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

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

Shewanella putrefaciens Fish pathogenic strains contain plasmids that are absent in the probiotic strain s. Putrefaciens pdp11

Probiotics are live microorganisms that confer a health benefit to the host when administered in adequate amounts. Shewanella putrefaciens Pdp11 has been described as a probiotic for farmed fish species such as Solea senegalensis and Sparus aurata. In contrast, other strains of S. putrefaciens have been described as pathogenic for other cultured fish damage of the mouth, extensive skin discoloration, exophthalmia, ascites and bad odour. The S. putrefaciens group was recovered from freshwater samples taken at the L′Albufera system, along autumn– winter 2015. Its counts significantly increased in freshwater parallel to hypoxia and temperature rising. Shewanellae strains were identified as S. putrefaciens and S. xiamenensis by 16S rRNA gene sequencing. These isolates recovered from sick eels or freshwater were virulent for European eel by IP challenge (LD50 106 CFU g−1 body weight. The plasmids plays an important role in the genes transfer and insertion then there can been implicated in antibiotics resistance, degradative pathway and pathogenicity characteristics. The genetic variation conducted by plasmid could induce an impact in probiotic proprieties. In this research, we searched the present or absent of plasmids in pathogenic and probiotic strains of S. putrefaciens. As well as, this plasmids implication in development of virulence factors. The probiotic strain S. putrefaciens Pdp11 did not present plasmid, which was only found in two of the five pathogenic strains. The results allowed us to discard the probiotic Pdp11 could present a pathogenic characteristic as the TA type II system as a virulence factor and its self-regulating characteristics, which may be behind its probiotic nature, making the Pdp11 strain unique in comparison to other S. putrefaciens strains

Changes in intestinal microbiota and gene expression of immune system parameters caused by dietary administration of alginate and Shewanella putrefaciens Pdp11 in Senegalese sole (Solea senegalensis Kaup, 1858) specimens

Dietary administration of sodium alginate single or mixed with Pdp11 modulated the intestinal microbiota diversity of sole juveniles. Both treatments promoted a change in the species present in the gut in comparison with control group. In addition, oral administration of alginate promoted the appearance of an intestinal microbiota highly influenced by the prebiotic presence. Synbiotic administration of alginate with Pdp11 promoted a more pronounced decreasing in the similarity index and in the species richness. Both treatments modulated the expression of genes related with immune system functions

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

ABSTRACT: 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 tnf?, il-6, lys-c1, c7, hsp70, and hsp90aa in the intestinal samples based on the diet. On the contrary, hsp90ab, gp96, cd4, cd8, il-1?, 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 dietThis study was supported by MINECO (grant no. AGL2014-51839-C5-2-R and AGL2017- 83370-C3-3-R) and FEDER funds

Microbiome network analysis in skin and gills of Sparus aurata fed with Nannochloropsis gaditana microalgae

1416 and 694 ASVs in gills and skin respectively were preserved in the taxonomic analysis after filtering. The predominant phylum in gills was Proteobacteria (~50%) in the control and hydrolyzed groups and reached 70.58% in the raw diet. The Bacteroidota phylum was the most represented and Firmicutes and Actinobacteria were close to 3% in all treatments in this tissue. However, in all three different treatments, the phyla abundance was similar in skin samples, ~90% of which corresponded to Proteobacteria. Bateroidota (5.5%–5.9%), Firmicutes (1.8%–2%) and Actinobacteria (<1%) constitute the other phyla. In gills, 13 significantly higher ASVs were obtained in the control (such as Achromobacter, Acidobacter) versus 4 whose abundance was higher in the hydrolyzed diet. The number of ASVs that differed significantly between the gill microbiota in the control vs. raw group amounted to 70, most of them being ASVs corresponding to the genus Shewanella (43) higher in the raw diet. Nevertheless, in the skin samples, Control group showed a significant increase of abundance related to Acinetobacter, Achromobacter, Pseudomonas, Shewanella, Vibrio and Sphingomonas among others. In the hydrolyzed group, the most significant abundance was associated with the genus Pseudomonas, Vibrio, Pseudoalteromonas, Ralstonia o Cutibacterium. In the case of the skin raw samples, there were ASVs corresponding to the genus Acinetobacter, Streptococcus or Pseudoalteromonas that were significantly different respect of control. Taking ASV abundance matrix for each diet and tissue, 6 co-occurrence networks were constructed. In all treatments in gills, Acinetobacter was a central genus in the network, and exhibited a negative correlation with Polaribacter. Besides, in the raw group, Polaribacter also showed negative correlation with Aeromonas, Pseudomonas and Francisellacea. This work shows that Acinetobacter has a key role in the balance of mucosa microbiota and was in co-exclusion with Polaribacter.This work was funded by research projects for young researchers, CEIMAR 2019 (Evaluation of hydrolysates of Nannochloropsis gaditana for use in high value-added finishing feed for farmed gilthead sea bream (Sparus aurata) (NAN2BREAM))

Biofilm inhibition of pathogenic strains by extracellular products (ECPS) of Shewanella.sp Probiotic

Shewanella putrefaciens Pdp11 and V.proteolyticus DCF 12.2 are strains isolated by our research group. Recent works have been focused in the identification of substances as alternative of anti-biofilm methods and their implication in surface attachment inhibition. In this research, S. putrefaciens Pdp11 and V.proteolyticus DCF 12.2 have been cultured under different growth conditions (temperature, culture media and during 24 and 48 hours of incubation) and their extracellular products (ECPs) have been extracted and tested as potential postbiotics that affect the biofilm formation of several fish pathogenic strains. This assay results evidence that Pdp11_Pmix_2324 ECPs have showed the most impact in the biofilm formation of pathogenic strains. Therefore, ECPs secreted by Pdp11 and V.proteolyticus DCF 12.2 are implicated in the inhibition to adhesion of pathogens on surfaces.Proyecto de investigación PID2020-113637RB-C22. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Identificación del probiótico Shewanella putrefaciens Pdp11 mediante PCR a través del transposón único que interrumpe al gen que codifica para la proteína fenazina.

Shewanella putrefaciens Pdp11 es una cepa descrita como probiótico en peces de importancia acuícola. La secuenciación de su genoma ha permitido establecer comparaciones a nivel genómico con otras cepas patógenas pertenecientes al mismo género. Como parte del estudio del genoma de Pdp11 se han identificado la presencia de 6 transposones y su ausencia en 7 cepas de Shewanella.sp (Pérez Gómez Olivia et al., 2021). En este trabajo se plantea el uso del transposón que interrumpe la proteína PhzE, implicada en la biosíntesis de la fenazina, para la identificación especifica de SpPdp11. Así como, la puesta a punto de la PCR para determinar la sensibilidad de los cebadores en la identificación del probiótico. En los cultivos acuícola, Shewanella putrefaciens Pdp11 se administra a los peces mediante su dieta en concentraciones de 10^9 ufc/gr de pienso, este trabajo permitiría la futura identificación y cuantificación del probiótico en muestras intestinales, así como el estudio del potencial de colonización del mismo.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Análisis de la microbiota de branquias de Sparus aurata tras la inclusión de la microalga Nannochloropsis gaditana en la dieta

The inclusion of microalgae in the diet of farmed fish such as sea bream (Sparus aurata) is proposed as a sustainable alternative to the traditional diet with fishmeal, since numerous benefits have been reported to the fish. The gills act as the first defence barrier against pathogenic organisms and have an autochthonous microbiota that contributes to this function, in the fact that it is important to know if diets supplemented with microalgae can cause any alteration. In this work, three diets (control, raw Nannochloropsis gaditana seaweed and hydrolyzed seaweed) were fed to juvenile sea bream for 90 days, later they were sacrificed and their organs frozen. 16S rRNA was sequenced on an Illumina® platform and bioinformatic analysis with MOTHUR was performed. A significant increase in the richness index (Chao1) was shown in the microbiota of the gills of the fish fed N. gaditana. The most significant changes occurred in the fish fed the raw algae, with a notable increase in the genus Shewanella in this group.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Assessment of in vitro postbiotic capabilities of the probiotic Shewanella putrefaciens pdp11 growing under different cultivation conditions containing microalgae dietary supplements widely used in aquaculture.

Probiotics have been established as potential tools to improve host health and environmental quality in aquaculture. For example,Shewanella putrefaciens Pdp11 (SpPdp11), a strain described as a probiotic for use in aquaculture (reviewedby Cámara et al. 2020). Despite the proven health benefits of probiotics, recent evidence suggests that bacterial viability is not necessary to attain effects. Thus, postbiotics have emerged as potential opportunities in the field of functional foods. The International Scientific Association for Probiotics and Prebiotics (ISAPP) convened a panel that defined postbiotics as a “preparation of inanimate microorganisms and/or their components that confer a health benefit to the host”. The present work evaluates the potential postbiotic, as extracellular products (ECPs), of SpPdp11 grown undern different cultivation conditions. These included different culture media composed of a blend of microalgae, to observe a possible synergistic effect. The ECPs obtained were evaluated for their cytotoxicity against different fish cell lines, enzymatic and antibacterial activity, and their effect on biofilm formation by several fish pathogensproyecto PID2020-113637RB-C22 de investigación financiado por MCIN/AEI/10.13039/ 50110001103