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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., Tis work was funded by the project IDE/2016/000282 from IDEPA (“Instituto de Desarrollo Empresarial of the Principality of Asturias”), which is partially supported by FEDER funds, SME-2017-0946-10 from MEIC (Ministry of Economy, Industry and competitiveness), SNEO 20171062 from CDTI (Centre for the Development of Industrial Technology) and a collaboration contract between Microviable Terapeutics SL. and the Consejo Superior de Investigaciones Científcas (Spanish National Research Council - CSIC).

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2020.103969., 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 Crohns 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., Our work is supported by the Spanish “Programa Estatal de Investigación. Desarrollo e Innovación Orientada a los Retos de la Sociedad” (grants AGL2013-44761-P and AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales”, Grant PS-2016), by the Asturias Regional Plan I + D + i for research groups (FYCYT-IDI/2018/000236) and by the Autonomic “Investigadores Emerxentes do Sistema Universitario de Galicia” (Grant EM2014/046). This work was partially supported by the Consellería de Educación. Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group. Finally, the authors wish to thank Jaume Morales and Rubén García form Agilent Technologies for technical support., info:eu-repo/semantics/publishedVersion

Lactobacillus genus includes both probiotic and representative strains of the human gut microbiota. Independent studies have reported on the anti-inflammatory properties of different Lactobacillus strains, although we are far from understanding the underlying molecular interplay. In this work we show that a daily administration of Lactobacillus acidophilus DSM20079T (DSM20079) to healthy piglets resulted in plasmatic increases of the anti-inflammatory IL10, whilst IL12 and the pro-inflammatory ratio IL12+TNF/IL10 decreased. The extracellular protein fraction of DSM20079 was identified as the responsible for the crosstalk interaction that elicited these tolerogenic effects. This strain was able to activate innate immune pathways in dendritic cells and to decrease the production of pro-inflammatory cytokines in both CD4+/CD8+ T cell subsets in healthy donors and in Crohns Disease patients. The tolerogenic effect exerted by the extracellular proteins of this strain suggests their potential use as coadjutant for therapeutic applications targeting chronic inflammatory illnesses., Our work is supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (grant AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer ColoRectal a partir de secuencias genéticas de microbiomas intestinales”, Grant PS-2016) and by the Asturias Regional Plan I+D+i for research groups (FYCYT-IDI/2018/000236). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). SING group thanks CITI (Centro de Investigación, Transferencia e Innovación) from University of Vigo for hosting its IT infrastructure. LJR was supported by the Principado de Asturias, PCTI 2018–2020 (GRUPIN: IDI2018-000237) and FEDER. This work was partially supported by the Consellería de Educación, Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group., info:eu-repo/semantics/publishedVersion

Cow's milk protein allergy (CMPA) is the most common allergy in the first year of life. Non-IgE mediated CMPA is characterized by digestive symptoms and tolerance development before the age of three. Gut microbiota composition in early life has been associated with food allergy. The ingestion of different foods/nutrients may mark different shifts in the microbial colonization of the infant intestine as well as the consumption of probiotics. Aim: To analyze changes in microbiota composition and metabolic and cytokine profiles in fecal samples from infants with non-IgE mediated CMPA after successful milk challenges, tolerance acquisition, and increasing dairy introduction in their diet. Methods: Twelve children with CMPA, aged between 1 and 2 years old, were recruited for the study. Participants were initially consuming hypoallergenic hydrolyzed formulas (four of them supplemented with the probiotic Lactobacillus rhamnosus GG), before being exposed to a standardized oral challenge (SOC) with cow's milk. Fecal samples were collected before, 1 week, and 1 month after performing the SOC. Changes in gut microbiota were determined by high-throughput amplicon sequencing of the 16S rRNA gene. Levels of lactobacilli were also determined by quantitative PCR (qPCR). Microbial metabolites were analyzed by chromatographic methods and fecal cytokines related to the Th1/Th2 balance were determined by immunoassay. Results: Lactic acid bacteria significantly increased in infants who outgrew non-IgE CMPA, after the introduction of milk. Microbial metabolites derived from the fermentation of proteins, such as branched chain fatty acids, and p-cresol, diminished. After the SOC, some cytokines related to inflammation (TNF-α, IFN-γ) increased. Accompanying the introduction of an unrestricted diet, we found significant differences in fecal microbial composition, metabolites, and cytokines between infants who did not consume the probiotic L. rhamnosus GG and those that did. Conclusions: These findings indicate that the introduction of intact milk proteins is followed by modifications in the infant gut environment through changes in immune mediators, microbiota, and its metabolic end-products. Consumption of probiotics during CMPA may contribute to gut homeostasis by fine-tuning these profiles., This research was funded by 2ª Convocatoria de Ayudas a Proyectos de Investigación Científica del Instituto Danone and grant IDI/2018/000236 from the Plan for Research, Development and Innovation of the Principado de Asturias 2018–2020 co-financed by the European Regional Development Funds (FEDER).

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., The MicroHealth group acknowledge the project AGL2015-64901-R (including FEDER European Union funds) from the Ministry of Economy, Industry and Competiveness (MINECO). N. Castro-Bravo thanks her FPI fellowship to the MINECO (BES2013-063984).

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 issue is to demonstrate the functionality of EPS in vivo, which will open an avenue of opportunities for the application of EPS-producing probiotics to improve health., In the MicroHealth group this research topic is currently financed by the project AGL2015-64901-R (including FEDER European Union funds) from the Ministry of Economy, Industry and Competiveness (MINECO). NC-B acknowledges her FPI fellowship to the MINECO (BES2013-063984).

Trabajo presentado en la IV European Conference Genomics of Complex Diseases: new challenges, celebrada en Granada (España) del 4 al 5 de octubre de 2018

Trabajo presentado en el 13th International dsRNA Virus Symposium, celebrado en Houffalize (Bélgica) del 24 al 28 de septiembre de 2018

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.

Trabajo presentado en el 7th International Human Microbiome Congress, IHMC, celebrado en County Kerry (Irlanda) del 28 al 26 de junio de 2018

Trabajo presentado en el 7th International Human Microbiome Congress, IHMC, celebrado en County Kerry (Irlanda) del 28 al 26 de junio de 2018, New studies and therapies based on gut microbiota sampling need from protocol normalization and standardization. During this process, keeping microbial viability from the sampling to the delivery, and the manipulation and storage of the faecal samples is crucial. This standardization requires a device/kit to avoid the toxic effect of oxygen on intestinal microbiota. [Objective] To develop and validate an anaerobic stool sample collection device: GutAliveR (PCT/ES2017/070087). [Methods] The efficiency of GutAliveRwas tested and compared with other stool collection systems. Maintenance of the anaerobic conditions was experimentally evaluated by means of total counts of anaerobic microorganisms. For this purpose, autoclaved faecal samples were inoculated with cultures of different anaerobic microorganisms. Also, a biodiversity index was calculated from faecal samples of healthy donors. Recounts were carried out at inoculation time and after 5/24/72 h incubation at room temperature (RT). [Results] In GutAliveR , anaerobic microorganisms inoculated showed a lower loss of viability after 24 and 72 h compared with conventional systems. Bifidobacterium bifidum remained viable and culturable up to 72 h at RT, while using a conventional system the bifidobacterial population was reduced 37% at 24 h and more than 99% at 72 h. The biodiversity index is higher when GutAliveR is used to collect and transport the faecal samples. [Conclusion] GutAliveR ensures an anoxic environment for the stool sampling and transport until processing at the laboratory. This could be critical in some applications, such as standardization and normalization of microbiota-based studies and the isolation of new probiotic strains from faeces.

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, because probiotic bacteria are used for human and animal consumption, one of the safety concerns over these bacteria is the presence of antibiotic resistance genes, since the human gut is a densely populated habitat that could favor the transfer of genetic material to potential pathogens. In this study, we analyzed the genetic basis responsible for the erythromycin and clindamycin resistance phenotype of B. breve CECT7263. We were able to identify and characterize a novel gene homologous to rRNA methylase genes which confers erythromycin and clindamycin resistance. This gene seems to be very uncommon in other bifidobacteria and in the gut microbiomes of both adults and infants. Even though conjugation experiments showed the absence of transferability under in vitro conditions, it has been predicted to be located in a putative genomic island recently acquired by specific bifidobacterial strains., This research was carried out thanks to the grant METASIN (Investigación, Desarrollo e Innovación en Nuevos Alimentos Multifuncionales para Síndrome Metabolic), funded by the Centro Para el Desarrollo Tecnológico Industrial (CDTI) and Biosearch S.A. This work was also funded by the EU Joint Programming Initiative A Healthy Diet for a Healthy Life (JPI HDHL, http://www.healthydietforhealthylife.eu/) and MIUR to M.V. This research benefits from the HPC (High-Performance Computing) facility of the University of Parma, Parma, Italy.

Trabajo presentado en el IX Workshop de la Sociedad Española de Probióticos y Prebióticos (SEPyP), celebrado en Zaragoza (España) el 15 y 16 de febrero de 2018

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., LR is a postdoctoral researcher supported by the Juan de la Cierva Postdoctoral Trainee Program of the Spanish Ministry of Economy and Competitiveness (MINECO; IJCI-2015-23196). BS and AM thanks MINECO for the funding of the project AGL2016-78311-R and PR-M for the project AGL2015-64901-R. SD is supported by a research contract associated to the project BIO2014-55019-JIN from the Spanish “Plan Estatal de I+D+i”.

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., This work was funded by the EU Joint Programming Initiative “A Healthy Diet for a Healthy Life” (JPI HDHL, http://www.healthydietforhealthylife.eu/) and MIUR to M.V. and by the Spanish MINECO to M.G. (project PCIN-2015-233). A.M. and S.D.P. are partially funded by grants from the Instituto Danone and by the Spanish Plan Estatal de I+D+I (reference AGL2016-78311-R). D.V.S. is a member of the APC Microbiome Institute funded by Science Foundation Ireland (SFI) through the Irish Government's National Development Plan (grant number SFI/12/RC/2273). J.M.R. is in receipt of AGL2016-75476-R (Ministerio de Economía y Competitividad (Spain). J.M. is in receipt of a Starting Investigator Research Grant (SIRG) (no. 15/SIRG/3430) funded by Science Foundation Ireland (SFI).

Trabajo presentado en el 7th Congress of European Microbiologists (FEMS 2017), celebrado en Valencia (España), del 9 al 13 de julio de 2017, [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

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., Research in our group is supported by grants AGL2015- 64901-R, BIO2014-55019-JIN, AGL2013-44761-P, and AGL2013-44039-R from the Spanish Ministry of Economy and Competitiveness

Trabajo presentado en el ESPGHAN 50th Annual Meeting, celebrado en Praga (República Checa) del 10 al 13 de mayo de 2017

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

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., Research in our lab is funded by Grants AGL2013‐44039‐R and AGL2013‐44761‐P from the Spanish “Plan Estatal de I+D+I.” Part of the authors is also partially funded by the [15VI013] Contract‐Programme from the University of Vigo and the Agrupamento INBIOMED from DXPCTSUG‐FEDER unha maneira de facer Europa (2012/273). B. S. was recipient of a Ramón y Cajal postdoctoral contract from the Spanish Ministry of Economy and Competitiveness.

Trabajo presentado en el 4th International Symposium on Propionibacteria and Bifidobacteria (PropioBifido 2016), celebrado en la Universidad de Cork (Irlanda) del 21 al 23 de septiembre de 2016, 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.

Trabajo presentado en la International Scientific Association for Probiotics and Prebiotics (ISAPP) Conference, celebrada en Turku (Finland) del 7 al 9 de junio de 2016