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

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

Use of in-vivo induced antigen technology to identify bacterial genes expressed during Solea senegalensis infection with Photobacterium damselae subsp. piscicida

The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important disease outbreaks affecting several fish species including flatfish Solea senegalensis (Kaup). Phdp is able to avoid host defences by invasion and intracellular survival in non-phagocytic cells, mainly epithelial cells. Virulence factors reported in Phdp include restricting complement-mediated activity, apoptosis of phagocytes caused by exotoxins secretion, iron acquisition mechanisms such as siderophores that enable the pathogen to obtain iron from transferrin and ability to bind haemin and antioxidant enzymatic activities capable to counteract superoxide radicals (Do Vale et al., 2005; Andreoni and Magnano, 2014). Commonly, genes expressed during pathogen infection are important for pathogenicity. In vivo-induced antigen technology (IVIAT) (Handfield et al., 2000) has been used to identify in vivo-induced genes using pooled sera from fish that have experienced photobacteriosis. Materials and methods Sera were obtained from surviving S. senegalensis specimens after sublethal infection with Phdp (Lg41/01) and subsequently pooled and adsorbed against in vitro grown Phdp Lg41/01 and Escherichia coli BL21 (DE3) cells and lysates according to Handfield et al. (2000). The efficiency of sera adsorption was evaluated based on the immunoreactivity after each adsorption step with whole and lysed Phdp cells grown in vitro. A genomic expression library of Phdp Lg41/01 was generated in E. coli BL21 (DE3) using pET-30 expression system (Novagen, San Diego, CA, USA). The expression library was probed with adsorbed and non-absorbed sera using immunoblot technique. Reactive clones of in vivo-induced and in vitro antigens were obtained, purified and their inserted DNA sequenced (Macrogen Europe, Amsterdam, The Netherlands). Nucleotide sequences were compared against the NCBI protein database using BLASTx. Results A progressive reduction in sera immunoreactivity against in vitro grown Phdp cells was detected after the adsorption rounds, especially after the first adsorption step. Thus, following adsorption steps substantially removed antibodies against in vitro expressed antigens and resulted in relative enrichment in antibodies recognizing in vivo expressed antigens. The library from Phdp Lg14/01 constructed in E. coli BL21 (DE3) consisted of approximately 6500 recombinants. A total of 117 clones were selected for their reactivity with pooled adsorbed and non-adsorbed sera from convalescent S. senegalensis specimens after a first round of screening. In a second screening, 14 out of 117 candidate clones showed positive reaction, among which two clones were clearly positive and two gave weak reaction against adsorbed sera. Predicted proteins codified by inserted sequences have intracellular and membrane cell location and are involved in virulence, synthesis of intermediary products, energy metabolism and gene replication. Inosine-5’-monophosphate dehydrogenase (IMPDH) and alkyl hydroperoxide reductase (AhpC) have been identified as in vivo induced antigens expressed during S. senegalensis infection with Phdp. Iron/manganese superoxide dismutase (Fe/Mn-SOD) and alanyl-tRNA synthetase (AlaRS) proteins have also been identified, though with weak signal. Discussion and conclusion Identification of immunogenic bacterial proteins during Phdp infection is essential for understanding bacterial pathogenesis and development of effective vaccines. AhpC peroxidase activity has a protective role by reducing hydrogen peroxide, peroxynitrite and organic hydroperoxides. Immunization with AhpC conferred protection against Helicobacter pylori infection (O’Riordan et al., 2012). IMPDH catalyzes the conversion of products essential in de novo synthesis of guanine nucleotides. Adequate levels of purine nucleotides are critical for cell proliferation, nucleic acid replication, cell signaling and as a biochemical energy source. This gene is an important therapeutic target against bacterial diseases (Shu and Nair,2008). In conclusion, different genes expressed during Phdp infection in S. senegalensis have been identified. Among them, IMPDH and AhpC have been identified as in vivo induced antigens expressed during S. senegalensis infection with Phdp. Thus, they are likely to play a role in the virulence of Phdp. The antigenic character of these proteins makes them potential targets for the development of new vaccines. References Andreoni, F., and Magnani, M., 2014. Photobacteriosis: Prevention and Diagnosis. Journal of Immunology Research, 2014: 1-7. Do Vale, A., Silva, M.T., dos Santos, N.M., Nascimento, D.S., Reis Rodrigues, P., Costa Ramos, C., Ellis, A.E., and Azevedo, J.E., 2005. AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils. Molecular Microbiology, 58: 1025-1038. Handfield, M., Brady, L.J., Progulske-Fox, A., and Hillman, J.D., 2000. IVIAT: a novel method to identify microbial genes expressed specifically during human infections. Trends in Microbiology, 8: 336-339. O’Riordan A.A., Morales V.A., Mulligan L., Faheem N., Windle H.J., and Kelleher D.P., 2012. Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine. Vaccine, 30:3876-3884. Shu, Q., and Nair, V., 2008. Inosine monophosphate dehydrogenase (IMPDH) as a target in drug discovery. Medicinal Research Reviews, 28:219-232.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Extractos del probiótico Shewanella putrefaciens Pdp11 protegen frente a la infección por RGNNV en cultivo celular

El agente etiológico de la retinopatía y encefalitis viral (VER) es el virus de la necrosis nerviosa (VNN). Aunque esta enfermedad puede causar una elevada mortalidad en numerosas especies de peces, no existen métodos efectivos para su tratamiento o prevención. La utilización de piensos funcionales es una estrategia novedosa para prevenir enfermedades de etiología infecciosa en acuicultura. El objetivo del presente trabajo ha sido evaluar in vitro la capacidad inmunoestimulante y antivírica frente a un aislado VNN de un extracto de la bacteria probiótica Shewanella putrefaciens Pdp11 (SpPdp11), aislada de dorada. En concreto, se ha cuantificado la transcripción de diferentes genes implicados en la respuesta inmune antiviral en células tratadas con este postbiótico, y se ha analizado su interferencia sobre distintas fases del proceso de infección vírica, analizándose tanto la supervivencia celular, como la multiplicación vírica. Los resultados muestran que el extracto de SpPdp11 es capaz de generar un estado antiviral en las células que las protege frente a la infección por VNN, por lo que este postbiótico es un buen candidato para incluir en los piensos de las especies susceptibles como estrategia de lucha frente a VNNProyecto de la Junta de Andalucía P18-RT-1067 y del Ministerio de Ciencia e Innovación PID2020-113637RB-C22 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

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

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

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

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