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[Introducción] Actualmente existe muy poca información acerca del efecto de la congelación sobre la viabilidad de la microbiota fecal, y hasta la fecha, no se ha realizado ninguna investigación para determinar las condiciones y compuestos más adecuados para criopreservar y maximizar la viabilidad microbiana durante la conservación de esta microbiota. La preservación de muestras complejas de microbiota puede ser de utilidad para recuperar microorganismos de interés, así como ahondar en estudios de microbiota intestinal mediante el empleo de modelos in vitro e in vivo sin necesidad de depender de muestras fecales frescas. [Objetivos] En el presente trabajo se analizó el papel de distintos criopreservantes y condiciones de congelación en muestras de microbiota intestinal extraída de heces, evaluándose el mantenimiento de la viabilidad y recuperación de la microbiota en su conjunto, así como de distintas poblaciones representativas a los 3 meses de su preservación. [Materiales y Métodos] A partir de 5 muestras de heces de donantes voluntarios sanos se obtuvo la microbiota fecal mediante un procedimiento de separación en gradiente de densidad por ultracentrifugación previamente descrito (Hevia y cols., 2015). Las muestras, procesadas en una estación de trabajo en anaerobiosis, se alicuotaron y se guardaron congeladas bajo 4 condiciones experimentales: A) en presencia de glicerol al 15% a -20°C; B) con glicerol al 20% a -80°C; C) con sacarosa al 10% a -80°C y D) con polietilenglicol (PEG) al 20% a -196°C. Se evaluó la viabilidad de la microbiota extraída antes y después de ser preservada mediante recuentos en placa en distintos medios, microscopia de fluorescencia (kit ¿Live/Dead BacLight¿) y mediante qPCR usando monoazida de propidio (PMA) para la detección de células no dañadas. [Resultados] Al comparar de forma global las cuatro condiciones de preservación se observó una diferencia significativa entre las distintas condiciones en cuanto a la reducción de los recuentos

Peptidome similarity analysis enables researchers to gain insights into differential peptide profiles, providing a robust tool to discriminate strain-specific peptides, true intra-species differences among biological replicates or even microorganism-phenotype variations. However, no in silico peptide fingerprinting software existed to facilitate such phylogeny inference. Hence, we developed the Peptidomes for Phylogenies (P4P) web tool, which enables the survey of similarities between microbial proteomes and simplifies the process of obtaining new biological insights into their phylogeny. P4P can be used to analyze different peptide datasets, i.e. bacteria, viruses, eukaryotic species or even metaproteomes. Also, it is able to work with whole proteome datasets and experimental mass-to-charge lists originated from mass spectrometers. The ultimate aim is to generate a valid and manageable list of peptides that have phylogenetic signal and are potentially sample-specific. Sample-to-sample comparison is based on a consensus peak set matrix, which can be further submitted to phylogenetic analysis. P4P holds great potential for improving phylogenetic analyses in challenging taxonomic groups, biomarker identification or epidemiologic studies. Notably, P4P can be of interest for applications handling large proteomic datasets, which it is able to reduce to small matrices while maintaining high phylogenetic resolution

The Mechanism of Action of the Human Microbiome (MAHMI) database is 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. Currently, MAHMI database contains over 300 hundred million peptide entries, with detailed information about peptide sequence, sources and potential bioactivity. The reference peptide data section is curated manually by domain experts. The in silico peptide data section is populated automatically through the systematic processing of publicly available exoproteomes of the human microbiome. Bioactivity prediction is based on the global alignment of the automatically processed peptides with experimentally validated immunomodulatory and antiproliferative peptides, in the reference section. MAHMI provides researchers with a comparative tool for inspecting the potential immunomodulatory or antiproliferative bioactivity of new amino acidic sequences and identifying promising peptides to be further investigated. Moreover, researchers are welcome to submit new experimental evidence on peptide bioactivity, namely, empiric and structural data, as a proactive, expert means to keep the database updated and improve the implemented bioactivity prediction method. Bioactive peptides identified by MAHMI have a huge biotechnological potential, including the manipulation of aberrant immune responses and the design of new functional ingredients/foods based on the genetic sequences of the human microbiome. Hopefully, the resources provided by MAHMI will be useful to those researching gastrointestinal disorders of autoimmune and inflammatory nature, such as Inflammatory Bowel Diseases. MAHMI database is routinely updated and is available free of charge.

Introducción: el lupus eritematoso sistémico es una enfermedad inflamatoria crónica en la que está implicado el estrés oxidativo.Objetivo: evaluar la concentración de antioxidantes de la dieta y sanguíneos, así como de la microbiota sobre las concentraciones de malondialdehído y proteína C reactiva en 21 pacientes de lupus y 21 controles pareados por edad y sexo.Métodos: los parámetros bioquímicos de rutina y proteína C reactiva se determinaron a través de métodos enzimáticos: cobre, zinc y selenio por espectrometría de masas, malondialdehído y capacidad antioxidante total por métodos espectrofotométricos, la microbiota fecal por técnicas metagenómicas y la dieta a través de cuestionarios de frecuencia de consumo.Resultados: no se han observado diferencias en la dieta en los pacientes con lupus respecto al grupo control, excepto en la ingesta de ácidos grasos trans, siendo mayor en el grupo de lupus. En estas pacientes se observaron mayores niveles circulantes de cobre y menores de zinc. La concentración de cobre en suero se relacionó directamente con los niveles de proteína C reactiva y esta proteína, a su vez, con la proporción de Lentisphaerae, Proteobacteria y Verrucomicrobia en heces. Además, mientras que los niveles de malondialdehído se asociaban inversamente con la proporción de Cyanobacteria y Firmicutes, con Actinobacteria se encontró una correlación positiva. La presencia de anti-SSA/Ro en lúpicas se relaciona con mayores concentraciones de zinc.Conclusión: estos resultados podrían ser útiles para profundizar en el futuro conocimiento de esta compleja enfermedad. Received

Scientific studies focused on the role of the human microbiome over human health have generated billions of gigabits of genetic information during the last decade. Nowadays integration of all this information in public databases and development of pipelines allowing us to biotechnologically exploit this information are urgently needed. Prediction of the potential bioactivity of the products encoded by the human gut microbiome, or metaproteome, is the first step for identifying proteins responsible for the molecular interaction between microorganisms and the immune system. We have recently published the Mechanism of Action of the Human Microbiome (MAHMI) database (http://www.mahmi.org), conceived as a resource compiling peptide sequences with a potential immunomodulatory activity. Fifteen out of the 300 hundred million peptides contained in the MAHMI database were synthesized. These peptides were identified as being encrypted in proteins produced by gut microbiota members, they do not contain cleavage points for the major intestinal endoproteases and displayed high probability to have immunomodulatory bioactivity. The bacterial peptides FR-16 and LR-17 encrypted in proteins from Bifidobacterium longum DJ010A and Bifidobacterium fragilis YCH46 respectively, showed the higher immune modulation capability over human peripheral blood mononuclear cells. Both peptides modulated the immune response toward increases in the Th17 and decreases in the Th1 cell response, together with an induction of IL-22 production. These results strongly suggest the combined use of bioinformatics and in vitro tools as a first stage in the screening of bioactive peptides encrypted in the human gut metaproteome.

Probiotics are live microorganisms that confer a health benefit on the host when administered in adequate amounts. Toxicity induced by molecular oxygen significantly affects the viability of probiotic bacteria during biomass generation, product manufacturing and storage also constitute a major challenge for their inclusion in dairy products. Oxygen toxicity is particularly critical in the case of bifidobacteria, which are strict anaerobes. This mini-review aims at compiling the knowledge on the response mechanisms, mainly in this bacterial group, focused on counteracting the deleterious effects of oxygen on probiotic cells. The effect of other technological and microbiological ways to attenuate oxygen toxicity, such as milk deaeration, microencapsulation or co-culture is reviewed. Potential uses of the isolation of better adapted strains of probiotic Bifidobacterium strains, as well as the next generation probiotics such as Faecalibacterium prausnitzii, are discussed.

The gastrointestinal tract of mammals hosts a high and diverse number of different microorganisms, known as intestinal microbiota. Many probiotics were originally isolated from the gastrointestinal tract, and they were defined by the Food and Agriculture Organization of the United Nations (FAO)/WHO as “live microorganisms which when administered in adequate amounts confer a health benefit on the host.” Probiotics exert their beneficial effects on the host through four main mechanisms: interference with potential pathogens, improvement of barrier function, immunomodulation and production of neurotransmitters, and their host targets vary from the resident microbiota to cellular components of the gut–brain axis. However, in spite of the wide array of beneficial mechanisms deployed by probiotic bacteria, relatively few effects have been supported by clinical data. In this regard, different probiotic strains have been effective in antibiotic‐associated diarrhea or inflammatory bowel disease for instance. The aim of this review was to compile the molecular mechanisms underlying the beneficial effects of probiotics, mainly through their interaction with the intestinal microbiota and with the intestinal mucosa. The specific benefits discussed in this paper include among others those elicited directly through dietary modulation of the human gut microbiota.

Bifidobacteria are members of the intestinal microbiota of mammals and other animals, and some strains are able to exert health-promoting effects. The genus Bifidobacterium belongs to the Actinobacteria phylum. Firmicutes, Bacteroidetes, and Actinobacteria constitute the most abundant phyla in the human intestinal microbiota, Firmicutes and Bacteroidetes being predominant in adults, and Actinobacteria in breast-fed infants, where bifidobacteria can reach levels higher than 90% of the total bacterial population. They are among the first microbial colonizers of the intestines of newborns, and play key roles in the development of their physiology, including maturation of the immune system and use of dietary components. Indeed, some nutrients, such as human milk oligosaccharides, are important drivers of bifidobacterial development. Some Bifidobacterium strains are considered probiotic microorganisms because of their beneficial effects, and they have been included as bioactive ingredients in functional foods, mainly dairy products, as well as in food supplements and pharma products, alone, or together with, other microbes or microbial substrates. Well-documented scientific evidence of their activities is currently available for bifidobacteria-containing preparations in some intestinal and extraintestinal pathologies. In this review, we focus on the role of bifidobacteria as members of the human intestinal microbiota and their use as probiotics in the prevention and treatment of disease.

Bifidobacterium represents a genus within the phylum Actinobacteria which is one of the major phyla in the healthy intestinal tract of humans. Bifidobacterium is one of the most abundant genera in adults, but its predominance is even more pronounced in infants, especially during lactation, when they can constitute the majority of the total bacterial population. They are one of the pioneering colonizers of the early gut microbiota, and they are known to play important roles in the metabolism of dietary components, otherwise indigestible in the upper parts of the intestine, and in the maturation of the immune system. Bifidobacteria have been shown to interact with human immune cells and to modulate specific pathways, involving innate and adaptive immune processes. In this mini-review, we provide an overview of the current knowledge on the immunomodulatory properties of bifidobacteria and the mechanisms and molecular players underlying these processes, focusing on the corresponding implications for human health. We deal with in vitro models suitable for studying strain-specific immunomodulatory activities. These include peripheral blood mononuclear cells and T cell-mediated immune responses, both effector and regulatory cell responses, as well as the modulation of the phenotype of dendritic cells, among others. Furthermore, preclinical studies, mainly germ-free, gnotobiotic, and conventional murine models, and human clinical trials, are also discussed. Finally, we highlight evidence supporting the immunomodulatory effects of bifidobacterial molecules (proteins and peptides, exopolysaccharides, metabolites, and DNA), as well as the role of bifidobacterial metabolism in maintaining immune homeostasis through cross-feeding mechanisms.

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis, and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum, and show that these sequences are useful for typing across three subspecies

Background: Some patients complain that eating lettuce, gives them gas and abdominal distention. Our aim was to determine to what extent the patients' assertion is sustained by evidence. Methods: An in vitro study measured the amount of gas produced during the process of fermentation by a preparation of human colonic microbiota (n = 3) of predigested lettuce, as compared to beans, a high gas-releasing substrate, to meat, a low gas-releasing substrate, and to a nutrient-free negative control. A clinical study in patients complaining of abdominal distention after eating lettuce (n = 12) measured the amount of intestinal gas and the morphometric configuration of the abdominal cavity in abdominal CT scans during an episode of lettuce-induced distension as compared to basal conditions. Key Results: Gas production by microbiota fermentation of lettuce in vitro was similar to that of meat (P = .44), lower than that of beans (by 78 ± 15%; P < .001) and higher than with the nutrient-free control (by 25 ± 19%; P = .05). Patients complaining of abdominal distension after eating lettuce exhibited an increase in girth (35 ± 3 mm larger than basal; P < .001) without significant increase in colonic gas content (39 ± 4 mL increase; P = .071); abdominal distension was related to a descent of the diaphragm (by 7 ± 3 mm; P = .027) with redistribution of normal abdominal contents. Conclusion and Inferences: Lettuce is a low gas-releasing substrate for microbiota fermentation and lettuce-induced abdominal distension is produced by an uncoordinated activity of the abdominal walls. Correction of the somatic response might be more effective than the current dietary restriction strategy., Peer Reviewed, Postprint (published version)

[EN] This PhD project aims to build the scientific basis to develop new functional foods based on bioactive peptides obtained from the genetic sequences of the human microbiome. These foods will be directed to the prevention of several gastrointestinal disorders of autoimmune and inflammatory nature, as well as to decrease the effects of the disease in patients. This will have a direct effect decreasing the economic burden of the treatments and increasing the quality of life of the patients. The proposed computational framework will be equipped to support the study of human gut microbiota interactions with the immune system and to manipulate aberrant immune responses. To this end, the PhD student will integrate both novel and state-of-the-art bioinformatics methods and tools for sequence analysis and protein characterisation with various well-established data integration and data mining methods. The PhD student will develop in silico tools to characterise encrypted peptides with potential bioactivity, firstly by studying their occurrence in the exoproteomes of the human microbiome, and thus obtain a database of peptides with different potential bioactivities. Moreover, functional analysis of proteome interactions will be pursued as means to reveal metabolic systems with greater prevalence among diseases progressing with inflammatory or autoimmune mechanisms, such as inflammatory bowel disease (IBD) and colo-rectal cancer (CRC). Further, this will help us to analyse nutrient-affected pathways in the human pathology. Most notably, the PhD student will investigate cross-feeding relationships between gut microbe enzymes and host carbohydrate metabolism enzymes, diet modulation and the use of probiotics in treating metabolic disorders. This project will shed light on our understanding of the human gut biology and cell cross-talk with food, probiotic and gut bacteria in the context of diseases such as IBD and CRC. Specifically, the new computational framework will equip r, [ES] Este proyecto de doctorado tiene como objetivo construir una base científica para el desarrollo de nuevos alimentos funcionales basados en péptidos bioactivos obtenidos de secuencias genéticas del microbioma humano. Estos alimentos serán dirigidos a la prevención de varios desordenes gastrointestinales de naturaleza autoinmune o inflamatoria, así como para disminuir el efecto de la enfermedad en los pacientes. Esto tendrá un efecto directo disminuyendo la carga económica de los tratamientos e incrementando la calidad de vida de los pacientes. El marco computacional propuesto estará equipado para apoyar el estudio de las interacciones de la microbiota intestinal humana con el sistema inmunológico y para manipular respuestas inmunes aberrantes. Para este fin, el estudiante de doctorado integrará métodos y herramientas de bioinformática innovadoras y avanzadas para el análisis de secuencias y la caracterización de proteínas con varios métodos bien establecidos de integración y minería de datos. El estudiante de doctorado desarrollará herramientas in silico para caracterizar péptidos encriptados con bioactividad potencial, primero estudiando su presencia en los exoproteomas del microbioma humano, obteniendo así una base de datos de péptidos con diferentes bioactividades potenciales. Además, el análisis funcional de las interacciones del proteoma se llevará a cabo como medio para revelar sistemas metabólicos con mayor prevalencia entre las enfermedades que evolucionan con mecanismos inflamatorios o autoinmunes, como la enfermedad inflamatoria intestinal (EII) y el cáncer colorrectal (CCR). Además, esto nos ayudará a analizar las vías afectadas por los nutrientes en la patología humana. En particular, el estudiante de doctorado investigará las relaciones de cross-feeding entre las enzimas de los microorganismos intestinales y las enzimas del metabolismo de los carbohidratos del huésped, la modulación de la dieta y el uso de probióticos en el tratamiento de los trasto

Background: There is increasing evidence of the key role that the gut microbiota plays in inflammatory diseases. Objectives: To identify differences in the faecal microbial composition of patients with psoriasis compared with healthy individuals in order to unravel the microbiota profiling in this autoimmune disease. Methods: 16S rRNA gene sequencing and bioinformatic analyses were performed with the total DNA extracted from the faecal microbiota of 19 patients with psoriasis and 20 healthy individuals from the same geographic location. Results: Gut microbiota composition of patients with psoriasis displayed a lower diversity and different relative abundance of certain bacterial taxa compared with healthy individuals. Conclusions: The gut microbiota profile of patients with psoriasis displayed a clear dysbiosis that can be targeted for microbiome-based therapeutic approaches.

Bioactive peptides secreted by probiotic Bifidobacterium longum (peptide B7) and opportunistic pathogen Bacteroides fragilis (peptide B12) modulate the intestinal cytokine milieu in health. Here, we characterized their capacity to modulate both the mucosal cytokine production and the phenotype of circulating antigen presenting cells (APCs) in active inflammatory bowel disease (IBD). The IBD mucosa produced higher levels of pro-inflammatory cytokines referred to healthy controls (HCs). Peptides B7 and B12, however, did not ameliorate the mucosal cytokine milieu in IBD. Human circulating APCs (B-cells, monocytes, plasmacytoid dendritic cells (pDCs), and conventional dendritic cells (cDCs)) were characterized by flow cytometry in presence/absence of the peptides. Circulating B-cells, monocytes, and cDCs from IBD patients were more activated than those from HCs. Peptide B7, but not B12, decreased CCR2 expression on all APC subsets from HC, but not IBD patients. Moreover, both peptides tend to further increase their pro-inflammatory profile in IBD. In summary, IBD patients display mucosal and circulating APC pro-inflammatory properties. Peptide B7 immunomodulatory capacity elicited over circulating APCs from HC, but not IBD patients, suggests the presence of disrupted modulatory mechanisms for this peptide in IBD. Future studies should address the effect of bacteria-derived immunomodulatory peptides in non-inflamed (quiescent) IBD patients.

[Background] The microbial populations of the human intestinal tract and their relationship to specific diseases have been extensively studied during the last decade. However, the characterization of the human bile microbiota as a whole has been hampered by difficulties in accessing biological samples and the lack of adequate methodologies to assess molecular studies. Although a few reports have described the biliary microbiota in some hepatobiliary diseases, the bile microbiota of healthy individuals has not been described. With this in mind, the goal of the present study was to generate fundamental knowledge on the composition and activity of the human bile microbiota, as well as establishing its potential relationship with human bile-related disorders., [Results] Human bile samples from the gallbladder of individuals from a control group, without any record of hepatobiliary disorder, were obtained from liver donors during liver transplantation surgery. A bile DNA extraction method was optimized together with a quantitative PCR (qPCR) assay for determining the bacterial load. This allows the selection of samples to perform functional metagenomic analysis. Bile samples from the gallbladder of individuals suffering from lithiasis were collected during gallbladder resection and the microbial profiles assessed, using a 16S rRNA gene-based sequencing analysis, and compared with those of the control group. Additionally, the metabolic profile of the samples was analyzed by nuclear magnetic resonance (NMR). We detected, for the first time, bacterial communities in gallbladder samples of individuals without any hepatobiliary pathology. In the biliary microecosystem, the main bacterial phyla were represented by Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Significant differences in the relative abundance of different taxa of both groups were found. Sequences belonging to the family Propionibacteriaceae were more abundant in bile samples from control subjects; meanwhile, in patients with cholelithiasis members of the families Bacteroidaceae, Prevotellaceae, Porphyromonadaceae, and Veillonellaceae were more frequently detected. Furthermore, the metabolomics analysis showed that the two study groups have different metabolic profiles., [Conclusions] Our results indicate that the gallbladder of human individuals, without diagnosed hepatobiliary pathology, harbors a microbial ecosystem that is described for the first time in this study. Its bacterial representatives and metabolites are different from those detected in people suffering from cholelithiasis. In this regard, since liver donors have been subjected to the specific conditions of the hospital’s intensive care unit, including an antibiotic treatment, we must be cautious in stating that their bile samples contain a physiologically normal biliary microbiome. In any case, our results open up new possibilities to discover bacterial functions in a microbial ecosystem that has not previously been explored.

In this work, we have used a metataxonomic analysis to study the bacterial biodiversity in three types of commercial dressing and ingredients habitually used in table olive packaging. The bioinformatics analysis showed that salt samples (marine and spring) had the highest biodiversity indexes, followed by the aromatic herbs (thyme, oregano, and fennel) and finally by fermented dressing samples (garlic, pepper, and pepper paste). In salt samples, a significant high presence of Cytophagia, Sphingobacteria, Deltaproteobacteria and Halobacteria classes was observed. On the contrary, the presence of Bacilli class in fermented dressing samples was significantly higher compared to the other samples. Finally, the abundance of Gammaproteobacteria was statistically higher in the samples of herbs. At genus or family taxonomic level, Pseudomonas and diverse non-assigned genus of the Enterobacteriaceae family predominated in many samples of herbs. In fermented dressing samples, diverse genus belonging to the Enterobacteriaceae family appeared in a high relative abundance in pepper paste and garlic, and presence of Vibrio sp. was detected in pepper. However, Lactobacillus, Weisella, and Pediococcus were the predominant genera found in many of these fermented samples. Salinibacter was the most representative genus in both types of salt samples. Multivariate analysis (PCA) of the metataxonomic data sets discriminated well the samples belonging to the three different groups, showing a high proportion of sequences shared by samples-group. This survey reports for the first time the presence of new bacterial species not previously related with table olive processing, as well as the presence of others which could compromise the stability of olive packaging.

Peptides encrypted in the intestinal microbial-exoproteome mediate the host-microbiota crosstalk, which is disrupted in inflammatory bowel disease (IBD). Here, the MAHMI database was used for the identification of 20 novel intestinal bacterial peptides. Our results revealed that serum IgA levels directed towards the peptides, but not IgG, discriminated healthy controls from IBD patients. Indeed, they also differentiated patients with ulcerative colitis from Crohńs disease and, within them, patients with and without intestinal inflammation. All peptides were immunomodulatory as they changed the intestinal cytokine milieu following human lamina propria mononuclear cells culture (with/out LPS), revealing a Bifidobacterium longum subsp. longum peptide with the highest tolerogenic properties. Therefore, bacterial peptides encrypted in the human gut metaproteome may have utility as non-invasive biomarkers to aid on IBD diagnosis and monitoring. These peptides also display immunomodulatory effects on the intestinal mucosa revealing them as novel functional compounds for non-drug therapeutic strategies in IBD.

This work reports on a large-scale potential neuropeptide activity screening in human gut microbiomes deposited in public databases. In our experimental approach, the sequences of the bioactive peptides collected in the MAHMI database, mainly predicted as immunomodulatory or antitumoral, were crossed with those of the neuroactive/digestive peptides. From 91,325,790 potential bioactive peptides, only 581 returned a match when crossed against the 5949 neuroactive peptides from the NeuroPep database and the 15 digestive hormones. Relevant bacterial taxa, such as Ruminococcus sp., Clostridium sp. were found among the main producers of the matching sequences, and many of the matches corresponded to adiponectin and the hormone produced by adipocites, which is involved in glucose homeostasis. These results show, for the first time, the presence of potentially bioactive peptides produced by gut microbiota members over the nervous cells, most notably, peptides with already predicted immunomodulatory or anti-inflammatory activity. Classical (Lactobacillus sp.) and next-generation (Faecalibacterium sp.) probiotics are shown to produce these peptides, which are proposed as a potential mechanism of action of psychobiotics. Our previous experimental results showed that many of these peptides were active when incubated with immune cells, such as dendritic cells, so their effect over the nervous system innervating the gut mucosa holds significant potential and should be explored.

Bifidobacteria administered in fermented dairy products have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to reach/colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in Bifidobacterium longum NCIMB8809 and its acidpH-resistant mutant B. longum 8809dpH were studied. Comparison of protein maps allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. Adaptation and response to low pH involved changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm. Remarkably, key proteins of the biosynthetic routes of cysteine and methionine were highly overrepresented. These results prompted us to perform a genomic and transcriptomic study in order to clarify the genetic mechanism behind the acquired acid resistance phenotype. Full genome sequencing and a transcriptomic analysis (microarray and RNAseq) allowed us to conclude that: i) a deletion of a large fragment of DNA in the mutant strain could be partially responsible for the acid resistance and ii) genes involved in the sulphur amino acid metabolism (metE, metB and cysD) were constitutively overexpressed in the mutant. Thus, the acquired acid resistance phenotype of B. longum 8809dpH could be related to a different cysteine and methionine metabolism in this strain.

Exopolysaccharides (EPS) are carbohydrate polymers that play an important role in survival and colonization of the gastrointestinal tract by the producing bacteria. An EPS producing Bifidobacterium animalis subsp. lactis strain has a ropy phenotype due to the synthesis of a high molar mass polymer, which seems to be involved in the anti-inflammatory properties induced by the bacterium. The ropy strain has a mutation in the wzz gene responsible for EPS chain length determination, which is absent in the non-ropy parental strain. To demonstrate the biological relevance of ropy-EPS production, we have obtained the mutant B. animalis subsp. lactis S89L derived from the non-ropy DSM10140 strain in which the wzz gene is replaced by the mutated one (wzzS89L). The ropy S89L mutant presented lower capability to adhere to HT29 intestinal cell monolayers. Additionally, both DSM10140 and S89L strains were labeled with fluorescent proteins (GFP and mCherry) harbored in plasmids containing the tuf promoter of this species. The fluorescence of the four strains obtained was quantified by flow cytometry and visualized under epifluorescence microscopy. The mCherry labeled strains were analyzed in the HT29 model, corroborating the lower adhesion capability of the ropy S89L with respect to its isogenic DSM10140. To the best of our knowledge, fluorescently-labeled B. animalis subsp. lactis strains have not been reported in literature. Given the industrial relevance of this species in the probiotic field, results presented here will open future opportunities to study the role of different EPS in the interaction of the producing bacteria with the host.

[Introducción] La fermentación de los carbohidratos no digeribles de la dieta en el colon por acción de la microbiota intestinal da lugar a la producción de ácidos grasos de cadena corta (AGCC). Los mayoritarios son los ácidos acético, propiónico y butírico, los cuales a su vez tienen un papel clave en el metabolismo lipídico y energético del hospedador. Recientes estudios han sugerido además que los polifenoles presentes en los alimentos ricos en fibra podrían influir en la composición y/o actividad metabólica de la microbiota intestinal [Objetivos] Realizar un estudio preliminar para detectar asociaciones entre el tipo de fibra ingerida en la dieta, los polifenoles presentes en estos alimentos y los cambios en la composición y actividad de la microbiota intestinal. [Materiales y Métodos] A partir de un total de 124 individuos sin diagnóstico previo de cáncer, enfermedad autoinmune o digestiva se ha realizado un cuestionario de frecuencias de consumo de alimentos para determinar la información dietética. A partir de las muestras fecales de estos individuos se ha estudiado la composición de la microbiota intestinal mediante qPCR y se ha determinado el contenido de AGCC mediante la técnica de CG-MS. [Resultados] El ácido acético fue el AGCC mayoritario en heces seguido de los ácidos propiónico y butírico. Se han identificado varios compuestos fenólicos y tipos de fibra ingeridos con la dieta como predictores de la producción fecal de los ácidos propiónico y butírico. Se han observado a su vez correlaciones positivas entre los polifenoles totales y varias subclases de estos compuestos así como de la fibra total y diferentes tipos de fibra con Bacteroides. El género Faecalibacterium se ha asociado también positivamente con la ingesta de fibra total y determinados tipos de fibra. [Conclusiones] Los resultados obtenidos sugieren la asociación entre la ingesta de fibra y polifenoles, procedentes de la dieta sobre los cambios en la producción de AGCC de la microbiota intes

The "Red Española de Bacterias Lácticas" -RedBAL- is a network of Spanish researchers working on different aspects of LAB related with the involvement of this bacterial group in food safety and quality, as well as in health. Our raison d'être is to stimulate the exchange of knowledge and the establishment of collaborations among the members, in order to improve our scientific achievements and favors the transfer of Science to Industry and Society. In addition, one of the new bets of redBAL is our opening to other scientific groups, with special emphasis in Latin American countries taking advantage of the use of a common language. Currently our RedBAL is constituted by 197 members, organized in 42 groups belonging to 21 Institutions, among others Universities and different Governmental Research Centers, spread around the Spanish geography. About 30% of the members are young Scientists (pre- and post-doctoral students) who constitute the future in the fascinating research field of LAB. The network started in 2002 and since them we have had several meetings to present our results; the next one will be the 11st and it will take place in Gijón (Asturias) from 28th-30th June (http://www.redbal.es). A number of groups in the RedBAL are devoted to study the interaction of commensal and probiotic bacteria with the human immune system. Scientific studies focused on the role of the human microbiome over human health have generated billions of gigabits of genetic information during the last decade. Prediction of the potential bioactivity of the products encoded by the human gut microbiome, or metaproteome, is the first step for identifying proteins responsible for the molecular interaction between microorganisms and the immune system. We have recently published the Mechanism of Action of the Human Microbiome (MAHMI) database (http://www.mahmi.org) (1), conceived as a resource compiling peptide sequences with a potential immunomodulatory activity. We have shown that the bacteria

Los probióticos están adquiriendo un gran interés en los últimos tiempos tanto por su uso para la elaboración de multitud de productos fermentados, fundamentalmente lácteos, como por su contribución al mantenimiento del estado de salud del consumidor. Las propiedades saludables que se les atribuyen y que sustentan su aplicación en la clínica, se relacionan con algunos mecanismos de acción sobre enfermedades relacionadas con el sistema digestivo y la función inmune. Ha sido evidenciado que afectan a la microbiota intestinal aumentando el número de bacterias beneficiosas y disminuyendo el de patógenas para el mantenimiento del equilibrio intestinal y prevención del desarrollo de enfermedades. Es necesario demostrar su eficacia en la salud humana mediante la realización de ensayos clínicos bien documentados que permitan seleccionar las poblaciones más apropiadas, siendo las bacterias probióticas más habituales las del género Lactobacillus y Bifidobacterium. Para la realización de éste trabajo fueron seleccionadas tres cepas pertenecientes a la colección del IPLA-CSIC: Lactobacillus casei subsp. rhamnosus GG (LGG), Lactobacillus acidophilus DSM 20079T y Bifidobacterium longum subsp. longum NCIMB 8809. Los efectos inmunomoduladores de estas bacterias y la influencia de las mismas sobre la respuesta inmunitaria llevan a estudiar el comportamiento de algunos de sus componentes (proteínas extracelulares, proteínas de superficie, ácido lipoteicoico y ADN) en individuos sanos, siendo el objetivo de este trabajo.

La microbiota intestinal es el conjunto de microorganismos que habita en inmunológicoel intestino humano de manera natural, estableciendo una relación estrecha con el hospedador que la alberga, al que le proporciona una serie de beneficios entre los que cabe destacar la función barrera contra bacterias potencialmente patógenas, el aprovechamiento de energía de alimentos no digeribles, y la maduración y desarrollo del sistema inmunológico. Hasta el momento, tanto estudios en humanos como en animales han demostrado que cambios en las poblaciones microbianas intestinales pueden tener consecuencias para la salud, y existen numerosos trabajos científicos que relacionan a una microbiota intestinal alterada (disbiosis intestinal), con procesos inflamatorios, asma alérgicos, enfermedades autoinmunes e incluso cáncer. El objetivo general de esta Tesis Doctoral es generar nuevo conocimiento sobre la composición de la microbiota intestinal en el marco de dos trastornos inmunológicos, el lupus eritematoso sistémico (LES) y el asma. A través del uso de técnicas independientes de cultivo, se analizaron muestras fecales y se determinó el perfil microbiano de grupos de individuos que padecían estas enfermedades, comparándolo con el de individuos sanos. De esta manera los resultados de esta Tesis Doctoral han contribuido a entender la relación entre la disbiosis intestinal que presenta la microbiota de estos individuos y su estado inmunológico. Para el caso del LES se estudiaron 20 mujeres de entre 34 y 68 años de edad, y se compararon con un grupo control sano. En asma alérgico se analizaron muestras de 21 pacientes de edades entre 24 y 57 años y se compararon con un grupo de 22 controles sanos de mismo sexo y rango de edad. Durante la primera parte de esta Tesis se optimizaron metodologías y protocolos de tratamiento de muestras fecales humanas.

Los organismos macroscópicos, tales como animales, plantas u hongos, suelen ser fáciles de distinguir gracias a diferencias morfológicas, características de su comportamiento o por el aislamiento reproductivo. Para los microorganismos pertenecientes a los dos dominios Prokaryota de la vida, las Bacteria y las Archaea, la clasificación de especies es una tarea mucho más complicada, dada la similitud genética existente.

Despite the existing evidence on the impact of olive oil and red wine on the intestinal microbiota, the effect of the global Mediterranean Diet (MD) has not been sufficiently studied. We explored the association between the adherence to a Mediterranean dietary pattern, and its components, with faecal microbiota in a cohort of adults with non-declared pathology. This transversal study involved 31 adults without a previous diagnosis of cancer, autoimmune or digestive diseases. Based on the data obtained by means of an annual food frequency questionnaire (FFQ), and the information existing in the literature, a Mediterranean Diet Score (MDS) was calculated. Dietary fibre was obtained from Marlett et al. tables and Phenol-Explorer Database was used for phenolic compounds intake. Quantification of microbial groups was performed by Ion Torrent 16S rRNA gene-based analysis and quantification of short-chain fatty acids (SCFAs) was performed using gas chromatography-mass spectrometry (MS). MDS was associated with a higher abundance of Bacteroidetes (p = 0.001), Prevotellacea (p = 0.002) and Prevotella (p = 0.003) and a lower concentration of Firmicutes (p = 0.003) and Lachnospiraceae (p = 0.045). Also, in subjects with MDS ≥ 4, higher concentrations of faecal propionate (p = 0.034) and butyrate (p = 0.018) were detected. These results confirm the complexity of the diet-microbiota interrelationship.

Typical bacterial strain differentiation methods are often challenged by high genetic similarity between strains. To address this problem, we introduce a novel in silico peptide fingerprinting method based on conventional wet-lab protocols that enables the identification of potential strain-specific peptides. These can be further investigated using in vitro approaches, laying a foundation for the development of biomarker detection and application-specific methods. This novel method aims at reducing large amounts of comparative peptide data to binary matrices while maintaining a high phylogenetic resolution. The underlying case study concerns the Bacillus cereus group, namely the differentiation of Bacillus thuringiensis, Bacillus anthracis and Bacillus cereus strains. Results show that trees based on cytoplasmic and extracellular peptidomes are only marginally in conflict with those based on whole proteomes, as inferred by the established Genome-BLAST Distance Phylogeny (GBDP) method. Hence, these results indicate that the two approaches can most likely be used complementarily even in other organismal groups. The obtained results confirm previous reports about the misclassification of many strains within the B. cereus group. Moreover, our method was able to separate the B. anthracis strains with high resolution, similarly to the GBDP results as benchmarked via Bayesian inference and both Maximum Likelihood and Maximum Parsimony. In addition to the presented phylogenomic applications, whole-peptide fingerprinting might also become a valuable complementary technique to digital DNA-DNA hybridization, notably for bacterial classification at the species and subspecies level in the future.

Bifidobacteria are members of the human gut microbiota, being numerically dominant in the colon of infants, while also being prevalent in the large intestine of adults. In this study, we determined and analyzed the pan-genome of Bifidobacterium adolescentis, which is one of many bacteria found in the human adult gut microbiota. In silico analysis of the genome sequences of eighteen B. adolescentis strains isolated from various environments, such as human milk, human feces and bovine rumen, revealed a high level of genetic variability, resulting in an open pan-genome. Compared to other bifidobacterial taxa such as Bifidobacterium bifidum and Bifidobacterium breve, the more extensive B. adolescentis pan-genome supports the hypothesis that the genetic arsenal of this taxon expanded so as to become more adaptable to the variable and changing ecological niche of the gut. These increased genetic capabilities are particularly evident for genes required for dietary glycan-breakdown