la microbiota la cual se enriquece en especies de Vibrio. Del mismo modo, la aplicación sólo durante la fase de metamorfosis (desde el día 10 al 30 después de la eclosión) produce una modulación de la microbiota desde el inicio y a la ausencia de patógenos como P. damselae subsp piscicida. Por tanto, Pdp11 se puede considerar como un probiótico adecuado para S. senegalensis, que limita la presencia de patógenos en el intestino, atenúa los efectos del estrés y protege en estadios larvarios

Use of water soluble extracts from ulva sp. by probiotics and fish bacterial pathogens

BACKGROUNDS The potential of seaweeds as dietary components is considered for a wide range of cultured fish species. In this context, Ulva is investigated as a good source of protein, minerals and vitamins. In addition, of probiotics are used to improve fish growth and modulate immune system and intestinal microbiota. To promote probiotics colonization and maintenance in the intestine, prebiotics are included in fish diets. Prebiotics are indigestible substrates used as energy sources for gastrointestinal microbiota, with a positive effect on the nutrition and health status of the host. In the present work, ability of selected probiotic and fish pathogen strains to use water soluble extracts from Ulva as nutrient source has been evaluated. MATERIALS AND METHODS Water-soluble extracts from Ulva sp. prepared by sonication of dehydrated samples were used to supplement minimum medium (M9). Probiotics and pathogens growth was evaluated based on the optical densities measured with a microplate reader. RESULTS AND CONCLUSIONS Probiotics were able to grow in minimum medium using water soluble extracts as nutrient source. On the other hand, P. damselae subsp. piscicida and V. harveyistrains were also able to grow with Ulvaextracts as nutrient source. However, incubation time to reach maximum growth was longer. Although Ulva extract may support growth of both probiotics and pathogen bacteria, faster growth of probiotics may help for the establishment of probiotic populations in the intestinal environment. In addition, beneficial effects on growth performance, gut microbiota, immunity and disease resistance of Ulva for Solea senegalensisare being studied. This work was funded by INIA, Ministerio de Economía y Competitividad and FEDER (RTA201400023 C0202).This work was funded by INIA, Ministerio de Economía y Competitividad and FEDER (RTA201400023 C0202).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Detection of transposons modifying genome background in probiotics

The study of probiotic microorganisms is very interesting in the aquaculture field. Administration of live microorganisms in adequate amounts confers some benefits to the host (Kechagia et al. 2013). Even if Shewanella putrafaciens include pathogens and saprophytic strains related to fish spoilage and fish infection (Esteve, Merchán, and Alcaide 2016). The Pdp11 strain of Shewanella putrefaciens has been proved to provide beneficial effects in Sparus aurata (Chabrillón et al. 2005) and Solea senegalensis (Rodrigáñez et al. 2008). Studies focused on Pdp11 could hed light on the origin of this probiotic character. We have designed a bioinformatic workflow to detect transposons in the newly sequenced Pdp11 genome (Tapia-Paniagua et al, in press). Their presence interrupting genes account for a contribution to its probiotic character due to the lost of virulence or the gain of probiotic effect. The workflow was developed in Ruby programming language and provides: the genomic localisation of known transposons, host coding regions disrupted by complete transposons or their repeated insertion sequences, and transposons and coding regions disrupted identifiers, to stablish the putative functions of Pdp11 that could be affect by the transposons disruption. These results would support new possible hypothesis about the Pdp11 probiotic character since 14 coding regions related to S. putrefaciens were disrupted by transposons, 4 of which are directly involved in pathogenic mechanisms. This work was supported by co-funding by the European Union through the European Regional Development Fund (ERDF) 2014-2020 "Programa Operativo de Crecimiento Inteligente" together with Spanish AEI "Agencia Estatal de Investigación" to grants RTA2013-00068-C03, AGL2017-83370-C3-3-R and RTA2017-00054-C03-03.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Intraspecifična karakterizacija izolata Vibrio alginolyticus prikupljenih iz sustava akvakulture i morskih biotopa u Tunisu korištenjem PCR-RFLP, ECP i OMPS profiliranja.

A total of 54 presumptive Vibrio alginolyticus strains isolated from a variety of Tunisian marine biotopes, such as seawater, sediment samples from some bathing areas, and aquaculture farms, were characterized and identified by several biochemical tests. DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, extracellular products (ECP) and outer membrane protein (OMP) profiling were used to evaluate their usefulness as a tool to investigate the Vibrio alginolyticus diversity within this complex group. Results showed that there is great heterogeneity in the diversity observed via the PCR-RFLP method related to the number of genotypes generated by the two enzymes SduI and FaqI tested. This heterogeneity was observed not only according to the origin (seawater, sediment, fish and bivalve aquaculture farms) but also within the same type of sample. The two other methods, ECP and OMP resulted in 32 and 26 profiles, respectively. The discriminatory index determined in this study highlighted the good ability of the PCR-RFLP method to discriminate similar V. alginolyticus strains.Ukupno 54 vjerojatna soja Vibrio alginolyticus izolirana su iz raznih morskih biotopa u Tunisu, iz morske vode i sedimenta s nekoliko kupališta i uzgajališta te su karakterizirani i identificirani pomoću nekoliko biokemijskih testova. Istraživana je prikladnost metoda kao što su analiza polimorfizma duljine restrikcijskih fragmenata PCR proizvoda (PCR-RFLP), profiliranje izvanstaničnih proizvoda (ECP) i proteina vanjske membrane (OMP) kao alata za istraživanje raznolikosti Vibrio alginolyticus unutar ove složene skupine. Rezultati su pokazali da postoji velika heterogenost u raznolikosti određenoj metodom PCR-RFLP koja se odnosila na broj genotipova generiranih s dva testirana enzima SduI i FaqI. Ova heterogenost je uočena ne samo prema podrijetlu (morska voda, sediment, uzgajališta riba i školjkaša), već i unutar istog tipa uzorka. Druge dvije metode, ECP i OMP rezultirale su s 32, odnosno 26 profila. Indeks diskriminacije utvrđen u ovoj studiji istaknuo je dobru sposobnost PCR-RFLP metode u razlikovanju sličnih sojeva V. alginolyticus

Characterization of Vibrio harveyi strains recovered from diseased farmed Senegalese sole (Solea senegalensis)

Aim: To characterize 16 Vibrio harveyi strains isolated from different epizootic outbreaks affecting farmed Senegalese sole. Materials and Results: The Vibrio harveyi strains tested have broad phenotypic diversity based on their biochemical and exoenzymatic patterns, outer membrane proteins (OMP), extracellular product (ECP) patterns and presence of prophages. Lethal dose 50 (LD50) of the strains and in vitro antagonism tests with two probiotic strains were also determined. The OMP analysis revealed three different patterns (A, M and V). The electrophoretic analysis of the ECP showed two different groups. All strains considered virulent based on their LD50 exhibited the same protein pattern in their ECP (pattern I), while all nonvirulent strains showed a different profile (pattern II). About 32% of the tested strains were positive for prophages, although a clear relationship between virulence and the presence of prophages has not been established. Conclusions: The results obtained have shown differences between virulent and avirulent strains isolated from diseased farmed Senegalese sole based on the protein patterns of their ECP. However, a clear relationship between virulence and presence of prophages has not been established. Significance and Impact of the Study: The differences observed between virulent and nonvirulent strains could be used to design prophylactic strategies against diseases caused by V. harveyi in farmed Senegalese sole

The effect of spray‐dried porcine plasma on gilthead seabream (Sparus aurata) intestinal microbiota

The effect of spray‐dried porcine plasma (SDPP) on the intestinal histological organization and autochthonous microbiota composition was evaluated in Sparus aurata. Fish were fed a basal diet (51 g/kg protein, 17 g/kg fat, 20.6 MJ/kg gross energy) and a diet containing 3 g/kg SDPP for 95 days (initial body weight, BW = 9.5 ± 0.2g, mean ± SD). The inclusion of SDPP promoted growth (p .05) between both groups. Intestinal microbiota was dominated by Proteobacteria (>85%) and Firmicutes (5%–12%), whereas Bacteroidetes never represented more than 1.5%. γ‐Proteobacteria, and Bacilli and Clostridia were the predominant classes. The short administration of SDPP (20 days) resulted in changes in microbiota diversity and richness associated with an increase in the sequences of the genus Lactobacillus and to a decrease in the genus Vibrio, whereas these changes were reverted at 95 days. Intestinal goblet cell density was not correlated to microbiota diversity and richness changes rather than to the immunostimulatory effect of the SDPP.info:eu-repo/semantics/acceptedVersio

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.This research was funded by Unicaja Foundation

Modulation of the intestinal microbiota and expression of gene of senegalese sole (Solea senegalensis, Kaup 1858) after dietary administration of oxytetracycline.

Oxitetracycline and probiotics (Pdp11) administration to sole juveniles showed important changes in the species composition of intestinal microbiota that established a relationship between these species and gene expression

Cambios en la microbiota intestinal de Sparus aurata tras la administración de dietas suplementadas con D. hansenii e INMUNOTEC

Abstract Effects on the intestinal microbiota of Sparus aurata after administration of diets supplemented with different doses of Debaryomyces hansenii or INMUNOTEC have been evaluated. The results revealed significant changes at metagenomical level and it could be related to metabolic changes, especially in the case of the diet supplemented with the highest dose of IMMUNOTEC. Resumen En este estudio se analizan los efectos que la inclusión en la dieta de distintas dosis de Debaryomyces hansenii e INMUNOTEC tienen sobre la microbiota intestinal de ejemplares de Sparus auratatras. Los resultados obtenidos revelaron cambios a nivel metagenómico, que podrían estar asociados con cambios metabólicos, especialmente en el caso de la dieta suplementada con la dosis más elevada de INMUNOTEC.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Proyecto DIETAplus, JACUMAR (Junta de Cultivos Marinos, MAPAMA). Cofinanciado fondos FEM

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.Funding for open access publishing: Universidad Málaga/CBUA. This work was funded by the proyectos de investigación PID2020-113637RB-C21 and PID2020-113637RB-C22 funded by MCIN/AEI/10.13039/ 501100011033 and is part of the ThinkInAzul programme supported by MCIN with funding from European Union Next Generation EU (PRTR-C17. I1) and by the Comunidad Autónoma de la Región de Murcia-Fundación Séneca (Spain)