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This work supports the massive presence of potential immunomodulatory peptides in the human gut metaproteome. These peptides were identified through the MAHMI database as potentially anti-inflammatory, and sixteen of them synthesized for characterize their mechanism of action. From them, peptide HM14 was encrypted in an extracellular protein produced by Bifidobacterium longum, a common member of the human microbiota, and displayed the highest anti-inflammatory capability. Molecular mechanism of action of HM14 pointed to a specific interaction between this immunomodulatory peptide and antigen presenting cells, which resulted in a higher formation of iTreg cells. Moreover, HM14 was effective in decreasing pro-inflammatory parameters in PBMCs isolated from a cohort of Crohn’s patients. Finally, non-targeted metabolomics confirmed the ability of HM14 to modulate the metabolic activity of PBMCs to fulfil its energy and biosynthetic requirements. Overall, our combined in silico/multiomics approach supports the human gut metaproteome as a source for immunomodulatory peptides.

Initial establishment of the human gut microbiota is generally believed to occur immediately following birth, involving key gut commensals such as bifidobacteria that are acquired from the mother. The subsequent development of this early gut microbiota is driven and modulated by specific dietary compounds present in human milk that support selective colonization. This represents a very intriguing example of host-microbe co-evolution, where both partners are believed to benefit. In recent years, various publications have focused on dissecting microbial infant gut communities and their interaction with their human host, being a determining factor in host physiology and metabolic activities. Such studies have highlighted a reduction of microbial diversity and/or an aberrant microbiota composition, sometimes referred to as dysbiosis, which may manifest itself during the early stage of life, i.e., in infants, or later stages of life. There are growing experimental data that may explain how the early human gut microbiota affects risk factors related to adult health conditions. This concept has fueled the development of various nutritional strategies, many of which are based on probiotics and/or prebiotics, to shape the infant microbiota. In this review, we will present the current state of the art regarding the infant gut microbiota and the role of key commensal microorganisms like bifidobacteria in the establishment of the first microbial communities in the human gut.

In recent years, bifidobacterial populations in the gut of various monkey species have been assessed in several ecological surveys, unveiling a diverse, yet unexplored ecosystem harbouring novel species. In the current study, we investigated the species distribution of bifidobacteria present in 23 different species of primates, including human samples, by means of 16S rRNA microbial profiling and internal transcribed spacer bifidobacterial profiling. Based on the observed bifidobacterial‐host co‐phylogeny, we found a statistically significant correlation between the Hominidae family and particular bifidobacterial species isolated from humans, indicating phylosymbiosis between these lineages. Furthermore, phylogenetic and glycobiome analyses, based on 40 bifidobacterial species isolated from primates, revealed that members of the Bifidobacterium tissieri phylogenetic group, which are typical gut inhabitants of members of the Cebidae family, descend from an ancient ancestor with respect to other bifidobacterial taxa isolated from primates.

[Background] Cow’s milk protein allergy (CMPA) is very common in infancy. Currently, the only therapeutic option is a dairy elimination diet. Standardized oral milk challenges are performed each 6 months to determine possible tolerance adquistion. [Aim] To analyze the intestinal changes in feces of infants with non-IgE mediated CMPA after successful milk challenges and introduction of dairy product in their diet. [Methods] Twelve allergic children (between 1 and 2 years old) that were initially consuming extensively hydrolyzed formulas provided stool samples before oral milk challenges, and a week and a month after. Changes in the intestinal microbiota populations were determined by high-throughput sequencing of the 16S rRNA gene, meanwhile diverse microbial metabolites (short chain fatty acids and indoles) were quantified by chromatographic methods. [Results] The introduction of milk in infants with outgrowing non-IgE CMPA increased significantly the levels of fecal lactic acid bacteria, in particular the genus Lactococcus. Microbial metabolites derived from the catabolism of proteins, such as escatol (produced from tryptophan) enhanced, meanwhile branched chain fatty acids diminished. [Conclusions]: The introduction of dairy products is accompanied by modifications in the infant gut environment through changes in the microbiota and protein metabolic end-products according to this kind of dietary change.

[Aim] The aim of this work was to evidence that the use of different stool collection strategies has a deep impact on the viability and diversity of the fecal microbiota that is recovered in the laboratory, notably if oxygen toxicity is not considered. This is particularly critical if the purpose of the sample is to envisage personalized biotherapeutic purposes, such as autologous fecal microbiota transplant or designing personalized biotherapeutics. [Methods] In order to analyze differences in the viability of fecal microbial populations during the whole delivery procedure, the same fecal specimens were sampled in conventional stool containers and GutAlive® devices, which minimize exposure of fecal microbiota to oxygen. Samples from five healthy donors were used and 150 differential colonies between the two devices were recovered. Differences included those associated to the two collection devices and individuals. All colonies were identified by 16S rRNA gene sequencing. Complete genomes of two extremely oxygen sensitive (EOS) bacteria recovered were sequenced with the Illumina MiSeq Sequencing System. [Results] Microbial diversity obtained was notably higher using GutAlive®. This device was able to maintain the viability of EOS species such as Akkermansia muciniphila, Faecalibacterium prausnitzii and a possible new member of the Clostridiales order. These obligate anaerobes were not recovered using the conventional stool container. Remarkably, GutAlive® allowed culturing and identifying an anaerobic isolate which may represent a new lineage within Clostridiales. This shows the importance of a personalized approach in microbiota-based therapies, as this novel isolate was recovered only from feces of one of the donors. [Conclusion] Using GutAlive® for stool sampling and transport allowed higher recovery of viable EOS bacteria by limiting oxygen exposure during the whole process. By standardizing the sampling and transport of the fecal specimens to the lab, GutAlive®

[Introduction] Milk is a complex food with 3.3% protein content and is fundamental in the diet throughout the all life and especially during childhood. However, cow`s milk protein allergy (CMPA) is very common in infancy and the only therapeutic option is a dairy elimination diet. The impact of the absence of milk proteins and dairy products in the gut ecosystem development of the infant has rarely been assessed. [Objective] To evaluate the intestinal changes in infants with non-IgE mediated CMPA after successful standardized oral challenges (SOCs) and introduction of milk in their diet. [Methodology] Twelve allergic children (between 1 and 2 years old) that were initially on a milk restriction diet provided stool samples before SOCs, and a week and a month after introduction of milk. Changes in the intestinal microbiota populations were determined by high-throughput sequencing of the 16S rRNA gene, meanwhile diverse microbial metabolites (short chain fatty acids and indoles) were quantified by chromatographic methods. [Main findings] The introduction of milk in infants that developed tolerance acquisition increased significantly the levels of fecal lactic acid bacteria. Microbial metabolites derived from the fermentation of proteins, such as branched chain fatty acids and p-cresol diminished. Statistical differences were observed between infants that were consuming probiotics (Lactobacillus rhamnosus GG) during the restriction diet period and those that were not. [Conclusion] The introduction of dairy products in the diet is accompanied by modifications in the infant gut environment through changes in the microbiota and protein metabolic end-products. Consumption of probiotics may affect the response to SOC with milk.

Gamma-amino butyric acid (GABA) is the main inhibitor neurotransmitter in the central nervous system, regulates multiple physiological processes in the human body and its dysfunctions has been linked to anxiety and depression disorders. In recent years, dietary supplementation with GABA has been associated with antihypertensive, analgesic and antidepressant properties and thus there is an increasing interest in identifying probiotic strains with GABA production capabilities, which could act as delivery vectors of this neurotransmitter in the human gut. Several lactic acid bacteria, including lactobacilli and streptococcal strains, have been reported capable to produce GABA, though this activity has been scarcely explored in members of the genus Bifidobacterium. In this work the GABA production capability was studied among members of the genus Bifidobacterium. First, an in silico analysis was performed on available Bifidobacterium genomes, to determine how widespread GABA production genes are within this group of bacteria. Based on the results, a screening of GABA production capability was performed in a collection of 58 Bifidobacterium strain, mainly comprised of members of the species B. adolescentis. For this purpose, the strains were grown overnight in the presence of the GABA precursor, monosodium glutamate and then, GABA and residual monosodium glutamate concentrations were determined through high-performance liquid chromatography in cell-free supernatants. The presence of the gadB and gadC genes, encoding a glutamate decarboxylase and a glutamate/GABA antiporter system, respectively, in the GABA producing strains was determined through PCR. The gadB and gadC genes were commonly detected in strains belonging to the species B. adolescentis. Besides, 25 % of the analyzed strains were capable to transform almost all monosodium glutamate provided, to GABA. In conclusion, GABA production is a relatively common trait in B. adolescentis strains, which could represent

[Objectives and Study] Cow's milk protein allergy (CMPA) is very common in infancy. Elimination diet is currently, the only therapeutic option available. Standardized oral milk challenges (SOC) are performed to determine tolerance development. The aim of our study was to analyze changes in feces of infants with non-IgE mediated CMPA after successful milk challenges (tolerance development) regarding microbiota composition and metabolic and cytokine profile [Methods]: Twelve non-IgE mediated CMPA children consuming extensively hydrolyzed formulas for at least 6 months ( 4 consumed formulas supplemented with Lactobacillus rhamnosus GG (LGG) ). In all cases a SOC was scheduled in order to determine tolerance acquisition. All 12 infants provided three consecutive stool samples: one before performing the SOC, one after a week and one after a month. Gut microbiota was determined through high-throughput sequencing. Microbial derived metabolites were analyzed by chromatographic methods. Fecal cytokines related to Th1/Th2 balance were also determined. [Results] With the re-introduction of milk, lactic-acid bacteria, in particular the genus Lactococcus, increased significantly. The content of some microbial metabolites, such as escatol, increased, meanwhile branched chain fatty acids diminished. After SOC, differences in TNF-a and butyrate levels were observed between infants consuming consume LGG supplemented formulas and those without probiotic supplementation. [Conclusions] These findings indicate that introduction of dairy products is followed by modifications in infant´s gut environment through maturation of the microbiota and its metabolic products. Consumption of probiotics may tune these profiles.

El ácido gamma-amino butírico (GABA) es el principal neurotransmisor inhibidor del sistema nervioso central, regula múltiples procesos fisiológicos en el cuerpo humano y su deficiencia se ha relacionado con trastornos de ansiedad y depresión. Además, en los últimos años, la suplementación dietética con GABA se ha asociado con propiedades antihipertensivas, analgésicas y antidepresivas y, por tanto, existe un interés creciente en la identificación de cepas probióticas con capacidad de producción de GABA, que podrían actuar como vectores para el suministro de este neurotransmisor en el intestino humano. Se sabe que algunas bacterias del ácido láctico, incluyendo algunas cepas de lactobacilos y estreptococos, son capaces de producir GABA, aunque esta actividad ha sido poco explorada en miembros del género Bifidobacterium. En este trabajo se estudió la capacidad de producción de GABA entre los miembros del género Bifidobacterium combinando aproximaciones in silico e in vitro. Se llevó a cabo un análisis in silico de los genomas de Bifidobacterium disponibles en la base de datos del NCBI, para determinar la ocurrencia de los genes responsables en la producción de GABA en este grupo. En base a los resultados, se determinó la capacidad de producción de GABA en una colección de 58 cepas de Bifidobacterium, principalmente compuestas por miembros de la especie B. adolescentis. Para ello, las cepas se cultivaron en presencia del precursor de GABA, glutamato monosódico, y las concentraciones de GABA y glutamato monosódico residual se determinaron en sobrenadantes libres de células mediante cromatografía líquida de alto rendimiento. La presencia de los genes gadB y gadC, que codifican una glutamato descarboxilasa y un sistema antiportador glutamato / GABA, respectivamente, en las cepas productoras de GABA se determinó mediante PCR. En el género Bifidobacterium, los genes gadB y gadC se encuentran fundamentalmente en cepas pertenecientes a la especie B. adolescentis. Además, el 2

[Background/Aims] The human gastrointestinal microbiota and its relationship with different physiological states has been characterized in detail in the last years. However, the microbiota of bile and gallbladder has been scarcely studied. Also, the functions of the autochthonous biliary microorganisms and the characteristics that allow the survival in this environment have not been investigated. In this context, we have constructed a metagenomic library from human bile of liver donors, in order to analyse the activities responsible for bacterial survival in presence of bile. [Methods] For the generation of the library we used total DNA isolated from human bile. Fragments with a size of approximately 20-30kb were cloned in the vector pCC1FOS™ (Epicentre®) and transformed into competent Escherichia coli. The pool of E. coli clones was tested in different concentrations of bile salts in order to detect highly resistant clones. [Results] Growth experiments allowed us to select five clones with a bile resistance phenotype. The selected clones were able to grow in a bile concentration at least 10 times higher than the E. coli strain containing the empty vector. The five fosmids were sequenced using Illumina technology and the bioinformatic analysis of the sequences showed the presence of a variety of potential genes that could play a role in bile resistance, coding for putative proteins involved in oxidative stress response, as well as DNA repair proteins, transmembrane pumps and lipopolysaccharide biosynthesis enzymes. Interestingly, the majority of DNA sequences displayed a low homology with genomic sequences of known microorganisms, suggesting that the native biliary microorganisms harbouring the DNA inserts could be new microbial taxons. [Conclusions] Five DNA fragments from biliary microorganisms, able to confer bile resistance in E. coli, were identified in our study. Further work is needed in order to unravel the specific DNA sequences responsible for the observed

Durante los últimos diez años ha habido avances en la compresión de cómo la microbiota intestinal humana contribuye a nuestra salud y enfermedad. La microbiota humana, especialmente la microbiota intestinal, es considerada un “órgano esencial” debido a que las comunidades microbianas que colonizan todas las superficies de nuestro cuerpo nos proporcionan funciones metabólicas, inmunológicas y fisiológicas esenciales. Tras el nacimiento, nuestra microbiota intestinal alcanza su configuración final a la edad de 2 a 3 años con la introducción de alimentos sólidos, estableciéndose una comunidad de microorganismos dominada por anaerobios obligados. Después del nacimiento, alrededor de 100 especies bacterianas colonizan el intestino, aumentando hasta 103 en la edad adulta. Su composición evoluciona constantemente, dependiendo de factores internos y externos como la edad, la raza, la dieta, la colonización materna, así como la exposición a antibióticos y xenobióticos. Además, otros factores como el estrés o los tratamientos farmacológicos conducen a rápidos cambios en la composición de nuestra microbiota. La complejidad de la microbiota intestinal hace difícil deducir la contribución individual de una especie/cepa a un fenotipo, enfermedad o condición. Por ello, el desarrollo de métodos para la modificación dirigida de una microbiota, especialmente enfocada en eliminar de una comunidad microbiana una bacteria específica, podría ser útil para descifrar como una especie contribuye a una enfermedad o a un proceso fisiológico, inmunológico o metabólico. En este estudio presentamos una tecnología que, si bien no es desconocida, sí que es pionera en cuanto a su aplicación. Hemos utilizado un anticuerpo policlonal dirigido a la proteína A de la capa S de Lactobacillus acidophilus DSM20079T para conseguir detectarlo en un consorcio bacteriano compuesto de 8 especies representativas de la microbiota intestinal humana. Además, se ha realizado una depleción y enriquecimiento positivo

This work presents a novel in silico approach to the prediction and characterization of the glycolytic capacities of the beneficial intestinal bacterium Faecalibacterium prausnitzii. Available F. prausnitzii genomes were explored taking the glycolytic capacities of F. prausnitzii SL3/3 and F. prausnitzii L2-6 as reference. The comparison of the generated glycolytic profiles offered insights into the particular capabilities of F. prausnitzii SL3/3 and F. prausnitzii L2-6 as well as the potential of the rest of strains. Glycoside hydrolases were mostly detected in the pathways responsible for the starch and sucrose metabolism and the biosynthesis of secondary metabolites, but this analysis also identified some other potentially interesting, but still uncharacterized activities, such as several hexosyltransferases and some hydrolases. Gene neighborhood maps offered additional understanding of the genes coding for relevant glycoside hydrolases. Although information about the carbohydrate preferences of F. prausnitzii is scarce, the in silico metabolic predictions were consistent with previous knowledge about the impact of fermentable sugars on the growth promotion and metabolism of F. prausnitzii. So, while the predictions still need to be validated using culturing methods, the approach holds the potential to be reproduced and scaled to accommodate the analysis of other strains (or even families and genus) as well as other metabolic activities. This will allow the exploration of novel methodologies to design or obtain targeted probiotics for F. prausnitzii and other strains of interest.

[Background] Gastrointestinal symptoms are frequent after pelvic radiotherapy and can greatly affect the quality of life of cancer survivors. The effect of radiation on the intestinal microbiome, and the implications of a radiotherapy-induced dysbiosis and the derived intestinal inflammation have received very little attention. [Aim] To perform a follow-up study in patients with prostate cancer for investigating alterations in gut microbiota and metabolites induced by pelvic radiotherapy and associations with inflammation and dietary changes. [Methods] Fourteen patients with prostate cancer undergoing pelvic radiotherapy were recruited and followed during the anti-cancer treatment until two months after finishing. Four stool samples were collected from each patient and changes in the bacterial communities were investigated by sequencing the V3-V4 region of the 16S rRNA gene with Illumina Technology (Miseq PE250), meanwhile short chain fatty acids (SCFAs) were analysed by gas chromatography. Additionally, calprotectin levels were determined using an ELISA kit and, evaluation of dietary intake was recorded by means of semi quantitative food frequency questionnaires. [Results] The composition of the gut communities changes along the radiation treatment, being the Bacteroidetes, the group more affected (p= 0.008, Wilcoxon test). Total SCFAs in feces was reduced with pelvic radiation. In particular, statistical differences were observed for butyrate and acetate excretion with respect to basal time. On the contrary, fecal calprotectin increased significantly during radiotherapy (p= 0.016). In addition, statistical differences in the energy intake were observed before and after two months of radiotherapy. Conclusions: An impact of pelvic radiotherapy on gut microbiota composition and metabolites was observed in prostate cancer patients. Intestinal inflammation occurs at the same time that the microbiome shifts. The effect of radiation was partially, but not completely, res

Introducción: La dispepsia funcional es una entidad clínica en la que no se encuentra una causa orgánica para los síntomas de molestias en la parte superior del tracto gastrointestinal (TGI). Su determinación se produce por exclusión de otras patologías, por lo que estos sujetos se someten normalmente a una endoscopia para tomar muestras del TGI superior, llegando hasta la primera porción del intestino delgado o duodeno. Aquí, las comunidades microbianas han sido muy poco estudiadas en comparación con las que habitan posiciones más distales en el intestino grueso. Sin embargo, es en el duodeno donde tiene lugar la secreción biliar y pancreática, y la digestión de las grasas. Una de las poblaciones más representativas en este nicho parece ser la de estreptococos, pero se han realizado muy pocos trabajos en sujetos sanos por la dificultad de acceso a las muestras. Objetivos: Llevar a cabo un estudio microbiológico mediantes diversas técnicas moleculares y de cultivo de muestras de aspirados duodenales en sujetos, aparentemente sanos, con el fin de identificar si los estreptococos son dominantes en este ecosistema, y asilar cepas comensales de este género de interés. Materiales y Métodos: Aspirados duodenales de sujetos con dispepsia se cultivaron en medio BHI incubándose a 37°C en condiciones de aerobiosis y microaerofilia. Además de los recuentos en placa y aislamiento de colonias para su posterior identificación (mediante secuenciación del gen SOD), se determinó la carga microbiana total mediante qPCR usando cebadores conservados para el ADNr 16S. Por otro lado, se determinaron las comunidades microbianas presentes mediante metagenómica filogenética. Resultados: En las muestras se obtuvieron recuentos en cultivo en torno a 104 ufc/ml, mientras que por qPCR los niveles fueron del orden de una unidad logarítmica superior (4,5x105 ). La asignación taxonómica de las secuencias obtenidas mediante tecnología Illumina reveló que las mayores abundancias relativas se corresp

The human gut microbiota is composed of the different microorganisms of our intestinal ecosystem. It has an important metabolic and nutritional role, and it is involved in the correct development of the gut and the maturation of our immune system. Currently, the application of fecal microbiota transplantation (FMT) has allowed us to establish causality in microbiome studies, and we know that microbiota can cure diseases or induce the appearance of disease symptoms, or even determine the success of some health treatments. But knowledge of the curative effects of feces has been documented many centuries ago. In the present article we want to make a brief review of the history of FMT, focusing on its effect on the human microbiome and its capacity to act on human physiology. We also want to place special emphasis on aspects that must be taken into account to translate microbiome research to medicine, related to safety, regulation and standardization of methods, as well as the importance of being able to predict the success of FMT.

Bile is a biological fluid synthesized in the liver, mainly constituted by bile acids and cholesterol, which functions as a biological detergent that emulsifies and solubilizes lipids, thereby playing an essential role in fat digestion. Besides, bile acids are important signaling molecules that regulate key functions at intestinal and systemic levels in the human body, affecting glucose and lipid metabolism, and immune homeostasis. Apart from this, due to their amphipathic nature, bile acids are toxic for bacterial cells and, thus, exert a strong selective pressure on the microbial populations inhabiting the human gut, decisively shaping the microbial profiles of our gut microbiota, which has been recognized as a metabolic organ playing a pivotal role in host health. Remarkably, bacteria in our gut also display a range of enzymatic activities capable of acting on bile acids and, to a lesser extent, cholesterol. These activities can have a direct impact on host physiology as they influence the composition of the intestinal and circulating bile acid pool in the host, affecting bile homeostasis. Given that bile acids are important signaling molecules in the human body, changes in the microbiota-residing bile biotransformation ability can significantly impact host physiology and health status. Elucidating ways to fine-tune microbiota-bile acids-host interplay are promising strategies to act on bile and cholesterol-related disorders. This manuscript summarizes the current knowledge on bile and cholesterol metabolism by intestinal bacteria, as well as its influence on host physiology, identifying knowledge gaps and opportunities to guide further advances in the field.

[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.

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.

Stool collection devices minimizing the exposure of gut bacteria to oxygen are critical for the standardization of further microbiota-based studies, analysis and developments. The aim of this work was to evidence that keeping anaerobiosis has a deep impact on the viability and diversity of the fecal microbiota that is recovered in the laboratory. Recovering certain microbial populations, such as obligate anaerobic bacteria, is particularly critical if the purpose of the study is to envisage personalized therapeutic purposes, such as autologous Fecal Microbiota Transplant. In this study the same fecal specimens were sampled in conventional stool containers and GutAlive, a disposable device that minimizes exposure of the gut microbiota to oxygen. Samples from five healthy donors were analysed and 150 differential colonies were recovered and identified by 16S rRNA gene sequencing. Globally, GutAlive maintained extremely oxygen sensitive (EOS) populations that were lost in conventional stool containers, and thus viability of species such as as Akkermansia muciniphila, Faecalibacterium prausnitzii and a novel member of the Clostridiales order was kept. These obligate anaerobes were not recovered using the conventional stool collection device. In conclusion, the use of GutAlive for stool collection and transport optimized the viability and recovery of EOS bacteria in the lab by diminishing oxygen toxicity.

The toll-like receptors involved in recognition of the exopolysaccharide produced by two isogenic, ropy and non-ropy, Bifidobacterium animalis subsp. lactis strains were investigated. Both strains interact with human embryonic kidney (HEK)-293 cells via TLR2, whereas purified EPSs specifically stimulate TLR4 regardless their molar mass.

Small intestinal bacterial overgrowth (SIBO) is a heterogeneous syndrome characterized by an increased number of bacteria in the small bowel. SIBO is diagnosed when bacterial cultures of upper intestine aspirates are ¿105 CFU/ml. Although investigation of these aspirates is considered the gold standard, non-invasive hydrogen breath test are commonly used for SIBO diagnosis. This syndrome has been related with a decrease of small bowel motility, an increase of intestinal permeability and bacterial translocation, being associated with serious diseases such as liver cirrhosis. To investigate the alterations in the bacterial communities of the small bowel from SIBO patients and the potential link with microbiota from other sites (saliva and feces). Duodenal aspirates from patients diagnosed with SIBO, control subjects and cirrhotic patients, with and without SIBO, were used to determine bacterial load (by quantitative PCR) and composition (by Illumina sequencing of 16S rDNA amplicons). Saliva and feces from cirrhotic patients were also included for comparative purposes. Bacterial quantification by qPCR revealed counts higher than 5 x 106 genome equivalents/ml of duodenal aspirates in SIBO patients whereas in the control subjects were below 106. Bacterial composition was represented by members of the Lactobacillales order, diverse families of protebacteria and genera belonging to Prevotellaceae, Veillonellaceae and Micrococcaceae families. No statistical differences were seen between the taxons presented in SIBO and non-SIBO cirrhotic patients. A further investigation with a broad number of patients is needed to confirm whether: i) the usual methods employed for SIBO diagnosis are reliable, and ii) the microbiota of duodenal aspirates in cirrhosis is more closely related to that of the oral cavity than the distal gut.

[Objectives and study] Cow`s milk protein allergy (CMPA) is the most common food allergy in the first year of life. A great proportion of children acquire tolerance before the age of 3 years , when a stable adult-like gut microbiota is being established. Associations between CMPA in infants and altered intestinal microbiota structure have been previously pointed out. However, non-IgE mediated forms remains poorly understood and are less studied than IgE mediated ones. Current guidelines recommend the use of extensively hydrolyzed formula (EHF) for CMPA as first choice, but other formulas based on vegetable proteins are also employed in daily practice. The aim of our study was to give clues on the role of type of feeding, microbiota composition and sensitization in non-IgE CMPA children. [Methods] Eighteen infants between 1 and 2 years old, diagnosed with non-IgE CMPA (clear positive oral challenge and a negative skin prick test) were recruited at different regional hospitals in Northern Spain (Asturias). They all were on exclusion diet and provided stool samples for the study. A detailed medical history, including type of feeding and formula used were recorded by the clinicians. After six months of milk exclusion a standardized oral challenge was performed under medical supervision. A control group of 10 age-matched healthy infants with normal diet consuming milk proteins were included in the study. Fecal samples of both groups were analyzed by high-throughput DNA sequencing of 16S rRNA gene amplicons (Illumina technology). [Results] Five of the eighteen CMPA infants were fed vegetable protein-based formulas. Of these, three were fed rice formula and, interestingly, none of them developed tolerance. These three children presented a clear distinct microbiota colonization pattern, characterized by a low abundancy of Bifidobacteria (less than 0.25% of assigned reads) and clustered separated from those CMPA infants who were consuming EHF and were tolerant to cow¿s milk

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.

La microbiota intestinal humana es el conjunto de microorganismos de nuestro intestino, con grupos de especies estables y otras variables. Esta microbiota establece una relación estrecha con su hospedador, al que proporciona beneficios nutricionales, metabólicos e inmunológicos, entre otros. Hoy sabemos que la gran mayoría de microorganismos intestinales no son responsables de causar enfermedad y su presencia, en cantidades adecuadas, es de gran importancia para mantener nuestro estado fisiológico. Sin embargo, cambios en las poblaciones microbianas intestinales pueden tener consecuencias negativas para la salud. Por ejemplo, numerosos estudios establecen una relación entre una microbiota intestinal alterada (disbiosis intestinal) y enfermedades autoinmunes, enfermedad inflamatoria intestinal o cáncer. Los probióticos son microorganismos vivos que cuando se administran en cantidades adecuadas confieren un beneficio a la salud del hospedador. La definición de probiótico no implica que necesariamente estos microorganismos tengan un origen intestinal, aunque muchos de ellos lo tienen y son instrumentos con los que podemos contar para mejorar las funciones de nuestra microbiota. Los microorganismos probióticos más utilizados en humanos son las bifidobacterias y los lactobacilos, aunque también se han utilizado otras bacterias, como Escherichia coli o Bacillus, y algunas levaduras. Las bifidobacterias, por ejemplo, tienen actividad inmunomoduladora y se han utilizado en la prevención o el tratamiento infecciones intestinales. El objetivo de esta comunicación es mostrar que hoy disponemos de información útil sobre probióticos y microbiota para mejorar la salud y la calidad de vida de las personas

Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli. This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve. In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island. IMPORTANCE Bifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, b

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.

Exopolysaccharides (EPS) are surface carbohydrate polymers present in most bacteria acting as a protective surface layer but also interacting with the surrounding environment. This review discusses the roles of EPS synthesized by strains of Lactobacillus and Bifidobacterium, many of them with probiotic characteristics, in the intestinal environment. Current knowledge on genetics and biosynthesis pathways of EPS in lactic acid bacteria and bifidobacteria, as well as the development of genetic tools, has created possibilities to elucidate the interplay between EPS and host intestinal mucosa. These include the microbiota that inhabits this ecological niche and the host cells. Several carbohydrate recognition receptors located in the intestinal epithelium could be involved in the interaction with bacterial EPS and modulation of immune response; however, little is known about the receptors recognizing EPS from lactobacilli or bifidobacteria and the triggered response. On the contrary, it has been clearly demonstrated that EPS play a relevant role in the persistence of the producing bacteria in the intestinal tract. Indeed, some authors postulate that some of the beneficial actions of EPS-producing probiotics could be related to the formation of a biofilm layer protecting the host against injury, for example by pathogens or their toxins. Nevertheless, the in vivo formation of biofilms by probiotics has not been proved to date. Finally, EPS produced by probiotic strains are also able to interact with the intestinal microbiota that populates the gut. In fact, some of these polymers can be used as carbohydrate fermentable source by some gut commensals thus being putatively involved in the release of bacterial metabolites that exert positive benefits for the host. In spite of the increasing knowledge about the role that these surface molecules play in the interaction of probiotic bacteria with the gut mucosal actors, both intestinal receptors and microbiota, the challenging i

[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.

En 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 debido fundamentalmente a la limitación que supone acceder al material biológico. En este trabajo se ha realizado el análisis metagenómico filogenético y funcional de muestras de bilis humana, con el fin de caracterizar el microbioma biliar de sujetos sanos y pacientes con colelitiasis, e identificar posibles disbiosis asociadas a esta patología. Se obtuvieron muestras de bilis de 28 individuos sanos y 16 pacientes con colelitiasis, de las que se extrajo el ADN biliar siguiendo un protocolo optimizado. A partir de las 44 muestras 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. 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 sujetos sanos, mientras que en pacientes con colelitiasis fueron más frecuentes las de las familias Bacteroidaceae, Prevotellaceae y Veillonellaceae. La secuenciación del ADN total biliar de tres sujetos sanos nos permitió realizar un análisis de sus metagenomas. El análisis funcional mostró que la asignación a las principales categorías funcionales del COG es similar a la del microbioma intestinal humano.

[Backgrounds] The exopolysaccharide (EPS) layer covering the surface of different bacteria, including Bifidobacterium animalis subsp. lactis, has been associated with the beneficial properties attributable to the producing bacterium. Indeed, EPS are one of the microbial associated molecular patterns that initiate the interaction with the host. [Objectives] Our aim was to obtain different EPS-producing B. animalis subsp. lactis variants, harboring plasmids with genes coding for fluorescent proteins, to study the role of EPS on the adherence of the producing strain. [Methods] One chromosomally stable EPS-producing variant (S89L strain) was obtained by means of a double crossover marker-less strategy. In the type strain DSM10140 a gene was replaced with another containing a nonsynonymous mutation associated with the ropy phenotype, yielding S89L. Besides, the type DSM10140 strain and the ropy S89L variant were labeled with two plasmids containing genes for mCherry or the green fluorescent proteins, thus yielding four fluorescent strains. These strains were used to check their adhesion to the human intestinal cell line HT29 as well as their capability to form biofilms in different abiotic surfaces (gold, polystyrene and glass) using different methodologies (real time monitoring and end-point crystal-violet staining). [Conclusions] This is the first report on the construction of fluorescent labeled B. animalis subsp. lactis strains; the qualitative and quantitative fluorescence analyses showed that the ropy S89L strain displayed a reduced capability to adhere to the intestinal epithelial monolayer and to form biofilms under abiotic surfaces. These results underline the usefulness of fluorescent labeling to elucidate the biological properties of bifidobacterial EPS

[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

[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

[Objetivos] La Alergia a Proteína a Leche de Vaca (APLV) es relativamente frecuente en niños pequeños. En las formas no mediadas por IgE, el diagnostico se basa en la historia clínica y una prueba de provocación positiva, siendo el tratamiento una dieta de exclusión. Una gran parte de los niños va a adquirir tolerancia antes de los 3 años, es por ello que, a partir del año, se valora la pauta de reintroducción de leche mediante pruebas de tolerancia. El objetivo de este trabajo fue analizar los cambios en la microbiota intestinal y parámetros asociados en heces de niños con APLV no mediada por IgE tras las pruebas de tolerancia a PLV. [Metodología] Se reclutó un grupo de niños entre 1 y 2 años y se recogieron muestras de heces antes de la prueba de tolerancia, a la semana y al mes de realización de la misma. Se estudió la microbiota intestinal mediante análisis metataxonómico del ADNr 16S, así como diversos marcadores inmunológicos y de inflamación, incluyendo la microbiota recubierta por IgA e IgG mediante citometría de flujo, la calprotectina fecal mediante ELISA y diversas citoquinas relacionadas con el balance Th1/Th2 mediante el sistema Bio-Plex. [Resultados] Tras la realización de las pruebas la mayoría de los niños superaron la tolerancia y solo en uno de los casos la provocación resultó positiva. El patrón de colonización microbiana en el niño en el que persiste la sensibilidad a PLV, a diferencia del resto, presentó una marcada presencia de los filos Verrucomicrobia y Proteobacteria. Se observó un aumento de los niveles de calprotectina, así como el grado de opsonización de la microbiota, a la semana de la introducción de la leche. [Conclusiones] En consonancia con la sintomatología gastrointestinal preponderante en estas formas de APLV, los resultados obtenidos apuntan a una respuesta intestinal predominantemente inflamatoria de distinto grado.

[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

The probiotic properties of Bifidobacterium breve CECT7263, as well as its safety, have been the focus of in several studies since 2008, including the sequencing of its genome in 2012. This study aims to complete the available genomic data to deepen the knowledge of some phenotypic characteristics of this strain.

The human gut microbiota is engaged in multiple interactions affecting host health during the host's entire life span. Microbes colonize the neonatal gut immediately following birth. The establishment and interactive development of this early gut microbiota are believed to be (at least partially) driven and modulated by specific compounds present in human milk. It has been shown that certain genomes of infant gut commensals, in particular those of bifidobacterial species, are genetically adapted to utilize specific glycans of this human secretory fluid, thus representing a very intriguing example of host-microbe coevolution, where both partners are believed to benefit. In recent years, various metagenomic studies have tried to dissect the composition and functionality of the infant gut microbiome and to explore the distribution across the different ecological niches of the infant gut biogeography of the corresponding microbial consortia, including those corresponding to bacteria and viruses, in healthy and ill subjects. Such analyses have linked certain features of the microbiota/microbiome, such as reduced diversity or aberrant composition, to intestinal illnesses in infants or disease states that are manifested at later stages of life, including asthma, inflammatory bowel disease, and metabolic disorders. Thus, a growing number of studies have reported on how the early human gut microbiota composition/development may affect risk factors related to adult health conditions. This concept has fueled the development of strategies to shape the infant microbiota composition based on various functional food products. In this review, we describe the infant microbiota, the mechanisms that drive its establishment and composition, and how microbial consortia may be molded by natural or artificial interventions. Finally, we discuss the relevance of key microbial players of the infant gut microbiota, in particular bifidobacteria, with respect to their role in health and disease