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The human gastrointestinal tract (GIT) is a very complex ecosystem involving a continuous interaction between nutrients, host cells and microorganisms. Recently, we have discovered a new family of immunomodulatory peptides that are encrypted within the sequence of a precise extracellular protein, which is secreted by the lactic acid bacterium Lactobacillus plantarum (Patent WO 2013034795 A1) (1). The genetic information about commensal/probiotic bacteria and gut microbiomes that is present in public repositories is huge, with only a list of 10 million unique bacterial proteins available mainly from the METAHIT and HMP projects (http://gigadb.org/dataset/100064). Our aim is to explore these 10 million unique proteins to obtain information on encrypted peptides which might have immunomodulatory bioactivity on host cells. With this in mind we have developed the MAHMI (Mechanism of Action of the Human Microbiome) database, a unique resource that provides comprehensive information about the sequence of potential immunomodulatory and antiproliferative peptides encrypted in the proteins produced by the human gut microbiota. Fifteen peptides were chosen by its potential immunomodulatory bioactivity. Selected peptides were assayed in vitro at a final concentration of 10 µg/mL using monocyte derived cells (Mo-DCs). Our bioinformatics/in vitro methodology allowed selection of new inmunomodulatory peptides encrypted in the human intestinal bacteria metaproteome. HM14 signaled through an unknown receptor, and imprinted anti-inflammatory traits in immune cells as evidenced by RNASeq and flow cytometry. The mechanisms used by this kind of peptides may be useful for the treatment of inflammatory diseases

[Introduction] Bifidobacteria and lactobacilli include both probiotic strains and representative microorganisms of the human gut microbiota, and their presence has been associated to a proper immune development and maturation. In addition, several independent works report the anti-inflammatory properties of several strains of those groups. However, we are far from understanding the molecular interplay behind/supporting these beneficial interactions. [Objective] To study the effect of different fractions of lactobacilli and bifidobacteria in human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors and patients with Inflammatory Bowel Disease (IBD) at the time of diagnosis. [Methods] The fractions were obtained from strains of Bifidobacterium longum NCIMB 8809, Lactobacillus rhamnosus GG and Lactobacillus acidophilus DSM 20079: DNA, surface-associated proteins, whole cell soluble extract and extracellular proteins. Different concentrations of the fractions were incubated with PBMCs, and key T-cell cytokines, T regulatory cell differentiation and RNASeq of CD4+ cells were performed. [Results] Among the extracts, extracellular proteins from L. acidophilus DSM 20079 increased IL-2 production by PBMCs isolated from healthy donors, and were able to increment the proportion of functional T regulatory cells. Data on the signaling mechanisms involved were obtained by RNASeq of CD4+ Th cell subpopulations. Finally, this fraction decreased drastically the production of the pro-inflammatory mediator TNF-alpha and induced increments of IL-12 in PBMCs isolated from IBD patients at the diagnosis. [Conclusions] Extracellular proteins from strain L. acidophilus DSM 20079 had a positive impact on the immunological status of both healthy donors and IBD patients. This fraction will be on the basis of new clinical studies focused in the immunomodulation of the human host targeting pro-inflammatory effectors and the T-cell regulatory response. This fraction may repres

n los últimos años se está revelando la relación entre el metabolismo de sales biliares y el colesterol, la microbiota intestinal y su implicación en la salud. A diferencia de otras localizaciones, el microbioma del tracto biliar apenas ha sido estudiado. En este trabajo se ha realizado un análisis metagenómico filogenético y funcional de muestras de bilis humana, además de un análisis metabolómico, con el fin de caracterizar el microbioma biliar de donantes de hígado y pacientes con colelitiasis, e identificar posibles disbiosis asociadas a esta patología. Se obtuvieron muestras de bilis de donantes de hígado y pacientes con colelitiasis, de las que se extrajo el ADN biliar siguiendo un protocolo optimizado. Se llevó a cabo la secuenciación masiva de amplicones del ADNr 16S mediante la tecnología Illumina. Además, se realizó secuenciación masiva por shotgun y análisis de metagenómica funcional de tres muestras de bilis de sujetos sanos. El análisis metabolómico se realizó utilizando técnicas de NMR. Los resultados mostraron una gran diversidad microbiana presente en este tipo de muestras, con presencia de 4 filos mayoritarios: Firmicutes, Bacteriodetes, Actinobacteria y Proteobacteria. El análisis estadístico mostró diferencias significativas en la abundancia relativa de diversos taxones presentes en la bilis de ambos grupos. En concreto, secuencias de la familia Propionibacteriaceae fueron más abundantes en la bilis de los donantes de hígado, mientras que en pacientes con colelitiasis fueron más frecuentes las de las familias Bacteroidaceae, Prevotellaceae, Porphyromonadaceae y Veillonellaceae. El análisis funcional mostró que la asignación a las principales categorías funcionales del COG es similar a la del microbioma intestinal humano. Por último, el análisis metabolómico nos permitió asociar diferentes perfiles metabólicos a los grupos de individuos objeto de estudio.

[ES] El estudio de los probióticos ha cobrado una gran importancia en los últimos años debido a su papel en el mantenimiento de una microbiota intestinal equilibrada, así como en el tratamiento de ciertas enfermedades, incluidos ciertos tipos de cáncer. En este trabajo, se ha analizado el efecto anti-proliferativo de un extracto bacteriano obtenido a partir de la cepa probiótica Lactobacillus acidophilus sobre la línea celular HT29. Este efecto se ha detectado con una técnica de impedancia no invasiva que permite cuantificar la proliferación celular. Además, se ha realizado un análisis del ciclo celular mediante citometría de flujo, ya que una posible hipótesis era que este extracto afectase a la división celular. Finalmente, se observó un efecto sobre la línea celular dependiente de la concentración. Aunque dicho efecto se atribuyó inicialmente a un descenso en la proliferación, los resultados de citometría no parecen reflejar esta idea y dan pie a nuevas hipótesis, incluidas una alteración en la morfología/adhesión celular o un efecto anti-proliferativo que no se detectaría debido a la aparición de mecanismos de resistencia. Además, un segundo análisis con citometría de flujo descarta su efecto sobre la viabilidad celular. Por otro lado, la elevada complejidad del extracto requiere de un fraccionamiento previo, por ejemplo mediante cromatografía líquida, para poder identificar qué moléculas son responsables del efecto observado. Antes de llevar a cabo esta operación, es necesaria una optimización de las condiciones de separación, lo cual se presenta en este trabajo utilizando la técnica de cromatografía en capa fina. De esta forma, se consiguió establecer condiciones óptimas tanto para cromatografía en fase reversa con mezclas de acetonitrilo y agua, como para fase normal con mezclas de hexano y acetato de etilo, acetato de etilo y metanol, y cloroformo y metanol., [EN] The global interest in probiotics has gained a great relevance over the last few years due to its importance in both the maintenance of a balanced gut microbiota, and the treatment of several diseases, including certain type of cancers. In the present study, the anti-proliferative effect from a bacterial extract which has been obtained from probiotic strain, Lactobacillus acidophilus, has been tested on the HT29 cell line, derived from a colon adenocarcinome. This effect could be observed thanks to a non-invasive impedance assay, which allows for cell proliferation quantification. Furthermore, a cell cycle analysis through flow cytometry has been also carried out in order to get a better idea about the action mechanism of the extract, as one of the main hypothesis would be its possible impact on the cell division. Finally it could be observed an effect over the cell line dependent on concentration. Although this effect had been previously associated with a decrease in cell proliferation, it seems that flow cytometry results do not reflect this idea and thus, several hypothesis have been purposed, including changes in cell morphology/adhesion or an anti-proliferative effect which was not detected because of resistance mechanisms. Additionally, a second flow cytometry analysis showed no effect on cell viability. On the other hand, the huge complexity of the extract requires a previous separation through liquid chromatography, in order to be able to identify which molecule or molecules are responsible for the observed effect. Before performing this technique, it is necessary to carry out an optimization through thin layer chromatography, which is included in this study. At the end, several optimal conditions have been reached for both reverse phase with mixtures of acetonitrile and water, and normal phase with mixtures of hexane and ethyl acetate, ethyl acetate

Probiotics are used to improve human health due to their capability to induce beneficial effects in gastrointestinal diseases and modulate the immune response. However, scarce information is known about the bacterial active compounds and the metabolic pathways of interaction with immune cells. In the present study, we analyzed the immune response elicited by surface-associated proteins, the whole surface extract (WSE) and the genomic DNA of three strains from the main representative probiotic species for human consumption, L. acidophilus DSM20079T, L. rhamnosus GG, B. longum NCIMB 8809, using an in vitro model of human PBMCs from healthy donors. Eighteen cytokines were quantified using Luminex Technology to understand the immune pathways modulate by bacterial extracts. Overall, the bacterial fractions elicited a different immune response in PBMCs, whereas the isolated DNA was able to induce a higher Th17-like response that could lead to a protective effect in the epithelial barrier function.

[Background] The wide range of potential applications has made the Basic Local Alignment Search Tool (BLAST) a ubiquitous tool in the field of Molecular Biology. Within this context, it is increasingly appealing to embed BLAST services within larger Web applications. [Results] This work introduces BlasterJS viewer, a new JavaScript library for the lightweight development of Web-based applications supporting the visualisation of BLAST outputs. BlasterJS detaches from similar data viewers by focusing on the visual and interactive display of sequence similarity results and being completely independent of BLAST services. BlasterJS is compatible with the text outputs generated by the BLAST family of programs, namely BLASTp, BLASTn, BLASTx, tBLASTn, and tBLASTx, and works in all major Web browsers. Furthermore, BlasterJS is available through the EBI’s BioJS registry 5, which extends its potential use to a wider scope of bioinformatics applications. [Conclusions] BlasterJS is new Javascript library that enables easy and seamless integration of visual and interactive representations of BLAST outputs in Web-based applications supporting sequence similarity search. BlasterJS is free accessible at http://sing-group.org/blasterjs/.

Introduction: The role of the human intestinal microbiota in the maintenance of a healthy physiological condition, as well as its relation to the development of disease, remains to be clarified. Current evidence suggests that intestinal microbes could be involved in the initiation and amplification of autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus (SLE). Despite recent progress in understanding how these microbes influence the pathophysiology of lupus, studies are still limited. Areas covered: In this review, we have tried to summarize the most relevant findings that have contributed to our understanding of the links between the human intestinal microbiota and the development of lupus. We also describe the potential role of individual microbial players in the physiology of lupus, and how they can shape relevant immune responses. Expert commentary: Culture-independent techniques based on massive sequencing represent a powerful tool to unravel the biological activity of gut microbes. Current data demonstrates that, depending on the pattern of intestinal microorganisms or the presence of specific bacteria, different responses related to lupus physiology can be triggered. Fecal microbiota transplantation, live biotherapeutics, or dietary interventions targeting the microbiota will likely become a treatment for SLE.

There are strong evidences that probiotics influence the immune status of the host, in a strain-specific manner, acting in the gastrointestinal tract. On the hypothesis that certain extracellular proteins and peptides from gut bacteria may mediate part of this immunomodulation and assuming they are able to diffuse through the mucus layer and interact with immune cells we have developed this work. Our study attempts to understand the immunomodulatory mechanisms of (i) Pext, the extracellular protein fraction of Lactobacillus acidophilus DSM20079T, (ii) HM14, a peptide encrypted in an extracellular glycoside hydrolase from Bifidobacterium longum NCIMB 8809 and (iii) Escherichia coli O111:B4 lipopolysaccharide (LPS), a well-known pro-inflammatory molecule, over human peripheral blood mononuclear cells (PBMCs). An untargeted LC-ESI-QTOF-MS metabolomics approach was applied to reveal intracellular changes in treated-PBMCs isolated from healthy donors. Differences in NADH arrest, NAD+ concentration reduction, as well as increases in palmitic acid and methanephrin were observed in HM14 and Pext treated-cells compared to those stimulated with LPS. This would support an anti-inflammatory molecular mechanism of action of such proteinaceous molecules. Moreover, this methodology has confirms the importance of metabolomics approaches to better understanding immune cell responses to gut bacterial-derived molecules.

Se sabe que las bacterias de la microbiota intestinal humana poseen efectos beneficiosos en el hospedador, entre ellos estimular el sistema inmunológico y proteger contra la actividad carcinogénica de elementos de la dieta. Por ello, se ha propuesto que los probióticos podrían tener un papel en la prevención del desarrollo del cáncer colorrectal. El objetivo de este trabajo fue estudiar el efecto de diferentes péptidos procedentes de bacterias de la microbiota humana sobre la proliferación de células de adenocarcinoma colónico humano. Para ello, se seleccionaron 41 péptidos del metaproteoma del tracto digestivo humano por presentar alta similitud (mayor del 60%) con péptidos de actividad antiproliferativa o inmunomoduladora ya conocida. Se incubaron células de adenocarcinoma colónico humano HT-29 en presencia de dichos péptidos cuantificando la proliferación mediante el dispositivo xCELLigence RTCA. Se compararon los resultados con controles negativos sin péptido y controles positivos con el quimioterápico 5-fluorouracilo. Los péptidos que mostraron mayor actividad antiproliferativa se ensayaron a distintas concentraciones para determinar su concentración inhibitoria del 50%. Como conclusión, 21 de los 41 péptidos estudiados presentaron actividad antiproliferativa, siendo más relevante el efecto observado con cuatro de ellos (DW15, GR16, TS13 y VV15). Este efecto fue de menor magnitud que utilizando un agente quimioterápico clásico y requirió un tiempo de incubación más largo.

[Purpose] Short-chain fatty acids (SCFAs) formation by intestinal bacteria is regulated by many different factors, among which dietary fibre is currently receiving most attention. However, since fibre-rich foods are usually good dietary sources of phenolic compounds, which are also known to affect the microbiota, authors hypothesize that the regular intake of these bioactive compounds could be associated with a modulation of faecal SCFA production by the intestinal microbiota. [Methods] In this work, food intake was recorded by means of a validated Food Frequency Questionnaire. Fibres were determined using Marlett food composition tables, and phenolic compounds were obtained from Phenol-Explorer Database. Analysis of SCFA was performed by gas chromatography–flame ionization/mass spectrometry and quantification of microbial populations in faeces by quantitative PCR. [Results] Klason lignin and its food contributors, as predictors of faecal butyrate production, were directly associated with Bacteroides and Bifidobacterium levels, as well as lignans with Bacteroides. Also, anthocyanidins, provided by strawberries, were associated with faecal propionate and inversely related to Lactobacillus group. [Conclusions] These results support the hypothesis we put forward regarding the association between some vegetable foods (strawberries, pasta, lentils, lettuce and olive oil) and faecal SCFA. More studies are needed in order to elucidate whether these associations have been mediated by the bacterial modulatory effect of the bioactive compounds, anthocyanins, lignans or Klason lignin, present in foodstuffs.

In this paper we bring together two perspectives onto one single issue: the relevance of fundamental research. We depart from the personal experience of a scientist to arrive at a philosophical reasoning, which allows us to detach ourselves from the question what is this for? We conclude that, even if there are plenty of examples of the utility of fundamental research, that question is of no use in identifying good scientific work.

In a recent excellent Review1 (Bile acid–microbiota crosstalk in gastrointestinal inflammation and carcinogenesis. Nat. Rev. Gastroenterol. Hepatol. 15, 111–128 (2018)), the connection between human and microbial bile acid (BA) metabolism was described. In this Review, the authors identified possible therapeutic targets from data published in the context of gastrointestinal inflammation and carcinogenesis. From the microbial point of view, BAs are one of the main factors determining probiotic survival through the human gastrointestinal tract, which indeed was the focus of my PhD work

Certain non-digestible oligosaccharides (NDO) are specifically fermented by bifidobacteria along the human gastrointestinal tract, selectively favoring their growth and the production of health-promoting metabolites. In the present study, the ability of the probiotic strain Bifidobacterium longum subsp. infantis CECT7210 (herein referred to as B. infantis IM-1®) to utilize a large range of oligosaccharides, or a mixture of oligosaccharides, was investigated. The strain was able to utilize all prebiotics screened. However, galactooligosaccharides (GOS), and GOS-containing mixtures, effectively increased its growth to a higher extent than the other prebiotics. The best synbiotic combination was used to examine the antimicrobial activity against Escherichia coli, Cronobacter sakazakii, Listeria monocytogenes and Clostridium difficile in co-culture experiments. C. difficile was inhibited by the synbiotic, but it failed to inhibit E. coli. Moreover, Cr. sakazakii growth decreased during co-culture with B. infantis IM-1®. Furthermore, adhesion experiments using the intestinal cell line HT29 showed that the strain IM-1® was able to displace some pathogens from the enterocyte layer, especially Cr. sakazakii and Salmonella enterica, and prevented the adhesion of Cr. sakazakii and Shigella sonnei. In conclusion, a new synbiotic (probiotic strain B. infantis IM-1® and GOS) appears to be a potential effective supplement for maintaining infant health. However, further studies are needed to go more deeply into the mechanisms that allow B.infantis IM-1® to compete with enteropathogens

Flow cytometry (FC) is a very popular technique for to the study of different eukaryotic cell phenotypes. In addition, FC has many applications in the field of food microbiology, although the few number of specific molecules that can be potentially targeted with fluorescence-conjugated antibodies limits its potential applications. The present work represents a step forward in the application of FC for detection of specific foodborne or probiotic bacteria by using fluorescent antibodies binding specific surface-associated protein. To illustrate this novel approach, we have used a monoclonal and polyclonal antibodies targeting cell-wall hydrolase (CWH), one of the major surface-associated proteins of the probiotic Lactobacillus rhamnosus GG. In order to verify the specific union of the antibodies to this surface protein, ten different bacterial strains belonging to different bacteria species taxonomically related and no related to L. rhamnosus, were tested. The optimal conditions for bacteria detection corresponded to a final suspension of 5E107 bacteria in exponential phase of growth using FC buffer, without freezing, without adding extra BSA to the FC buffer and without fixing bacteria. Our results showed that GG strain was detected using the polyclonal anti-CWH serum in a higher extent (91.62%) than the rest of strains (L. casei 393: 64.49%; L. amylovorus: 46.13%, E. coli: 45.13%; L. acidophilus DSM20079T: 35.83%; B. longum NCIMB8809: 33.68%; L. gasseri BM7/10: 32.41%; L. reuteri DSM21016: 29.26%; L. plantarum NCIMB8826: 26.33% and L. delbruekii: 22.52%). The percentage of L. rhamnosus labeled with the monoclonal antibody was 53.06, which contrasted with the 91.62% labeled with the polyclonal antibody. The low affinity of the monoclonal antibody was perhaps due to the lack of immunogenicity of the single epitope chosen for antibody generation (CWHp). The high efficiency observed for the polyclonal anti-CWH serum is maybe a consequence of being constituted by a mixt

In recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The aim of this study was to characterize the EVs released by the dairy isolate Lactiplantibacillus plantarum BGAN8 and to gain an insight into the putative mechanism of EVs uptake by intestinal epithelial cells. The cryo-TEM observation undoubtedly demonstrated the release of EVs (20 to 140 nm) from the surface of BGAN8, with exopolysaccharides seems to be part of EVs surface. The proteomic analysis revealed that the EVs are enriched in enzymes involved in central metabolic pathways, such as glycolysis, and in membrane components with the most abundant proteins belonging to amino acid/peptide ABC transporters. Putative internalization pathways were evaluated in time-course internalization experiments with non-polarized HT29 cells in the presence of inhibitors of endocytic pathways: chlorpromazine and dynasore (inhibitors of clathrin-mediated endocytosis—CME) and filipin III and nystatin (disrupting lipid rafts). For the first time, our results revealed that the internalization was specifically inhibited by dynasore and chlorpromazine but not by filipin III and nystatin implying that one of the entries of L. plantarum vesicles was through CME pathway.

Bacteria-host interactions are mediated by different microbial associated molecular patterns which are most often surface structures such as, among others, exopolysaccharides (EPSs). In this work, the capability of two isogenic EPS-producing Bifidobacterium animalis subsp. lactis strains to modulate the gut microbiota of healthy mice, was assessed. Each strain produces a different type of polymer; the ropy strain S89L synthesized a rhamnose-rich, high-molecular weight EPS in highest abundance than the non-ropy DMS10140 one. BALB/c mice were orally fed for 10 days with milk-bifidobacterial suspensions and followed afterward for 7 post-intervention days (wash-out period). The colonic content of mice was collected in several sampling points to perform a metataxonomic analysis. In addition, the influence of specific microbial clades, apparently stimulated by the ropy and non-ropy strains, on mouse plasmatic cytokine levels was investigated through hierarchical association testing. Analysis of 16S rRNA gene sequences showed that the abundance of Firmicutes phylum significantly increased 7 days after cessing the treatment with both strains. The relative abundance of Alloprevotella genus also rose, but after shorter post-treatment times (3 days for both DMS10140 and S89L strains). Some bacterial clades were specifically modulated by one or another strain. As such, the non-ropy DMS10140 strain exerted a significant influence on Intestinomonas genus, which increased after 4 post-administration days. On the other hand, feeding with the ropy strain S89L led to an increase in sequences of Faecalibaculum genus at 4 post-treatment days, while the abundance of Erysipelotrichaceae and Lactobacillaceae families increased for prolonged times. Association testing revealed that several lactobacilli and bifidobacterial significantly stimulated by ropy S89L strain were positively associated with the levels of certain cytokines, including IL-5 and IL-27. These results highlight relevant c

Bifidobacteria are among the most abundant microorganisms inhabiting the intestine of humans and many animals. Within the genus Bifidobacterium, several beneficial effects have been attributed to strains belonging to the subspecies Bifidobacterium longum subsp. longum and Bifidobacterium longum subsp. infantis, which are often found in infants and adults. The increasing numbers of sequenced genomes belonging to these two subspecies, and the availability of novel computational tools focused on predicting glycolytic abilities, with the aim of understanding the capabilities of degrading specific carbohydrates, allowed us to depict the potential glycoside hydrolases (GH) of these bacteria, with a focus on those GH profiles that differ in the two subspecies. We performed an in silico examination of 188 sequenced B. longum genomes and depicted the commonly present and strain-specific GHs and GH families among representatives of this species. Additionally, GH profiling, genome-based and 16S rRNA-based clustering analyses showed that the subspecies assignment of some strains does not properly match with their genetic background. Furthermore, the analysis of the potential GH component allowed the distinction of clear GH patterns. Some of the GH activities, and their link with the two subspecies under study, are further discussed. Overall, our in silico analysis poses some questions about the suitability of considering the GH activities of B. longum subsp. longum and B. longum subsp. infantis to gain insight into the characterization and classification of these two subspecies with probiotic interest.

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. The probiotic microorganisms most commonly used in the food and pharmacy industry belong to Lactobacillus and Bifidobacterium, and several strains of these genera have demonstrated beneficial attributes. In addition, some other intestinal bacteria inhabiting the human microbiota, such as Faecalibacterium prausnitzii and Akkermansia muciniphila, have recently been discovered and are able to display health-promoting effects in animal and human trials. The beneficial properties of probiotics have been known for a long time, although little is known about the molecular mechanisms and the molecules responsible for their effects. However, in recent years, advances in microbiome studies, and the use of novel analytical and molecular techniques have allowed a deeper insight into their effects at the molecular level. This review summarizes the current knowledge of some of the molecules of probiotics and other intestinal commensal bacteria responsible for their immunomodulatory effect, focusing on those with more solid scientific evidence.

[Backgrounds] Infections caused by Clostridium difficile are increasing and constitute a problem due to the high morbidity and mortality rates in groups of risk, being the main aetiological agent of antibiotic-associated diarrhoea. This opportunistic pathogen is a common inhabitant of the intestinal microbiota but starts to proliferate after a microbiota dysbiosis caused by antibiotic treatment. The major virulence factor is the production of different toxins, mainly A and B. In previous works we have selected the strain Bifidobacterium longum IPLA20022 as a probiotic candidate being able to in vitro reduce the toxicity of C. difficile LMG21717 producing both types of toxins. [Objectives] Our aim is to gain insight into the potential mechanisms involved in the ability of IPLA20022 strain to reduce the cytotoxic effect of C. difficile upon an intestinal epithelial monolayer obtained from HT29 cells. [Methods] Different cellular fractions were obtained from B. longum IPLA20022 and were incubated with C. difficile. Afterwards, the cell-free supernatants were tested for their anti-clostridial capability upon HT29 cells using the RTCA (real time cell analyser) technology following the integrity of the intestinal monolayer; the remaining toxins were analyzed by means of ELISA tests. Those bifidobacterial fractions showing capability to reduce the toxicity of C. difficile were analyzed by means of proteomic techniques. [Conclusions] Preliminary results show that extracellular factors from B. longum IPLA20022 are involved in the reduction of clostridial cytotoxicity. This opens new opportunities for the therapeutically application of the strain, or their metabolites, for attenuating symptoms of C. difficile infection

[Objetivos] Los exopolisacáridos (EPS) son polímeros de carbohidratos localizados en la superficie bacteriana que desempeñan un papel relevante en la supervivencia del tracto gastrointestinal de la bacteria productora. Estudios previos de nuestro grupo han demostrado que los EPS de elevado peso molecular, los cuales confieren un fenotipo ¿ropy¿ (filante), están implicados en las propiedades anti-inflamatorias de ciertas cepas de Bifidobacterium animalis subsp. Lactis (B. Lactis). El objetivo de este estudio fue obtener cepas de B. Lactis con marcaje fluorescente para demostrar sus propiedades funcionales así como la implicación de los EPS en las mismas. [Metodología] Hemos construido plásmidos portadores de genes que codifican proteínas fluorescentes (GFP and mCherry), cuya expresión está bajo el control del promotor del factor de elongación Tu específico de esta especie. Los plásmidos se han introducido en dos cepas: DSM10140 (parental, no ropy) y S89L (recombinante, ropy); esta última ha adquirido el fenotipo ropy mediante la inclusión del gen que lo confiere por doble sobre-cruzamiento. [Resultados] Se ha detectado y cuantificado la fluorescencia de las cuatro cepas portadoras de proteínas fluorescentes. Se ha comprobado que la cepa ropy S89L-mCherry se adhiere en menor proporción que la cepa no-ropy 10140-mCherry a superficies bióticas (línea intestinal humana HT29) y abióticas (vidrio, plástico y oro). Este hecho indica que la presencia de polímeros de gran tamaño puede reducir la adherencia de la cepa productora al epitelio intestinal. [Conclusiones] Hemos aplicado, por primera vez, con éxito la técnica de doble-sobre-cruzamiento para incluir un gen en bifidobacterias y también hemos conseguido el marcaje fluorescente en cepas de B. Lactis, las cuales no habían sido descritas hasta el momento. Dada la gran aplicación de esta especie en el campo de los probióticos, la disponibilidad de cepas fluorescentes supone una nueva oportunidad para su estudio en modelos

Clostridium difficile es la causa más común de diarrea nosocomial en los países industrializados siendo los principales grupos de riesgo pacientes hospitalizados y ancianos, pero la incidencia está aumentando en otras poblaciones. Los factores de virulencia más importantes son las exotoxinas, sobre todo la A y la B, ya que incrementan la permeabilidad intestinal e inducen una respuesta inmune inflamatoria; todo ello conduce a la formación de pseudomembranas en el colon y en casos extremos se puede producir perforación intestinal, sepsis y muerte. El tratamiento convencional de la infección asociada a C. difficile consiste en la administración de antibióticos (metronidazol, vancomicina y fidaxomicina); sin embargo, el alto porcentaje de recurrencia de esta infección hace que se estén estudiando terapias alternativas y tratamientos adyuvantes para reducir los efectos causados por este patógeno, entre estas alternativas está el uso de probióticos. En nuestro grupo hemos desarrollado un modelo in vitro basado en la monitorización continua del comportamiento de líneas celulares intestinales, a través de medidas de impedancia empleando la tecnología ¿Real Time Cell Analyzer¿ (RTCA), que es eficaz para estudiar la cinética de citotoxicidad de los sobrenadantes procedentes de un cultivo toxigénico de C. difficile1. Estudios previos con este modelo nos permitieron evaluar el potencial probiótico de distintas cepas de bifidobacterias y lactobacillos para contrarrestar in vitro el efecto citotóxico de C. difficile LMG21717 (ribotipo 001), productor de toxinas A y B, sobre la línea intestinal HT29. La cepa que mostró la mayor capacidad para reducir el efecto tóxico fue Bifidobacterium longum IPLA200222. El objetivo del presente trabajo se centró en analizar la capacidad de esta bifidobacteria para atenuar el efecto tóxico de otras cepas de C. difficile. Para ello se utilizaron aislados del patógeno, procedentes de colección así como aislados clínicos, entre las que se incluyen

[Introducción] La fibrosis quística (FQ) es una enfermedad autosómica recesiva atribuible a una mutación en el gen regulador de la conductancia transmembranal de la Fibrosis Quística (CFTR). Como resultado, existe una alteración en el transporte transepitelial de iones, lo que resulta en una deficiencia en la producción de la capa de mucus que recubre los epitelios respiratorio y gastrointestinal. Esta disfunción es un factor crítico que condiciona la calidad y esperanza de vida en los pacientes con FQ, generando además unas condiciones muy particulares en la mucosa intestinal y respiratoria, que pueden condicionar la composición de la microbiota asociada a dichos ambientes. [Objetivos] Identificar marcadores microbianos a nivel de la microbiota intestinal, asociados a la FQ así como caracterizar la microbiota asociada a muestras de esputo en los mismos pacientes. Materiales y Métodos: Se ha estudiado la composición de la microbiota en muestras fecales y de esputo de 21 pacientes con FQ, mediante secuenciación masiva de productos de amplificación del gen del ARN ribosomal 16S mediante la plataforma Ion Torrent. Las posibles disbiosis intestinales asociadas a la FQ han sido analizadas mediante comparación con los perfiles metagenómicos obtenidos en 22 individuos sanos equiparables en distribución de sexo y edad. [Resultados] El perfil microbiano obtenido en muestras de heces de pacientes de FQ presentó una diversidad alfa significativamente menor al observado en el grupo control de individuos sanos, observándose además una reducción significativa de la ratio Firmicutes/Bacteroidetes. A nivel de Familia, los grupos cuya abundancia mostro cambios significativos en el grupo de FQ respecto al grupo control han sido las familias de Lachnospiraceae y Verrucomicrobia. Asimismo, se han determinado los perfiles microbianos en las muestras de esputo de los pacientes de FQ. Cambios en el perfil microbiano asociado a los pacientes con FQ han podido asociarse con el número de cic

Bifidobacterium longum strains are amongst the most widely used probiotics for human consumption due to their ability to modulate the host immune response, and withstand passage through the human gastrointestinal tract. Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR, occur widely in bacteria and provide adaptive immunity via acquisition of DNA sequences from viruses and plasmids that constitute unique genetic records of immunization in each bacterium (1). CRISPR-Cas systems comprise a variety of cas genes and a repeat-spacer array, where the repeat sequence (diamonds) flanked the spacers (squares). The spacers are short sequences homologous to the invasive DNA that the bacteria recorded to be immunize against a future challenge with the same virus. Spacers represent the immune background of each strain and are consider a barcode for genotyping each strain