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Specific microbial profiles and changes in intestinal microbiota have been widely demonstrated to be associated with the pathogenesis of a number of extra-intestinal (obesity and metabolic syndrome) and intestinal (inflammatory bowel disease) diseases as well as other metabolic disorders, such as non-alcoholic fatty liver disease and type 2 diabetes. Thus, maintaining a healthy gut ecosystem could aid in avoiding the early onset and development of these diseases. Furthermore, it is mandatory to evaluate the alterations in the microbiota associated with pathophysiological conditions and how to counteract them to restore intestinal homeostasis. This review highlights and critically discusses recent literature focused on identifying changes in and developing gut microbiota-targeted interventions (probiotics, prebiotics, diet, and fecal microbiota transplantation, among others) for the above-mentioned pathologies. We also discuss future directions and promising approaches to counteract unhealthy alterations in the gut microbiota. Altogether, we conclude that research in this field is currently in its infancy, which may be due to the large number of factors that can elicit such alterations, the variety of related pathologies, and the heterogeneity of the population involved. Further research on the effects of probiotics, prebiotics, or fecal transplantations on the composition of the human gut microbiome is necessary., Ongoing research is funded by grant PI-0538-2017, Junta de Andalucía, Spain (to L.F.).

Objectives: There are many differences in the fecal infant microbiota associated with various feeding methods. The aim of this study was to examine the major differences in the fecal microbiota of breast-fed (BF) and formula-fed (FF) infants and to describe the principal bacterial components that would explain the variability in the predominant bacterial families and genus clusters. Methods: Fecal samples from 58 infants, 31 of whom were exclusively BF and 27 of whom were exclusively FF with a standard formula in agreement with the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition recommendations, were analyzed by fluorescent in situ hybridization combined with flow cytometry. Principal component analysis was used to maximize the information gained for the predominant bacterial families and genus clusters using a minimal number of bacterial groups. Results: The predominant detected group was Bifidobacterium, followed by Enterobacteriaceae and Bacteroides in both BF and FF infants. The Lactobacillus group was the only independent variable associated with FF infants. We also found that 3 principal components were sufficient to describe the association between the bacterial group, genus, and species studied in BF and FF infants; however, these components differed between BF and FF infants. For the former, the 3 factors found were Bifidobacterium/ Enterobacteriaceae, Lactobacillus/Bacteroides, and Clostridium coccoides/ Atopobium; for the latter, Bifidobacterium/Enterobacteriaceae, Bacteroides and C coccoides were observed. Conclusions: There is a clear clustering of components of infant microbiota based on the feeding method.

Ideally, cell models should resemble thein vivoconditions; however, in mostin vitroexperimental models, epithelial cells are cultivated as monolayers, in which the establishment of functional epithelial features is not achieved. To overcome this problem, co-culture experiments with probiotics, dendritic cells and intestinal epithelial cells and three-dimensional models attempt to reconcile the complex and dynamic interactions that existin vivobetween the intestinal epithelium and bacteria on the luminal side and between the epithelium and the underlying immune system on the basolateral side. Additional models include tissue explants, bioreactors and organoids. The present review details thein vitromodels used to study host–microbe interactions and explores the new tools that may help in understanding the molecular mechanisms of these interactions.

The aim of the present study was to isolate, identify and characterise novel strains of lactic acid bacteria and bifidobacteria with probiotic properties from the faeces of exclusively breast-fed infants. Of the 4680 isolated colonies, 758 exhibited resistance to low pH and tolerance to high concentrations of bile salts; of these, only forty-two exhibited a strong ability to adhere to enterocytesin vitro.The identities of the isolates were confirmed by 16S ribosomal RNA (rRNA) sequencing, which permitted the grouping of the forty-two bacteria into three different strains that showed more than 99 % sequence identity withLactobacillus paracasei,Lactobacillus rhamnosusandBifidobacterium breve, respectively. The strain identification was confirmed by sequencing the 16S–23S rRNA intergenic spacer regions. Strains were assayed for enzymatic activity and carbohydrate utilisation, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur and namedL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036. The strains were susceptible to antibiotics and did not produce undesirable metabolites, and their safety was assessed by acute ingestion in immunocompetent and immunosuppressed BALB/c mouse models. The three novel strains inhibitedin vitrothe meningitis aetiological agentListeria monocytogenesand human rotavirus infections.B. breveCNCM I-4035 led to a higher IgA concentration in faeces and plasma of mice. Overall, these results suggest thatL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036 should be considered as probiotic strains, and their human health benefits should be further evaluated.

Probiotics are live microorganisms that, when ingested in adequate amounts, provide health benefits to the host. The strains most frequently used as probiotics include lactic acid bacteria and bifidobacteria, which are isolated from traditional fermented products and the gut, faeces and breast milk of human subjects. The identification of microorganisms is the first step in the selection of potential probiotics. The present techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods, such as denaturing gradient gel electrophoresis/temperature gradient gel electrophoresis and fluorescencein situhybridisation, are employed to identify and characterise probiotics. The ability to examine fully sequenced genomes has accelerated the application of genetic approaches to elucidate the functional roles of probiotics. One of the best-demonstrated clinical benefits of probiotics is the prevention and treatment of acute and antibiotic-associated diarrhoea; however, there is mounting evidence for a potential role for probiotics in the treatment of allergies and intestinal, liver and metabolic diseases. These positive effects are generally attributed to the ability of probiotics to regulate intestinal permeability, normalise host intestinal microbiota, improve gut immune barrier function and equilibrate the balance between pro-inflammatory and anti-inflammatory cytokines. However, the positive effects of probiotics are not always substantiated by findings from properly conducted clinical trials. Notably, even when the results from randomised, placebo-controlled trials support the beneficial effects of a particular probiotic for a specific indication, the benefits are generally not translatable to other probiotic formulations.

Probiotics are live microorganisms that provide health benefits to the host when ingested in adequate amounts. The strains most frequently used as probiotics include lactic acid bacteria and bifidobacteria. Probiotics have demonstrated significant potential as therapeutic options for a variety of diseases, but the mechanisms responsible for these effects have not been fully elucidated yet. Several important mechanisms underlying the antagonistic effects of probiotics on various microorganisms include the following: modification of the gut microbiota, competitive adherence to the mucosa and epithelium, strengthening of the gut epithelial barrier and modulation of the immune system to convey an advantage to the host. Accumulating evidence demonstrates that probiotics communicate with the host by pattern recognition receptors, such as toll-like receptors and nucleotide-binding oligomerization domain-containing protein-like receptors, which modulate key signaling pathways, such as nuclear factor-ĸB and mitogen-activated protein kinase, to enhance or suppress activation and influence downstream pathways. This recognition is crucial for eliciting measured antimicrobial responses with minimal inflammatory tissue damage. A clear understanding of these mechanisms will allow for appropriate probiotic strain selection for specific applications and may uncover novel probiotic functions. The goal of this systematic review was to explore probiotic modes of action focusing on how gut microbes influence the host.

Background The action of probiotics has been studied in vitro in cells isolated from both mice and humans, particularly enterocytes (IECs), dendritic cells (DCs) and co-cultures of peripheral DCs and IECs. Peripheral DCs and murine DCs differ from human gut DCs, and to date there are no data on the action of any probiotic on co-cultured human IECs and human intestinal DCs. To address this issue, a novel transwell model was used. Human IECs (Caco-2 cells) grown in the upper chamber of transwell filters were co-cultured with intestinal-like human DCs grown in the basolateral compartment of the transwells. The system was apically exposed for 4 h to live probiotic L. paracasei CNCM I-4034 obtained from the faeces of breastfed infants or to its cell-free culture supernatant (CFS) and challenged with Salmonella typhi. The secretion of pro- and anti-inflammatory cytokines in the basolateral compartment was determined by immunoassay, and the DC expression pattern of 20 TLR signaling pathway genes was analysed by PCR array. Results The presence of the live probiotic alone significantly increased IL-1β, IL-6, IL-8, TGF-β2, RANTES and IP-10 levels and decreased IL-12p40, IL-10, TGF- β1 and MIP-1α levels. This release was correlated with a significant increase in the expression of almost all TLR signaling genes. By contrast, incubation of the co-culture with CFS increased IL-1β, IL-6, TGF-β2 and IP-10 production only when Salmonella was present. This induction was correlated with an overall decrease in the expression of all TLR genes except TLR9, which was strongly up-regulated. Conclusions The data presented here clearly indicate that L. paracasei CNCM I-4034 significantly increases the release of pro-inflammatory cytokines, enhances TLR signaling pathway activation and stimulates rather than suppresses the innate immune system. Furthermore, our findings provide evidence that the effects of probiotics in the presence of IECs and DCs differ from the effects of probiotics on cultures of each cell type alone, as reported by us earlier. Thus, co-culture systems such as the one described here are needed to characterise the effects of probiotics in vitro, highlighting the potential utility of such co-cultures as a model system. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0408-6) contains supplementary material, which is available to authorized users.

The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance to the commensal microbiota and food antigens. Different strains of probiotics possess the ability to finely regulate the activation of dendritic cells (DC), polarising the subsequent activity of T-cells. Nevertheless, information about their underlying mechanisms of action is scarce. In the present study, we investigated the immunomodulatory effects of a potentially probiotic strain,Lactobacillus rhamnosusCNCM I-4036, and its cell-free culture supernatant (CFS) on human DC challenged withEscherichia coli. The results showed that the levels of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8 and IL-12p70 were higher in the cells treated with liveL. rhamnosusthan in the cells treated with the CFS. In the presence ofE. coli, the supernatant was more effective than the probiotic bacteria in reducing the secretion of pro-inflammatory cytokines. In addition, liveL. rhamnosuspotently induced the production of transforming growth factor (TGF)-β1 and TGF-β2, whereas the CFS increased the secretion of TGF-β1. However, in the presence ofE. coli, both treatments restored the levels of TGF-β. The probiotic strainL. rhamnosusCNCM I-4036 and its CFS were able to activate the Toll-like receptor signalling pathway, enhancing innate immunity. The two treatments induced gene transcription ofTLR-9. LiveL. rhamnosusactivated the expression ofTLR-2andTLR-4genes, whereas the CFS increased the expression ofTLR-1andTLR-5genes. In response to the stimulation with probiotic/CFS andE. coli, the expression of each gene tested was notably increased, with the exception ofTNF-αandNFKBIA. In conclusion, the CFS exhibited an extraordinary ability to suppress the production of pro-inflammatory cytokines by DC, and may be used as an effective and safer alternative to live bacteria.

We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Leprfa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Leprfa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Leprfa/fa rats that received probiotics than in rats fed the placebo. Zucker-Leprfa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Leprfa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean+/fa rats. Serum TNF-α levels decreased in the Zucker-Leprfa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Leprfa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats., Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3545 managed by the Fundacion General Empresa-Universidad de Granada.

Numerousin vitroandin vivostudies conducted using different probiotic micro-organisms have demonstrated their ability to interfere with the growth and virulence of a variety of enteropathogens. The reported beneficial effects of the use of probiotics to complement antibiotic therapy or prevent diarrhoea or gastrointestinal infection in infants have increased in recent years. In the present study, we demonstrated the capacity of supernatants obtained from three novel probiotics (Lactobacillus paracaseiCNCM I-4034,Bifidobacterium breveCNCM I-4035 andLactobacillus rhamnosusCNCM I-4036) isolated from the faeces of breastfed infants to inhibit the growth of enterotoxigenic and enteropathogenic (EPEC) bacteria, such asEscherichia coli,SalmonellaandShigella. To assess their potential antimicrobial activity, the 17 and 24 h cell-free supernatants broth concentrates (10 × ) having 1, 2 or 4 % of the three probiotics were incubated with EPEC bacteria strains. After 17 h of co-culture, the supernatants were able to inhibit the growth ofE. coli,SalmonellaandShigellaup to 40, 55 and 81 %, respectively. However, the inhibitory capacity of some supernatants was maintained or completely lost when the supernatants (pH 3·0) were neutralised (pH 6·5). Overall, these results demonstrated thatL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036 produce compounds that exhibited strain-specific inhibition of enterobacteria and have the potential to be used as probiotics in functional foods.

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro-inflammatory pathways., This study was supported by Hero Spain S. A. through a number 3143 contract signed with the Fundación General Universidad de Granada Empresa and co-sponsored by a CDTI project, a public entity of the Ministry of Economy and Competitiveness of the Spanish Government.

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs) are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs) and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells) harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants, Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS) decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-β2 secretion, whereas the supernatant increased the secretion of TGF-β1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR) signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods., This work was funded by Hero Spain S.A. through the contract n° 3143 signed with the Fundación General Universidad de Granada Empresa and co-sponsored by the CDTI, a public entity of the Ministry of Economy and Competitiveness of the Spanish Government. Carolina Gomez-Llorente received a postdoctoral fellowship from Plan Propio of University of Granada.