Study of the intestinal microbiota of Solea senegalensis specimens after the administration of the probiotic Shewanella putrefaciens SpPdp11 by Next Generation Sequencing

Introduction Probiotics are defined as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host [1]. The use of probiotics is a key tool to protect farmed fish, in many cases predisposed to stress and/or infection under intensive culture conditions. In this way, Shewanella putrefaciens Ppd11 (SpPdp11) is a microorganism applied to farmed fish such as Solea senegalensis and Sparus aurata that has demonstrated probiotic effect such as promotes the growth and a better efficiency of feed utilization, stimulating the immune system of S. senegalensis and S. aurata, and the stress tolerance of S. senegalensis specimens to high stocking densities [2]. In addition, its capability to modulate the intestinal microbiota of these farmed fish has also been demonstrated using Denaturing Gradient Gel Electrophoresis (DGGE). At present, the Next Generation Sequencing (NGS) methodology is a better and more sensitive way to evaluate the composition of the microbiota and to analyze the effects on it of different factors, such as the dietary supplementation with a probiotic. In this context, this is the first time that the effect of the probiotic on the intestinal microbiota of S. senegalensis is analyzed using the NGS methodology.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Inhibición en la formación de biofilm por efecto de productos extracelulares de la potencial cepa probiótica shewanella putrefaciens Pdp11

Extracellular products from Shewanella putrefaciens Pdp11 have been obtained under different conditions of temperature and time of incubation, demonstrating its capability to exert an inhibitory effect in biofilm formation of fish pathogens such as Tenacibaculum soleae, Tenacibaculum gallaecium, Vibrio anguillarum and Aeromonas hydrophila. No effect has been observed for Tenacibaculum maritimum biofilm formation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The probiotic Shewanella putrefaciens PDP11 target virulence factors by modulating quorum sensing inhibition

Bacteria communicate with each other by producing signal molecules and regulating the production of virulence factors that have importance in pathogenicity. Quorum sensing (QS) is a bacterial communication mechanism based on the perception of population density and secretion of determining signal molecules called autoinducers (AI) such as the case of Acylhomoserine lactones (AHLs). AHLs-mediated QS processes seem to be common in the marine environment and among marine pathogenic bacteria, which pathogenesis could be mitigated by probiotics, among others. Probiotics are defined as live microbial cells that confer health benefits to the host and some of their mechanisms include the production of antagonistic compounds that are inhibitory toward pathogens. In this sense, Shewanella putrefaciens Pdp11, a strain described as a probiotic for use in aquaculture, has been analysed to mediate QS processes by quorum-quenching assays using synthetic AHLs. The enzymatic activity is estimated at around 80% and 30% for C8- and C10-HSL, respectively, while the rest of AHLs tested were not degraded by the Pdp11 strain. It would be an interesting feature of the probiotic Pdp11 strain since these AHLs are related to facilitating microbial adhesion by promoting biofilm formation among other virulence factors related to pathogens. On the other hand, a distinctive feature of AHL inactivated by lactonase is that it can be reactivated by acid treatment. In this way, little C8-AHL was recovered when it is extracted to pH2, which indicates the enzyme activity is not derived from the hydrolysis of the lactone ring derived from the action of lactonases, suggesting the enzyme activity in Pdp11 could be an AHL-acylase. The potential QQ activity of Pdp11 was unknown so, these preliminary studies led to a further as another promising probiotic QQ tool for aquaculture

Adsorption of salmonella in clay minerals and clay-based materials

A series of clay minerals and clay-based materials have been tested to eliminate one of the most dangerous bacteria we can find in the water: Salmonella. It has been proven that the use of clays and their PCH materials can be a suitable method for removing Salmonella from water. The results of this initial study show that all the materials analyzed have great salmonella adsorption capacities ranging from the lowest value observed in the mont-PCH sample (0.29 × 1010 CFU g−1 ) to the highest value observed in the natural palygorskite sample (1.52 × 1010 CFU g−1 ). Macroporosity, accessible external surface area, and the presence of silanol groups in the external surface of the particles appears to be the controlling factors for Salmonella adsorption capacity while it seems that the structural characteristics of the clay minerals and their respective PCH does not affect the adsorption capacityThe authors wish to thank the project BIA2017-82391-R for the financial suppor

Potential postbiotic activities of extracellular products of probiotic bacteria from gilthead seabream gastrointestinal tract

Probiotics have been established as a potential tool for improving gut health and environmental quality in aquaculture. However, recent evidence suggests that bacterial viability is not necessary to attain the beneficial-promoting effects. In this way, postbiotics have emerged providing a potential opportunity in the field of functional foods. They are soluble factors resulting from the metabolic activity of a probiotic or any released molecule, including short-chain fatty acids, enzymes, extracellular products, etc. that can have interesting properties inducing biological responses on health similar to probiotics while avoiding the administration of live microorganisms. Interestingly, its production can be affected by different factors such as the components of the culture media among others. Information on this type of postbiotic activities is very scarce, especially in the case of aquaculture, so the evaluation of the nutraceutical use of postbiotics to improve health management in fish and other cultivated aquatic organisms is an emerging area of research in aquaculture. In a previous work, we characterized four potential probiotics that were isolated from the gastrointestinal tract of Sparus aurata specimens fed with a diet containing a blend of microalgae. This diet involves a selection pressure on the intestinal microbiota of the fish that will be used to achieve enrichment in bacteria with a set of extracellular enzymatic activities capable of metabolizing and mobilizing the components of the diet enriched with microalgae. Here, we investigate the postbiotic potential of the extracellular products (ECPs) obtained from the four candidate probiotics grown on different microalgae-supplemented medium, and evaluate its enzymatic and antibacterial activity, and its cytotoxicity against the SAF-1 cell line. Our objective is to select different ECPs with a variety of activities that help the digestive process of seabream, with the aim to be included in aquafeeds.This work was funded by Junta de Andalucía (grant no. P20_00085) and co-financed by FEDER funds

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)

In vitro assessment of antagonistic activities of isolates from gilthead seabream (Sparus aurata) gastrointestinal tract fed microalgae supplemented diet

The use of probiotics has emerged as a sustainable alternative to antibiotics in the control of infectious diseases, favouring fish health management, growth performance and feed utilisation, among others. microalgae represent an interesting source of nutrients and functional ingredients for aquafeeds. However, their digestibility is often limited by the presence of anti-nutritional factors or absence of appropriate enzymatic activities in the fish gastrointestinal tract (GIT). The aims of the present work were to isolate potential probiotics from the GIT of Sparus aurata fed with a diet containing 25 % microalgae and characterize their antagonism against fish pathogens. Altogether, 117 strains were isolated from juvenile seabream (146.8 ± 16.4 g) and screened for hydrolytic enzyme activities. Results showed that 48 %, 41 %, 77 % and 30 % of isolates were able to hydrolyse protein, lipids, collagen and starch, respectively. Moreover, 46 %, 8 % and 57 % of isolates exhibited the ability to degrade phytate, tannins and cellulose, respectively. Based on these results, a total of 32 isolates were selected for inhibitory activity against several fish pathogens assessment. Inhibition against Aeromonas hydrophila and Vibrio anguillarum was detected in 38 % of the isolates, whilst 44 % and 47 % inhibited P damselae subsp. damselae and P. damselae subsp. piscicida, respectively. Inhibition abilities were detected in the isolates when tested against Tenacibaculum species. Thus, 56 % inhibited Tenacibaculum maritimum; 63 % T. soleae and 22 % T. gallaecium. Overall, results showed that three strains display ability to hydrolyse 4 of the assayed substrates and produce inhibition against 8 fish pathogens, and two strains are capable to hydrolyse 5 substrates and inhibit 8 fish pathogens. Selected strains show characteristics to be considered for further characterization as potential probiotics in gilthead seabream aquaculture and microalgae-supplemented aquafeeds.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

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

Expresión de genes del sistema inmune innato del lenguado (Solea senegalensis) alimentados con Ulva ohnoi durante la infección con P. damselae subsp. piscicida

Las algas son fuente de proteínas y numerosas sustancias biológicamente activas. En el caso del género Ulva, son muy escasos los estudios sobre los efectos en el sistema inmune. En el presente trabajo se ha evaluado la respuesta a la infección por P. damselae subsp. piscicida de genes del sistema inmune innato en lenguados (Solea senegalensis) alimentados con una dieta suplementada con Ulva ohnoi. Los ejemplares de lenguado con un peso inicial de 10,7g se alimentaron durante 3 meses con una dieta control y otra conteniendo 5% de polvo seco de U. ohnoi. Transcurrido este periodo, los peces (54,17 g) se sometieron a infección frente a P. damselae subsp. piscicida y se determinó la expresión relativa de los genes que codifican para componentes del sistema del complemento, y citoquinas inflamatorias en hígado y riñón de los lenguados infectados. Los resultados obtenidos muestran, tras la infección, un incremento en la expresión de los genes que codifican para los componentes C1q4, C3, C4/1 y C9 así como TNF en el hígado de los peces que recibieron el alga. Por el contrario, se observaron niveles inferiores de expresión de los genes que codifican para las citoquinas IL1beta, IL6 e IL8b en el hígado de los ejemplares que recibieron el alga. A nivel del riñón, los lenguados alimentados con la dieta conteniendo Ulva sólo presentaron diferencias respecto al grupo control en la expresión de los genes que codifican para IL8b y C9. La activación del sistema del complemento da como resultado la formación del complejo de ataque de membrana, que tiene un papel esencial en la eliminación de microorganismos patógenos. Por otro lado, una respuesta inflamatoria controlada puede contribuir a limitar el daño derivado de la infección por P. damselae subsp. piscicida, en peces alimentados con la dieta conteniendo Ulva.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Ministerio de Ciencia de Innovación y FEDER (Ref. RTA2014 00023 C02