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Table 1: Experimental procedures in mice Primers used in RT-PCRFigure S1: Colitis-associated carcinogenesis is more severe in CX3CR1GFP/GFP (KO mice) Figure S2: Macrophages from CX3CR1GFP/GFP mice are able to produce and sense IL10 Figure S3: CX3CR1 signaling induces STAT3 phosphorylation Figure S4: Sequencing of fecal microbiota of CX3CR1GFP/ (WT) and CX3CR1GFP/GFP (KO) mice (Figure S5: Effect of pharmacological treatments with the HMOX-1 inhibitor znPP (red) or the HMOX-1 stimulator coPP (blue) on the inflammation induced by 1 administration of DSS in WT mice Figure S6: Hematoxylin and eosin staining of CX3CR1GFP/ (WT) and CX3CR1GFP/GFP (KO) colon treated with coPP Figure S7: Double immunofluorescence on sections of adenomatous polyps from mice undergoing the AOM-DSS protocol

CX3CR1+ macrophages in the intestinal lamina propria contribute to gut homeostasis through the immunomodulatory interleukin IL10, but there is little knowledge on how these cells or the CX3CR1 receptor may affect colorectal carcinogenesis. In this study, we show that CX3CR1-deficient mice fail to resolve gut inflammation despite high production of IL10 and have increased colitis and adenomatous polyps in chemical and genetic models of colon carcinogenesis. Mechanistically, CX3CL1-mediated engagement of the CX3CR1 receptor induced upregulation of heme-oxygenase-1 (HMOX-1), an antioxidant and anti-inflammatory enzyme. CX3CR1-deficient mice exhibited significantly lower expression of HMOX-1 in their adenomatous colon tissues. Combining LPS and CX3CL1 displayed a strong synergistic effect in vitro, but HMOX-1 levels were significantly lower in KO macrophages. Cohousing of wild-type and CX3CR1−/− mice during the AOM/DSS treatment attenuated disease severity in CX3CR1−/− mice, indicating the importance of the microbiome, but did not fully reinstate HMOX-1 levels and did not abolish polyp formation. In contrast, pharmacologic induction of HMOX-1 in vivo by cobalt protoporphyrin-IX treatment eradicated intestinal inflammation and fully protected KO mice from carcinogenesis. Taken together, our results establish an essential role for the receptor CX3CR1 in gut macrophages in resolving inflammation in the intestine, where it helps protects against colitis-associated cancer by regulating HMOX-1 expression. Cancer Res; 77(16); 4472–85. ©2017 AACR.

Reportedly, Western-type diets may induce the loss of key microbial taxa within the gastrointestinal microbiota, promoting the onset of noncommunicable diseases. It was hypothesized that the consumption of raw vegetables could contribute to the maintenance of the intestinal microbial community structure. In this context, we explored bacteria associated with commercial rocket salads produced through different farming practices: traditional (conventional, organic, and integrated) and vertical farming. Viable counts of mesophilic bacteria and lactic acid bacteria (LAB) were performed on plate count agar (PCA) and de Man-Rogosa-Sharpe (MRS) agar at pH 5.7, whereas metataxonomics through 16S rRNA gene sequencing was used to profile total bacteria associated with rocket salads. We found that rocket salads from vertical farming had much fewer viable bacteria and had a bacterial community structure markedly different from that of rocket salads from traditional farming. Furthermore, although α- and β-diversity analyses did not differentiate rocket samples according to farming techniques, several bacterial taxa distinguished organic and integrated from conventional farming salads, suggesting that farming practices could affect the taxonomic composition of rocket bacterial communities. LAB were isolated from only traditional farming samples and belonged to different species, which were variably distributed among samples and could be partly associated with farming practices. Finally, the INFOGEST protocol for

Purpose Aging can be characterized by increased systemic low-grade inflammation, altered gut microbiota composition, and increased intestinal permeability (IP). The intake of polyphenol-rich foods is proposed as a promising strategy to positively affect the gut microbiota-immune system-intestinal barrier (IB) axis. In this context, we tested the hypothesis that a PR-dietary intervention would affect the presence of bacterial factors in the bloodstream of older adults. Methods We collected blood samples within a randomized, controlled, crossover intervention trial in which older volunteers (n = 51) received a polyphenol-enriched and a control diet. We quantified the presence of bacterial DNA in blood by qPCR targeting the 16S rRNA gene (16S; bacterial DNAemia). Blood DNA was taxonomically profiled via 16S sequencing. Results Higher blood 16S levels were associated with higher BMI and markers of IP, inflammation, and dyslipidemia. PR-intervention did not significantly change bacterial DNAemia in the older population (P = 0.103). Nonetheless, the beneficial changes caused by the polyphenol-enriched diet were greatest in participants with higher bacterial DNAemia, specifically in markers related to IP, inflammation and dyslipidemia, and in fecal bacterial taxa. Finally, we found that the bacterial DNA detected in blood mostly belonged to γ-Proteobacteria, whose abundance significantly decreased after the polyphenol-rich diet in subjects with higher bacterial DNAemia at baseline. Conclusions This study shows that older subjects with higher bacterial DNAemia experienced a beneficial effect from a polyphenol-rich diet. Bacterial DNAemia may be a further relevant marker for the identification of target populations that could benefit more from a protective dietary treatment. Registration This trial was retrospectively registered at www.isrctn.org (ISRCTN10214981) on April 28, 2017.

Probiotic microorganisms may benefit the host by influencing diverse physiological processes, whose nature and underlying mechanisms are still largely unexplored. Animal models are a unique tool to understand the complexity of the interactions between probiotic microorganisms, the intestinal microbiota, and the host. In this regard, in this pilot study, we compared the effects of 5-day administration of three different probiotic bacterial strains (Bifidobacterium bifidum MIMBb23sg, Lactobacillus helveticus MIMLh5, and Lacticaseibacillus paracasei DG) on three distinct murine intestinal sites (ileum, cecum, and colon). All probiotics preferentially colonized the cecum and colon. In addition, probiotics reduced in the ileum and increased in the cecum and colon the relative abundance of numerous bacterial taxonomic units. MIMBb23sg and DG increased the inducible nitric oxide synthase (iNOS) in the ileum, which is involved in epithelial homeostasis. In addition, MIMBb23sg upregulated cytokine IL-10 in the ileum and downregulated the cyclooxygenase COX-2 in the colon, suggesting an anti-inflammatory/regulatory activity. MIMBb23sg significantly affected the expression of the main gene involved in serotonin synthesis (TPH1) and the gene coding for the serotonin reuptake protein (SERT) in the ileum and colon, suggesting a potential propulsive effect toward the distal part of the gut, whereas the impact of MIMLh5 and DG on serotonergic genes suggested an effect toward motility control. The three probiotics decreased the expression of the permeability marker zonulin in gut distal sites. This preliminary in vivo study demonstrated the safety of the tested probiotic strains and their common ability to modulate the intestinal microbiota. The probiotics affected host gene expression in a strain-specific manner. Notably, the observed effects in the gut were site dependent. This study provides a rationale for investigating the effects of probiotics on the serotonergic system, which is a topic still widely unexplored.

Chlorogenic acid (CGA) and caffeic acid (CA) are bioactive compounds in whole grains, berries, apples, some citrus fruits and coffee, which are hypothesized to promote health-beneficial effects on the cardiovascular system. This study aimed to evaluate the capacity of CGA and CA to reduce lipid accumulation in macrophages, recognized as a critical stage in the progression of atherosclerosis. Furtherly, the modulation of CCAAT/enhancer-binding protein β (C/EBPβ) and peroxisome proliferator-activated receptor- γ1 (PPAR-γ1), as transcription factors involved in lipid metabolism, was evaluated. THP-1-derived macrophages were treated for 24 h with 0.03, 0.3, 3 and 30 μM of CGA and CA, tested alone or in combination, and a solution of oleic/palmitic acid (500 μM, 2:1 ratio). Lipid storage was assessed spectrophotometrically through fluorescent staining of cells with Nile red. C/EBPβ and PPAR-γ1 mRNA and protein levels were evaluated by RT-PCR and enzyme-linked immunosorbent assay, respectively. The mix of CGA + CA (1:1 ratio) reduced lipid accumulation at all concentrations tested, except for the highest one. The greatest effect ( − 65%; p

We appreciate the intent by Salminen et al. to clarify and reorganize the nomenclature regarding the use of inactivated bacteria and their products as health-promoting factors (Salminen, S. et al. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. 18, 649–667 (2021))1. Nonetheless, we found several major caveats in the consensus statement (Box 1) that might generate ambiguity.

Oral consumption of probiotics is practical and can be an effective solution to preserve vaginal eubiosis. Here, we studied the ability of orally administered Lactobacillus paracasei LPC-S01 (DSM 26760) to affect the composition of the vaginal microbiota and colonize the vaginal mucosa in nondiseased adult women. A total of 40 volunteers took oral probiotic (24 billion CFU) or placebo capsules daily for 4 weeks, and after a 4-week washout, they switched to placebo or probiotic capsules according to the crossover design. A total of 23 volunteers completed the study according to the protocol. Before and after capsule ingestion, vaginal swabs were collected for qPCR quantification to detect L. paracasei LPC-S01 and for 16S rRNA gene sequencing. Vaginal swabs were grouped according to their bacterial taxonomic structure into nine community state types (CSTs), four of which were dominated by lactobacilli. Lactobacillus paracasei LPC-S01 was detected in the vagina of two participants. Statistical modeling (including linear mixed-effects model analysis) demonstrated that daily intake of probiotic capsules reduced the relative abundance of Gardnerella spp. Quantitative PCR with Gardnerella vaginalis primers confirmed this result. Considering the pathogenic nature of G. vaginalis, these results suggest a potential positive effect of this probiotic capsule on the vaginal microbial ecosystem., This study aims to assess whether a lactobacillus of vaginal origin can benefit the eubosis of the vaginal mucosa of childbearing age woman upon oral intake.

The increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from a group of forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all the older volunteers contained detectable amounts of bacterial DNA in their blood. The total amount of 16S rRNA gene copies varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly correlated with the serum levels of zonulin, an emerging marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected after approximately four months from the same subjects. Analyses of 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of Pseudomonadaceae and Enterobacteriaceae. Several control samples were also analyzed to assess the influence exerted by contaminant bacterial DNA potentially originating from reagents and materials. The date reported here suggest that para-cellular permeability of epithelial (and potentially also endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

The consumption of probiotic products is continually increasing, supported by growing scientific evidence of their efficacy. Considering that probiotics may primarily affect health (either positively or negatively) through gut microbiota modulation, the first aspect that should be evaluated is their impact on the intestinal microbial ecosystem. In this study, we longitudinally analyzed the bacterial taxonomic composition and organic acid levels in four fecal samples collected over the course of four weeks from 19 healthy adults who ingested one capsule a day for two weeks of a formulation containing at least 70 billion colony-forming units, consisting of 25% lactobacilli and 75% Bifidobacterium animalis subsp. lactis. We found that 16S rRNA gene profiling showed that probiotic intake only induced an increase in a single operational taxonomic unit ascribed to B. animalis, plausibly corresponding to the ingested bifidobacterial strain. Furthermore, liquid chromatography/mass spectrometry revealed a significant increase in the lactate and acetate/butyrate ratio and a trend toward a decrease in succinate following probiotic administration. The presented results indicate that the investigated probiotic formulation did not alter the intestinal bacterial ecosystem of healthy adults and suggest its potential ability to promote colonization resistance in the gut through a transient increase in fecal bifidobacteria, lactic acid, and the acetate/butyrate ratio.

Polyphenols are bioactive molecules widely distributed in numerous foods such as fruits, vegetables, tea, coffee, cocoa and beverages. Their main classification include flavonoids (i.e. flavonols, flavones, flavanones, flavanols, anthocyanins, and isoflavones), non-flavonoids (i.e. lignans and stilbens) and phenolic acids (i.e. hydroxycinnamic and hydroxybenzoic acids)(1). Caffeic acid (CA) and chlorogenic acid (CGA; an ester of CA and quinic acid) are the major representatives of hydroxycinnamic acids. Accumulating evidence has demonstrated that CA and CGA may exert different biological activities, including antioxidant, anti-inflammatory, antidiabetic, and antihypertensive(2). Despite these promising and diverse anti-atherosclerotic actions, investigations addressing the effect of CA and CGA on atherogenesis are scarce.The present study evaluated the capacity of CA and CGA to reduce lipid accumulation in macrophages derived from monocytic THP-1 cells. THP-1-derived macrophages were incubated with fatty acids (500 μM oleic/palmitic acid, 2:1 ratio) and different concentrations (from 0.03 to 3 μM) of CA and CGA, alone or in combination. Lipid accumulation was quantified spectrophotometrically (excitation: 544 nm, emission: 590 nm) with the fluorescent dye, Nile red. The fold increase compared to the control (without fatty acids) was calculated. In addition, the expression of several transcription factors including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein (CEBP), as potential mechanisms involved in the regulation of lipid accumulation, was evaluated by real time PCR.Analysis of variance (ANOVA) was used to assess the effect of the different concentrations of CA and CGA on lipid accumulation in THP-1 macrophages following stimulation with FA.The preliminary results obtained have shown a significant increase in lipid accumulation following fatty acid exposure (p < 0.0001). Incubation with CA and CGA did not reduce lipid accumulation in THP-1 derived macrophages, while the combination of CA + CGA at 0.03, 0.3 and 3 μM (p < 0.01) decreased cellular fatty acid uptake at all concentrations tested by -28%, -32%, -23%, respectively. An apparent modulation of the transcriptional activity of PPARγ, but not CEBP, was observed following the combination of phenolic acids.In conclusion, the incubation of CA + CGA at physiologically relevant concentrations, but not the single compounds, seem to reduce the uptake of fatty acids in THP-1-derived macrophages. Further experiments are ongoing in order to confirm the findings obtained and to better identify the mechanisms of action involved in the reduction of lipid accumulation as a key phenomenon of atherogenesis.

Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is classified according to bowel habits as IBS with constipation (IBS-C), with diarrhea (IBS-D), with alternating constipation and diarrhea (IBS-M), and unsubtyped (IBS-U). The mechanisms leading to the different IBS forms are mostly unknown. This study aims to evaluate whether specific fecal bacterial taxa and/or short-chain fatty acids (SCFAs) can be used to distinguish IBS subtypes and are relevant for explaining the clinical differences between IBS subcategories. We characterized five fecal samples collected at 4-weeks intervals from 40 IBS patients by 16S rRNA gene profiling and SCFA quantification. Finally, we investigated the potential correlations in IBS subtypes between the fecal microbial signatures and host physiological and clinical parameters. We found significant differences in the distribution of Clostridiales OTUs among IBS subtypes and reduced levels of SCFAs in IBS-C compared to IBS-U and IBS-D patients. Correlation analyses showed that the diverse representation of Clostridiales OTUs between IBS subtypes was associated with altered levels of SCFAs; furthermore, the same OTUs and SCFAs were associated with the fecal cytokine levels and stool consistency. Our results suggest that intestinal Clostridiales and SCFAs might serve as potential mechanistic biomarkers of IBS subtypes and represent therapeutic targets.

CX3CR1+ macrophages in the intestinal lamina propria contribute to gut homeostasis through the immunomodulatory interleukin IL10, but there is little knowledge on how these cells or the CX3CR1 receptor may affect colorectal carcinogenesis. In this study, we show that CX3CR1-deficient mice fail to resolve gut inflammation despite high production of IL10 and have increased colitis and adenomatous polyps in chemical and genetic models of colon carcinogenesis. Mechanistically, CX3CL1-mediated engagement of the CX3CR1 receptor induced upregulation of heme-oxygenase-1 (HMOX-1), an antioxidant and anti-inflammatory enzyme. CX3CR1-deficient mice exhibited significantly lower expression of HMOX-1 in their adenomatous colon tissues. Combining LPS and CX3CL1 displayed a strong synergistic effect in vitro, but HMOX-1 levels were significantly lower in KO macrophages. Cohousing of wild-type and CX3CR1−/− mice during the AOM/DSS treatment attenuated disease severity in CX3CR1−/− mice, indicating the importance of the microbiome, but did not fully reinstate HMOX-1 levels and did not abolish polyp formation. In contrast, pharmacologic induction of HMOX-1 in vivo by cobalt protoporphyrin-IX treatment eradicated intestinal inflammation and fully protected KO mice from carcinogenesis. Taken together, our results establish an essential role for the receptor CX3CR1 in gut macrophages in resolving inflammation in the intestine, where it helps protects against colitis-associated cancer by regulating HMOX-1 expression. Cancer Res; 77(16); 4472–85. ©2017 AACR.

Group A streptococci (GAS) cause 20-30% of pediatric pharyngitis episodes, which are a major cause of ambulatory care visits. Therefore, a strategy to prevent GAS dissemination in children could significantly benefit public healthcare. Contextually, we assessed the possibility of employing alternative food-grade strategies to be used with the oral probiotic L. helveticus MIMLh5 for the prevention of pharyngeal infections. First, we demonstrated through an antagonism-by-exclusion assay that guaran may potentially prevent S. pyogenes adhesion on pharyngeal cells. Subsequently, we showed that an anthocyanin-rich fraction extracted from wild blueberry (BbE) exerts anti-inflammatory effects on the human macrophage cell line U937. Finally, we showed that BbE reduces interferon-β expression in MIMLh5-stimulated murine dendritic cells, resulting in a reduction in the pro-inflammatory cytokines IL-12 and TNF-α. In conclusion, this proof-of-concept study indicates that different food-grade strategies may be concomitantly adopted to potentially prevent GAS colonization and modulate local immune defences.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

The mannose receptor (MR) is a C-type lectin involved in endocytosis and with a poorly defined ability to modulate cellular activation. We investigated the effect of mannan treatment prior to stimulation of murine bone marrow-derived dendritic cells with the Gram-positive bacteria Lactobacillus acidophilus NCFM (L. acidophilus) on the induction of Interleukin (IL)-12. Mannan enhanced the IL-12 production induced by L. acidophilus in a dose dependent manner (up to 230% enhancement). Additionally, mannan-enhanced IL-12 induction was also demonstrated with another Gram-positive bacteria, Staphylococcus aureus (S. aureus), while an IL-12 reducing effect was seen on Escherichia coli stimulated cells. Furthermore, the expression of Interferon β (Ifnb) was increased in cells treated with mannan prior to stimulation with L. acidophilus. The addition of mannan but not of bacteria led to endocytosis of MR, while addition of mannan prior to L. acidophilus or S. aureus resulted in increased endocytosis of bacteria, a faster killing of endocytosed bacteria, and increased reactive oxygen species production. Expression of signaling lymphocytic activation molecule (SLAMF)1 shown previously to be involved in the facilitation of endosomal degradation was upregulated by mannan but not by L. acidophilus and S. aureus. The IL-12 enhancement by mannan but not the IL-12 induced by the bacteria was abrogated by addition of inhibitors of clathrin coated pits (chlorpromazine and monodansylcadaverine). Furthermore, the addition of acid sphingomyelinase, a facilitator of ceramide raft formation, prior to addition of L. acidophilus enhanced the IL-12 production and the endocytosis of bacteria. In summary, our results show that mannan increases the IL-12 production induced by some Gram-positive bacteria through MR-endocytosis, which increases bacterial endocytosis and endosomal killing. The differential effect of MR activation on the IL-12 production induced by Gram-positive and Gram-negative bacteria may influence the immune response toward allergens and other glycoproteins.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

Rigoni et al. report that hypomorphic Rag2R229Q mutation is associated with altered microbiota composition and defects in the gut–blood barrier and suggest that intestinal microbes may play a critical role in the distinctive immune dysregulation of Omenn syndrome., Omenn syndrome (OS) is caused by hypomorphic Rag mutations and characterized by a profound immunodeficiency associated with autoimmune-like manifestations. Both in humans and mice, OS is mediated by oligoclonal activated T and B cells. The role of microbial signals in disease pathogenesis is debated. Here, we show that Rag2R229Q knock-in mice developed an inflammatory bowel disease affecting both the small bowel and colon. Lymphocytes were sufficient for disease induction, as intestinal CD4 T cells with a Th1/Th17 phenotype reproduced the pathological picture when transplanted into immunocompromised hosts. Moreover, oral tolerance was impaired in Rag2R229Q mice, and transfer of wild-type (WT) regulatory T cells ameliorated bowel inflammation. Mucosal immunoglobulin A (IgA) deficiency in the gut resulted in enhanced absorption of microbial products and altered composition of commensal communities. The Rag2R229Q microbiota further contributed to the immunopathology because its transplant into WT recipients promoted Th1/Th17 immune response. Consistently, long-term dosing of broad-spectrum antibiotics (ABXs) in Rag2R229Q mice ameliorated intestinal and systemic autoimmunity by diminishing the frequency of mucosal and circulating gut-tropic CCR9+ Th1 and Th17 T cells. Remarkably, serum hyper-IgE, a hallmark of the disease, was also normalized by ABX treatment. These results indicate that intestinal microbes may play a critical role in the distinctive immune dysregulation of OS.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production. IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

Modulation of the intestinal microbial ecosystem (IME) is a useful target to establish probiotic efficacy in a healthy population. We conducted a randomized, double-blind, crossover, and placebo-controlled intervention study to determine the impact of Bifidobacterium bifidum strain Bb on the IME of adult healthy volunteers of both sexes. High-throughput 16S rRNA gene sequencing was used to characterize the fecal microbiota before and after 4 weeks of daily probiotic cell consumption. The intake of approximately one billion live B. bifidum cells affected the relative abundance of dominant taxa in the fecal microbiota and modulated fecal butyrate levels. Specifically, Prevotellaceae ( P = 0.041) and Prevotella ( P = 0.034) were significantly decreased, whereas Ruminococcaceae ( P = 0.039) and Rikenellaceae ( P = 0.010) were significantly increased. We also observed that the probiotic intervention modulated the fecal concentrations of butyrate in a manner dependent on the initial levels of short-chain fatty acids (SCFAs). In conclusion, our study demonstrates that a single daily administration of Bifidobacterium bifidum strain Bb can significantly modify the IME in healthy (not diseased) adults. These findings demonstrate the need to reassess the notion that probiotics do not influence the complex and stable IME of a healthy individual. IMPORTANCE Foods and supplements claimed to contain health-promoting probiotic microorganisms are everywhere these days and mainly intended for consumption by healthy people. However, it is still debated what actual effects probiotic products may have on the healthy population. In this study, we report the results of an intervention trial aimed at assessing the modifications induced in the intestinal microbial ecosystem of healthy adults from the consumption of a probiotic product. Our results demonstrate that the introduction of a probiotic product in the dietary habits of healthy people may significantly modify dominant taxa of the intestinal microbiota, resulting in the modulation of short-chain fatty acid concentrations in the gut. The overall changes witnessed in the probiotic intervention indicate a mechanism of microbiota modulation that could have potential effects on human health.

The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1β levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-β1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo.

Background Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn syndrome (OS). Objective The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified. Methods We analyzed a cohort of 15 patients with OS and the 129Sv/C57BL/6 knock-in Rag2R229Q/R229Q (Rag2R229Q) mouse model. Homing phenotypes of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt. Results We show that memory/activated T cells from patients with OS and from the Rag2R229Q mouse model of OS abundantly express the skin homing receptors cutaneous lymphocyte associated antigen and CCR4 (Ccr4), associated with high levels of chemokine C-C motif ligands 17 and 22. Serum levels of LPS are also elevated. A broad Th1/Th2/Th17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2R229Q mice is associated with enhanced cutaneous inflammation on local and systemic administration of LPS. Likewise, boosting colitis in Rag2R229Q mice results in increased frequency of Ccr4+ splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation, and dermal infiltration by Th1 effector T cells. Conclusions These results support the existence of an interplay between gut and skin that can sustain skin inflammation in OS., Graphical abstract

Alteration of the gut microbiota has been associated with different gastrointestinal disorders. Normobiosis restoration by faecal microbiota transplantation (FMT) is considered a promising therapeutic approach, even if the mechanisms underlying its efficacy are at present largely unknown. Here we sought to elucidate the functional effects of therapeutic FMT administration during experimental colitis on innate and adaptive immune responses in the intestinal mucosa. We show that therapeutic FMT reduces colonic inflammation and initiates the restoration of intestinal homeostasis through the simultaneous activation of different immune-mediated pathways, ultimately leading to IL-10 production by innate and adaptive immune cells, including CD4+ T cells, iNKT cells and Antigen Presenting Cells (APC), and reduces the ability of dendritic cells, monocytes and macrophages to present MHCII-dependent bacterial antigens to colonic T cells. These results demonstrate the capability of FMT to therapeutically control intestinal experimental colitis and poses FMT as a valuable therapeutic option in immune-related pathologies., Faecal microbiota transplantation (FMT) is becoming a therapeutic option in several gastrointestinal disorders. Here, Burrello et al. study the immunological mechanisms by which FMT reduces colonic inflammation and initiates the restoration of intestinal homeostasis in a mouse model of colitis.

Hyperlipidemia starts at a pediatric age and represents an unquestionable risk factor for cardiovascular disease. Modulation of the intestinal microbial ecosystem (IME), in principle, can ameliorate lipid profiles. In this study, we characterized the IME of children and adolescents with primary hyperlipidemia by analyzing fecal samples through 16S rRNA gene profiling (n = 15) and short chain fatty acid (SCFA) quantification (n = 32). The same analyses were also carried out on age-matched normolipidemic controls (n = 15). Moreover, we evaluated the modulatory effect of regular hazelnut intake (approximately 0.43 g of hazelnuts with skin per kg of body weight) on the IME of 15 children and adolescents with hyperlipidemia for eight weeks. We found alterations of numerous operational taxonomic units potentially associated with SCFA-producing bacteria and reductions in the fecal levels of acetate, butyrate and propionate in hyperlipidemic subjects. Furthermore, we observed that an eight-week hazelnut intervention may induce limited changes in fecal microbiota composition but can significantly modulate the fecal levels of predominant intestinal SCFAs, such as acetate. Finally, correlation analyses indicated that changes in lipidemic parameters are linked to modifications of the abundance of specific bacterial taxa, such as the families Lachnospiraceae and Ruminococcaceae and the genera Akkermansia, Bacteroides, Roseburia, and Faecalibacterium. This study suggests that children and adolescents with primary hyperlipidemia possess an altered IME. The promising results presented here support the need for future dietary interventions aimed at positively modulating the IME of hyperlipidemic subjects.

Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is classified according to bowel habits as IBS with constipation (IBS-C), with diarrhea (IBS-D), with alternating constipation and diarrhea (IBS-M), and unsubtyped (IBS-U). The mechanisms leading to the different IBS forms are mostly unknown. This study aims to evaluate whether specific fecal bacterial taxa and/or short-chain fatty acids (SCFAs) can be used to distinguish IBS subtypes and are relevant for explaining the clinical differences between IBS subcategories. We characterized five fecal samples collected at 4-weeks intervals from 40 IBS patients by 16S rRNA gene profiling and SCFA quantification. Finally, we investigated the potential correlations in IBS subtypes between the fecal microbial signatures and host physiological and clinical parameters. We found significant differences in the distribution of Clostridiales OTUs among IBS subtypes and reduced levels of SCFAs in IBS-C compared to IBS-U and IBS-D patients. Correlation analyses showed that the diverse representation of Clostridiales OTUs between IBS subtypes was associated with altered levels of SCFAs; furthermore, the same OTUs and SCFAs were associated with the fecal cytokine levels and stool consistency. Our results suggest that intestinal Clostridiales and SCFAs might serve as potential mechanistic biomarkers of IBS subtypes and represent therapeutic targets.

Background: Evidence suggests a role of intestinal microbiota-host interactions in the pathophysiology and symptoms of irritable bowel syndrome (IBS). Objective: The objective of this article is to assess the effects of Lactobacillus paracasei CNCM I-1572 on clinical and gut microbiota-related factors in IBS. Methods: We conducted a multicenter, randomized, double-blind, cross-over, 18-week, placebo-controlled, pilot trial assessing the effect of Lactobacillus paracasei CNCM I-1572 on symptoms, gut microbiota composition, fecal short chain fatty acid (SCFA), immunoglobulin A, and cytokines in IBS. The intestinal microbial ecosystem was characterized by 16S rRNA gene profiling. Results: Forty IBS patients were enrolled from five Italian centers. Lactobacillus paracasei CNCM I-1572 did not significantly improve IBS symptoms, including primary efficacy variables worst abdominal pain/discomfort and IBS degree of relief. Interestingly, Lactobacillus paracasei CNCM I-1572 induced a significant reduction in genus Ruminococcus, dominated by taxa related to Ruminococcus bromii and Ruminococcus callidus, a significant increase in the SCFAs acetate and butyrate, and a significant reduction in the pro-inflammatory cytokine interleukin-15. Conclusions: This pilot study shows that Lactobacillus paracasei CNCM I-1572 is able to modulate gut microbiota structure/function and reduce immune activation in IBS. As no statistically significant effect on IBS-symptoms was found, further studies are necessary to determine the role of this probiotic in IBS. The study was registered at ClinicalTrials.gov registry under identifier NCT02371499.

Lactobacillus casei group (Lcs) consists of three phylogenetically closely related species (L. casei, L. paracasei, and L. rhamnosus), which are widely used in the dairy and probiotic industrial sectors. Strategies to easily and rapidly characterize Lcs are therefore of interest. To this aim, we developed a method according to a technique known as high resolution melting analysis (HRMa), which was applied to a 150 bp groEL gene fragment. The analysis was performed on 53 Lcs strains and 29 strains representatives of species that are commonly present in dairy and probiotic products and can be most probably co-isolated with Lcs strains. DNA amplification was obtained only from Lcs strains, demonstrating the specificity of the groEL primers designed in this study. The HRMa clustered Lcs strains in three groups that exactly corresponded to the species of the L. casei group. A following HRMa separated the 39 L. paracasei strains in two well distinct intraspecific groups, indicating the possible existence of at least two distinct genotypes inside the species. Nonetheless, the phenotypic characterization demonstrated that the genotypes do not correspond to the two L. paracasei subspecies, namely paracasei and tolerans. In conclusion, the melting curve analysis developed in this study is demonstrably a simple, labor-saving, and rapid strategy obtain the genotyping of a bacterial isolate and simultaneously potentially confirm its affiliation to the L. casei group of species. The application of this method to a larger collection of strains may validate the possibility to use the proposed HRMa protocol for the taxonomic discrimination of L. casei group of species. In general, this study suggests that HRMa can be a suitable technique for the genetic typization of Lactobacillus strains.

Evidence suggests a role of intestinal microbiota-host interactions in the pathophysiology and symptoms of irritable bowel syndrome (IBS).The objective of this article is to assess the effects ofWe conducted a multicenter, randomized, double-blind, cross-over, 18-week, placebo-controlled, pilot trial assessing the effect ofForty IBS patients were enrolled from five Italian centers.This pilot study shows that

Lactobacillus paracasei DG is a bacterial strain with recognized probiotic properties and is used in commercial probiotic products. However, the mechanisms underlying its probiotic properties are mainly unknown. In this study, we tested the hypothesis that the ability of strain DG to interact with the host is at least partly associated with its ability to synthesize a surface-associated exopolysaccharide (EPS). Comparative genomics revealed the presence of putative EPS gene clusters in the DG genome; accordingly, EPS was isolated from the surface of the bacterium. A sample of the pure EPS from strain DG (DG-EPS), upon nuclear magnetic resonance (NMR) and chemical analyses, was shown to be a novel branched hetero-EPS with a repeat unit composed of l -rhamnose, d -galactose, and N -acetyl- d -galactosamine in a ratio of 4:1:1. Subsequently, we demonstrated that DG-EPS displays immunostimulating properties by enhancing the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and particularly that of the chemokines IL-8 and CCL20, in the human monocytic cell line THP-1. In contrast, the expression of the cyclooxygenase enzyme COX-2 was not affected. In conclusion, DG-EPS is a bacterial macromolecule with the ability to boost the immune system either as a secreted molecule released from the bacterium or as a capsular envelope on the bacterial cell wall. This study provides additional information about the mechanisms supporting the cross talk between L. paracasei DG and the host. IMPORTANCE The consumption of food products and supplements called probiotics (i.e., containing live microbial cells) to potentially prevent or treat specific diseases is constantly gaining popularity. The lack of knowledge on the precise mechanisms supporting their potential health-promoting properties, however, greatly limits a more appropriate use of each single probiotic strain. In this context, we studied a well-known probiotic, Lactobacillus paracasei DG, in order to identify the constitutive molecules that can explain the documented health-promoting properties of this bacterium. We found a novel polysaccharide molecule, named DG-EPS, that is secreted by and covers the bacterium. We demonstrated that this molecule, which has a chemical structure never identified before, has immunostimulatory properties and therefore may contribute to the ability of the probiotic L. paracasei DG to interact with the immune system.

BACKGROUND The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. OBJECTIVE This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. METHODS High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. RESULTS Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system.

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1 -like gene, named tgaA . Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.

Intestinal inflammation is a natural process crucial for the maintenance of gut functioning. However, abnormal or prolonged inflammatory responses may lead to the onset of chronic degenerative diseases, typically treated by means of pharmacological interventions. Dietary strategies for the prevention of inflammation are a safer alternative to pharmacotherapy. Anthocyanins and other polyphenols have been documented to display anti-inflammatory activity. In the present study, three bioactive fractions (anthocyanin, phenolic, and water-soluble fractions) were extracted from a wild blueberry powder. The Caco-2 intestinal model was used to test the immunomodulatory effect of the above fractions. Only the anthocyanin-rich fraction reduced the activation of NF-κB, induced by IL-1β in intestinal epithelial Caco-2 cells. Specifically, concentrations of 50 and 100 μg mL(-1) decreased NF-κB activation by 68.9 and 85.2%, respectively (p ≤ 0.05). These preliminary results provide further support for the role of food bioactives as potential dietary anti-inflammatory agents.

Dairy products are an important part of daily food in the Himalayan country of Nepal. A wide variety of traditional fermented milk products are obtained in relation to different geo-climatic conditions of the country and different ethnic groups. Locally called Dahi is the most popular product, but little data are available on the autochthonous lactic acid bacteria (LAB) characterizing this yogurt-like product. Thirty-two replicate samples of indigenous Dahi were collected from four districts of Nepal at different altitude. In total of 193 isolates were obtained. Of these, the 120 rod-shaped isolates representing the dominant LAB population (62%) were further identified at the species level by using internal transcribed spacers (ITS) amplification, 16S rRNA gene sequence determination, and species-specific PCR. A further characterization at the strain level was carried out by combining analysis of repetitive elements and randomly amplified polymorphic DNA (RAPD) typing. Based on these analyses, the isolates were grouped in ten different species, among which Lactobacillus delbrueckii subsp. bulgaricus, L. paracasei, and L. rhamnosus represented the dominant species. A high degree of intraspecies diversity was also observed for all Lactobacillus species, except for L. rhamnosus isolates, which proved to give a single typing profile. Bacterial isolates represent a source of novel potential probiotics and starter cultures. The strain collection obtained from this study is a first step in the preservation of the natural biodiversity of bacterial population of the traditional Nepalese fermented Dahi.

Here, we describe data obtained from transcriptome profiling of human cell lines and intestinal cells of a murine model upon exposure and colonization, respectively, with Bifidobacterium bifidum PRL2010. Significant changes were detected in the transcription of genes that are known to be involved in innate immunity. Furthermore, results from enzyme-linked immunosorbent assays (ELISAs) showed that exposure to B. bifidum PRL2010 causes enhanced production of interleukin 6 (IL-6) and IL-8 cytokines, presumably through NFκB activation. The obtained global transcription profiles strongly suggest that Bifidobacterium bifidum PRL2010 modulates the innate immune response of the host. © 2014, american Society for Microbiology. All Rights Reserved., We thank GenProbio srl for financial support of the Laboratory of Probiogenomics. This work was financially supported by Fondazione Cariplo (grant 2010-0678 to S.G. and V.T.) and by a FEMS Jensen Award to F.T. This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under grant SFI/12/RC/2273. The grant ILINK2010-0122, funded by CSIC, is also acknowledged.

Most intervention studies on probiotics or prebiotics consist of several-week feeding periods and focus on specific host dysfunctions. However, probiotics are often consumed by healthy people and time can be limited; the efficacy of such dietary intervention, however, has only rarely been considered. In this paper, we report the results of a short-term (1 week) study, in which 11 healthy subjects consumed a commercial probiotic food supplement, resulting in a daily intake of about 6 billion viable cells of 10 bacterial species. We measured by quantitative real-time PCR (qPCR) the impact that this product had on faecal Bifidobacterium and Lactobacillus spp. Faecal lactobacilli and bifidobacteria, whose number in the gut is a commonly considered parameter to assess the efficacy of a probiotic/prebiotic intervention, were not significantly affected by the intervention. The only significant increase was observed for intestinal Lactobacillus acidophilus group. However, the significance of this change disappeared after only few days, indicating that it was plausibly due to DNA carryover from the ingestion of a large number of L. acidophilus cells. According to this study, short-term intake of a quite high number of live probiotics can be ineffective in healthy humans on the faecal concentration of bifidobacteria and lactobacilli.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.