Reduction of product composition variability using pooled microbiome ecosystem therapy and consequence in two infectious murine models.

Growing evidence demonstrates the key role of the gut microbiota in human health and disease. The recent success of microbiotherapy products to treat recurrent Clostridioides difficile infection has shed light on its potential in conditions associated with gut dysbiosis, such as acute graft-versus-host disease, intestinal bowel diseases, neurodegenerative diseases, or even cancer. However, the difficulty in defining a "good" donor as well as the intrinsic variability of donor-derived products' taxonomic composition limits the translatability and reproducibility of these studies. Thus, the pooling of donors' feces has been proposed to homogenize product composition and achieve higher taxonomic richness and diversity. In this study, we compared the metagenomic profile of pooled products to corresponding single donor-derived products. We demonstrated that pooled products are more homogeneous, diverse, and enriched in beneficial bacteria known to produce anti-inflammatory short chain fatty acids compared to single donor-derived products. We then evaluated pooled products' efficacy compared to corresponding single donor-derived products in Salmonella and C. difficile infectious mouse models. We were able to demonstrate that pooled products decreased pathogenicity by inducing a structural change in the intestinal microbiota composition. Single donor-derived product efficacy was variable, with some products failing to control disease progression. We further performed in vitro growth inhibition assays of two extremely drug-resistant bacteria, Enterococcus faecium vanA and Klebsiella pneumoniae oxa48, supporting the use of pooled microbiotherapies. Altogether, these results demonstrate that the heterogeneity of donor-derived products is corrected by pooled fecal microbiotherapies in several infectious preclinical models.IMPORTANCEGrowing evidence demonstrates the key role of the gut microbiota in human health and disease. Recent Food and Drug Administration approval of fecal microbiotherapy products to treat recurrent Clostridioides difficile infection has shed light on their potential to treat pathological conditions associated with gut dysbiosis. In this study, we combined metagenomic analysis with in vitro and in vivo studies to compare the efficacy of pooled microbiotherapy products to corresponding single donor-derived products. We demonstrate that pooled products are more homogeneous, diverse, and enriched in beneficial bacteria compared to single donor-derived products. We further reveal that pooled products decreased Salmonella and Clostridioides difficile pathogenicity in mice, while single donor-derived product efficacy was variable, with some products failing to control disease progression. Altogether, these findings support the development of pooled microbiotherapies to overcome donor-dependent treatment efficacy.

Intestinal Microbiota Increases Cell Proliferation of Colonic Mucosa in Human-Flora-Associated (HFA) Mice.

Intestinal epithelium renewal strictly depends on fine regulation between cell proliferation, differentiation, and apoptosis. While murine intestinal microbiota has been shown to modify some epithelial cell kinetics parameters, less is known about the role of the human intestinal microbiota. Here, we investigated the rate of intestinal cell proliferation in C3H/HeN germ-free mice associated with human flora (HFA, n = 8), and in germ-free (n = 15) and holoxenic mice (n = 16). One hour before sacrifice, all mice were intraperitoneally inoculated with 5-bromodeoxyuridine (BrdU), and the number of BrdU-positive cells/total cells (labelling index, LI), both in the jejunum and the colon, was evaluated by immunohistochemistry. Samples were also observed by scanning electron microscopy (SEM). Moreover, the microbiota composition in the large bowel of the HFA mice was compared to that of of human donor's fecal sample. No differences in LI were found in the small bowels of the HFA, holoxenic, and germ-free mice. Conversely, the LI in the large bowel of the HFA mice was significantly higher than that in the germ-free and holoxenic counterparts (p = 0.017 and p = 0.048, respectively). In the holoxenic and HFA mice, the SEM analysis disclosed different types of bacteria in close contact with the intestinal epithelium. Finally, the colonic microbiota composition of the HFA mice widely overlapped with that of the human donor in terms of dominant populations, although Bifidobacteria and Lactobacilli disappeared. Despite the small sample size analyzed in this study, these preliminary findings suggest that human intestinal microbiota may promote a high proliferation rate of colonic mucosa. In light of the well-known role of uncontrolled proliferation in colorectal carcinogenesis, these results may deserve further investigation in a larger population study.

A Pulmonary Lactobacillus murinus Strain Induces Th17 and RORγt+ Regulatory T Cells and Reduces Lung Inflammation in Tuberculosis.

The lungs harbor multiple resident microbial communities, otherwise known as the microbiota. There is an emerging interest in deciphering whether the pulmonary microbiota modulate local immunity, and whether this knowledge could shed light on mechanisms operating in the response to respiratory pathogens. In this study, we investigate the capacity of a pulmonary Lactobacillus strain to modulate the lung T cell compartment and assess its prophylactic potential upon infection with Mycobacterium tuberculosis, the etiological agent of tuberculosis. In naive mice, we report that a Lactobacillus murinus (Lagilactobacillus murinus) strain (CNCM I-5314) increases the presence of lung Th17 cells and of a regulatory T cell (Treg) subset known as RORγt+ Tregs. In particular, intranasal but not intragastric administration of CNCM I-5314 increases the expansion of these lung leukocytes, suggesting a local rather than systemic effect. Resident Th17 and RORγt+ Tregs display an immunosuppressive phenotype that is accentuated by CNCM I-5314. Despite the well-known ability of M. tuberculosis to modulate lung immunity, the immunomodulatory effect by CNCM I-5314 is dominant, as Th17 and RORγt+ Tregs are still highly increased in the lung at 42-d postinfection. Importantly, CNCM I-5314 administration in M. tuberculosis-infected mice results in reduction of pulmonary inflammation, without increasing M. tuberculosis burden. Collectively, our findings provide evidence for an immunomodulatory capacity of CNCM I-5314 at steady state and in a model of chronic inflammation in which it can display a protective role, suggesting that L. murinus strains found in the lung may shape local T cells in mice and, perhaps, in humans.

The microbiota plays a critical role in the reactivity of lung immune components to innate ligands.

The gut microbiota contributes to shaping efficient and safe immune defenses in the gut. However, little is known about the role of the gut and/or lung microbiota in the education of pulmonary innate immune responses. Here, we tested whether the endogenous microbiota in general can modulate the reactivity of pulmonary tissue to pathogen stimuli by comparing the response of specific-pathogen-free (SPF) and germ-free (GF) mice. Thus, we observed earlier and greater inflammation in the pulmonary compartment of GF mice than that of SPF mice after intranasal instillation to lipopolysaccharide (LPS), a component of Gram-negative bacteria. Toll-like receptor 4 (TLR4) was more abundantly expressed in the lungs of GF mice than those of SPF mice at steady state, which could predispose the innate immunity of GF mice to strongly react to the environmental stimuli. Lung explants were stimulated with different TLR agonists or infected with the human airways pathogen, respiratory syncytial virus (RSV), resulting in greater inflammation under almost all conditions for the GF explants. Finally, alveolar macrophages (AM) from GF mice presented a higher innate immune response upon RSV infection than those of SPF mice. Overall, these data suggest that the presence of microbiota in SPF mice induced a process of innate immune tolerance in the lungs by a mechanism which remains to be elucidated. Our study represents a step forward to establishing the link between the microbiota and the immune reactivity of the lungs.

Influenza Virus Infection Impairs the Gut's Barrier Properties and Favors Secondary Enteric Bacterial Infection through Reduced Production of Short-Chain Fatty Acids.

Along with respiratory tract disease per se, viral respiratory infections can also cause extrapulmonary complications with a potentially critical impact on health. In the present study, we used an experimental model of influenza A virus (IAV) infection to investigate the nature and outcome of the associated gut disorders. In IAV-infected mice, the signs of intestinal injury and inflammation, altered gene expression, and compromised intestinal barrier functions peaked on day 7 postinfection. As a likely result of bacterial component translocation, gene expression of inflammatory markers was upregulated in the liver. These changes occurred concomitantly with an alteration of the composition of the gut microbiota and with a decreased production of the fermentative, gut microbiota-derived products short-chain fatty acids (SCFAs). Gut inflammation and barrier dysfunction during influenza were not attributed to reduced food consumption, which caused in part gut dysbiosis. Treatment of IAV-infected mice with SCFAs was associated with an enhancement of intestinal barrier properties, as assessed by a reduction in the translocation of dextran and a decrease in inflammatory gene expression in the liver. Lastly, SCFA supplementation during influenza tended to reduce the translocation of the enteric pathogen Salmonella enterica serovar Typhimurium and to enhance the survival of doubly infected animals. Collectively, influenza virus infection can remotely impair the gut's barrier properties and trigger secondary enteric infections. The latter phenomenon can be partially countered by SCFA supplementation.

Comparative methods for fecal sample storage to preserve gut microbial structure and function in an in vitro model of the human colon.

In vitro gut models, such as the mucosal artificial colon (M-ARCOL), provide timely and cost-efficient alternatives to in vivo assays allowing mechanistic studies to better understand the role of human microbiome in health and disease. Using such models inoculated with human fecal samples may require a critical step of stool storage. The effects of preservation methods on microbial structure and function in in vitro gut models have been poorly investigated. This study aimed to assess the impact of three commonly used preserving methods, compared with fresh fecal samples used as a control, on the kinetics of lumen and mucus-associated microbiota colonization in the M-ARCOL model. Feces from two healthy donors were frozen 48 h at - 80 °C with or without cryoprotectant (10% glycerol) or lyophilized with maltodextrin and trehalose prior to inoculation of four parallel bioreactors (e.g., fresh stool, raw stool stored at - 80 °C, stool stored at - 80 °C with glycerol and lyophilized stool). Microbiota composition and diversity (qPCR and 16S metabarcoding) as well as metabolic activity (gases and short chain fatty acids) were monitored throughout the fermentation process (9 days). All the preservative treatments allowed the maintaining inside the M-ARCOL of a complex and functional microbiota, but considering stabilization time of microbial profiles and activities (and not technical constraints associated with the supply of frozen material), our results highlighted 48 h freezing at - 80 °C without cryoprotectant as the most efficient method. These results will help scientists to determine the most accurate method for fecal storage prior to inoculation of in vitro gut microbiome models. KEY POINTS: • In vitro ARCOL model reproduces luminal and mucosal human microbiome. • Short-term storage of fecal sample influences microbial stabilization and activity. • 48 h freezing at - 80°C: most efficient method to preserve microbial ecosystem. • Scientific and technical requirements: influencers of preservation method.

Binding activity to intestinal cells and transient colonization in mice of two Lactobacillus paracasei subsp. paracasei strains with high aggregation potential.

Surface properties like hydrophobicity, aggregation ability, adhesion to mucosal surfaces and epithelial cells and transit time are key features for the characterization of probiotic strains. In this study, we used two Lactobacillus paracasei subsp. paracasei strains (BGNJ1-64 and BGSJ2-8) strains which were previously described with very strong aggregation capacity. The aggregation promoting factor (AggLb) expressed in these strains showed high level of binding to collagen and fibronectin, components of extracellular matrix. The working hypothesis was that strains able to aggregate have an advantage to resist in intestinal tract. So, we assessed whether these strains and their derivatives (without aggLb gene) are able to bind or not to intestinal components and we compared the transit time of each strains in mice. In that purpose parental strains (BGNJ1-64 and BGSJ2-8) and their aggregation negative derivatives (BGNJ1-641 and BGSJ2-83) were marked with double antibiotic resistance in order to be tracked in in vivo experiments in mice. Comparative analysis of binding ability of WT and aggregation negative strains to different human intestinal cell lines and mucin revealed no significant difference among them, excluding involvement of AggLb in interaction with surface of intestinal cells and mucin. In vivo experiments showed that surviving and transit time of marked strains in mice did not drastically depend on the presence of the AggLb aggregation factor.

Mucus and microbiota as emerging players in gut nanotoxicology: The example of dietary silver and titanium dioxide nanoparticles.

Given the growing use of nanotechnology in many common consumer products, including foods, evaluation of the consequences of chronic exposure to nanoparticles in humans has become a major public health issue. The oral route of exposure has been poorly explored, despite the presence of a fraction of nanosized particles in certain food additives/supplements and the incorporation of such particles into packaging in contact with foods. After their ingestion, these nanoparticles pass through the digestive tract, where they may undergo physicochemical transformations, with consequences for the luminal environment, before crossing the epithelial barrier to reach the systemic compartment. In this review, we consider two examples, nanosilver and nanotitanium dioxide. Despite the specific features of these particles and the differences between them, both display a close relationship between physicochemical reactivity and bioavailability/biopersistence in the gastrointestinal tract. Few studies have focused on the interactions of nanoparticles of silver or titanium dioxide with the microbiota and mucus. However, the microbiota and mucus play key roles in intestinal homeostasis and host health and are undoubtedly involved in controlling the distribution of nanoparticles in the systemic compartment.

Risk factors and impact of allergic bronchopulmonary aspergillosis in Pseudomonas aeruginosa-negative CF patients.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a major complication in cystic fibrosis (CF) patients. Risk factors for ABPA and clinical deterioration in CF patients, negative for Pseudomonas aeruginosa (Pa), were explored. METHODS: We performed a retrospective case-control study in 73 Pa-negative patients. Each patient was matched with 2 controls for age, gender, pancreas sufficiency, DeltaF508 mutation (homozygous or heterozygous), and Pa colonization. RESULTS: Median FEV1 at the year of diagnosis (index year) was significantly lower in patients with ABPA. The median of cumulative values of FEV1 and FVC before the index year was not significantly different. After the index year, the median of cumulative data for FEV1 and FVC was significantly lower; there were significantly more hospitalization days and more IV antibiotic days compared to controls. Comparing pre- and post-index year data in patients with ABPA, significantly more hospitalization days and more IV antibiotic days were observed after the index year. During the period preceding the index year, significantly more ABPA patients were treated with rhDNase and inhaled corticosteroids. CONCLUSIONS: Bronchial damage cannot be considered as a facilitating factor for ABPA. ABPA causes a significant increase in bronchial damage. In patients with ABPA, further bronchial damage can be controlled by an increase in hospitalization days and use of IV antibiotics. rhDNase and inhaled corticosteroids were associated with the development of ABPA.

Food-grade TiO2 is trapped by intestinal mucus in vitro but does not impair mucin O-glycosylation and short-chain fatty acid synthesis in vivo: implications for gut barrier protection.

BACKGROUND: Titanium dioxide (TiO2) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial. RESULTS: We tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO2 particles accumulated inside "patchy" regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular "islands" located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO2 particles was attributed to this mucus patchy structure. We compared TiO2-mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment. CONCLUSIONS: Food-grade TiO2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under "healthy gut" conditions.

Fecal microbiota manipulation prevents dysbiosis and alcohol-induced liver injury in mice.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) is a leading cause of liver failure and mortality. In humans, severe alcoholic hepatitis is associated with key changes to intestinal microbiota (IM), which influences individual sensitivity to develop advanced ALD. We used the different susceptibility to ALD observed in two distinct animal facilities to test the efficiency of two complementary strategies (fecal microbiota transplantation and prebiotic treatment) to reverse dysbiosis and prevent ALD. METHODS: Mice were fed alcohol in two distinct animal facilities with a Lieber DeCarli diet. Fecal microbiota transplantation was performed with fresh feces from alcohol-resistant donor mice to alcohol-sensitive receiver mice three times a week. Another group of mice received pectin during the entire alcohol consumption period. RESULTS: Ethanol induced steatosis and liver inflammation, which were associated with disruption of gut homeostasis, in alcohol-sensitive, but not alcohol resistant mice. IM analysis showed that the proportion of Bacteroides was specifically lower in alcohol-sensitive mice (p<0.05). Principal coordinate analysis showed that the IM of sensitive and resistant mice clustered differently. We targeted IM using two different strategies to prevent alcohol-induced liver lesions: (1) pectin treatment which induced major modifications of the IM, (2) fecal microbiota transplantation which resulted in an IM very close to that of resistant donor mice in the sensitive recipient mice. Both methods prevented steatosis, liver inflammation, and restored gut homeostasis. CONCLUSIONS: Manipulation of IM can prevent alcohol-induced liver injury. The IM should be considered as a new therapeutic target in ALD. LAY SUMMARY: Sensitivity to alcoholic liver disease (ALD) is driven by intestinal microbiota in alcohol fed mice. Treatment of mice with alcohol-induced liver lesions by fecal transplant from alcohol fed mice resistant to ALD or with prebiotic (pectin) prevents ALD. These findings open new possibilities for treatment of human ALD through intestinal microbiota manipulation.

The microbiota shifts the iron sensing of intestinal cells.

The amount of iron in the diet directly influences the composition of the microbiota. Inversely, the effects of the microbiota on iron homeostasis have been little studied. So, we investigate whether the microbiota itself may alter host iron sensing. Duodenal cytochrome b and divalent metal transporter 1, involved in apical iron uptake, are 8- and 10-fold, respectively, more abundant in the duodenum of germ-free (GF) mice than in mice colonized with a microbiota. In contrast, the luminal exporter ferroportin is 2-fold less abundant in GF. The overall signature of microbiota on iron-related proteins is similar in the colon. The colonization does not modify systemic parameters as plasma transferrin saturation (20%), plasma ferritin (150 ng/L), and liver (85 µg/g) iron load. Commensal organisms (Bacteroides thetaiotaomicron VPI-5482 and Faecalibacterium prausnitzii A2-165) and a probiotic strain (Streptococcus thermophilus LMD-9) led to up to 12-fold induction of ferritin in colon. Our data suggest that the intestinal cells of GF mice are depleted of iron and that following colonization, the epithelial cells favor iron storage. This study is the first to demonstrate that gut microbes induce a specific iron-related protein signature, highlighting new aspects of the crosstalk between the microbiota and the intestinal epithelium.

Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of Lactococcus lactis IBB477 strain.

The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells. The results indicate that two plasmids, pIBB477a and b, are involved in adhesion of the IBB477 strain. One of the genes localised on plasmid pIBB477b (AJ89_14230), which encodes cell wall-associated peptidase S8 (PrtP), mediates adhesion of the IBB477 strain to bare, mucin- and fibronectin-coated polystyrene, as well as to HT29-MTX cells. Interactions between bacteria and mucus secreted by HT29-MTX cells were further investigated by fluorescent staining and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus. Finally, the ability of IBB477 strain and its ΔprtP deletion mutant to colonise the gastrointestinal tract of conventional C57Bl/6 mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut.

A proposed framework for an appropriate evaluation scheme for microorganisms as novel foods with a health claim in Europe.

This paper concerns the procedure and the scientific approach to obtain market authorization for a microorganism to be recognized as a novel food with a health claim. Microorganisms that have not been traditionally used during food production in Europe prior to 1997 are considered as novel foods, which should undergo an in-depth characterization and safety assessment before being authorized on the European market. If a novel food bacterium is claimed to provide a beneficial effect on health, these claims must also be investigated before they can be authorized. Some requirements to obtain novel food certification are shared with those required to obtain a health claim. Although regulation exists that deals with these issues for foods in general, bacteria in food raise a specific set of questions that are only minimally addressed in official documentation. We propose a framework and suggest a list of criteria that should be assessed to obtain marketing authorization and health claim for a bacterium in accordance with European health policy.

Primocolonization is associated with colonic epithelial maturation during conventionalization.

Interaction between the gut microbiota and the host starts immediately after birth with the progressive colonization of the sterile intestine. Our aim was to investigate the interactions taking place in the colonic epithelium after the first exposure to gut microbiota. Germ-free (GF) rats were inoculated with two different microbiotas: the first, obtained from suckling rats, was rich in primocolonizing bacteria and the second, obtained from adult rats, was representative of a mature microbiota. Once transferred into GF rats, these two microbiotas evolved such that they converged, and recapitulated the primocolonization pattern, mimicking the chronological scheme of implantation following birth. The two microbiotas induced common responses in the colonic epithelium: a transitory proliferative phase followed by a compensatory phase characterized by increases in the abundance of p21(Cip1) and p27(Kip1) and in the number of goblet cells. The effects of the two microbiotas diverged only through their effects on colonic transporters. Analyses of solute carriers and aquaporins revealed that functional maturation was more pronounced following exposure to adult microbiota than suckling microbiota. The colon matured in parallel with the evolution of the microbiota composition, and we therefore suggest a link between intestinal events regulating homeostasis of the colon and modulation of microbial composition.

Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent.

BACKGROUND: The intestinal mucus layer plays a key role in the maintenance of host-microbiota homeostasis. To document the crosstalk between the host and microbiota, we used gnotobiotic models to study the influence of two major commensal bacteria, Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, on this intestinal mucus layer. B. thetaiotaomicron is known to use polysaccharides from mucus, but its effect on goblet cells has not been addressed so far. F. prausnitzii is of particular physiological importance because it can be considered as a sensor and a marker of human health. We determined whether B. thetaiotaomicron affected goblet cell differentiation, mucin synthesis and glycosylation in the colonic epithelium. We then investigated how F. prausnitzii influenced the colonic epithelial responses to B. thetaiotaomicron. RESULTS: B. thetaiotaomicron, an acetate producer, increased goblet cell differentiation, expression of mucus-related genes and the ratio of sialylated to sulfated mucins in mono-associated rats. B. thetaiotaomicron, therefore, stimulates the secretory lineage, favoring mucus production. When B. thetaiotaomicron was associated with F. prausnitzii, an acetate consumer and a butyrate producer, the effects on goblet cells and mucin glycosylation were diminished. F. prausnitzii, by attenuating the effects of B. thetaiotaomicron on mucus, may help the epithelium to maintain appropriate proportions of different cell types of the secretory lineage. Using a mucus-producing cell line, we showed that acetate up-regulated KLF4, a transcription factor involved in goblet cell differentiation. CONCLUSIONS: B. thetaiotaomicron and F. prausnitzii, which are metabolically complementary, modulate, in vivo, the intestinal mucus barrier by modifying goblet cells and mucin glycosylation. Our study reveals the importance of the balance between two main commensal bacteria in maintaining colonic epithelial homeostasis via their respective effects on mucus.

The microbiota and the host organism switch between cooperation and competition based on dietary iron levels.

The microbiota significantly impacts digestive epithelium functionality, especially in nutrient processing. Given the importance of iron for both the host and the microbiota, we hypothesized that host-microbiota interactions fluctuate with dietary iron levels. We compared germ-free (GF) and conventional mice (SPF) fed iron-containing (65 mg/Kg) or iron-depleted (<6 mg/Kg) diets. The efficacy of iron privation was validated by iron blood parameters. Ferritin and Dmt1, which represent cellular iron storage and transport respectively, were studied in tissues where they are abundant: the duodenum, liver and lung. When the mice were fed an iron-rich diet, the microbiota increased blood hemoglobin and hepcidin and the intestinal ferritin levels, suggesting that the microbiota helps iron storage. When iron was limiting, the microbiota inhibited the expression of the intestinal Dmt1 transporter, likely via the pathway triggered by Hif-2α. The microbiota assists the host in storing intestinal iron when it is abundant and competes with the host by inhibiting Dmt1 in conditions of iron scarcity. Comparison between duodenum, liver and lung indicates organ-specific responses to microbiota and iron availability. Iron depletion induced temporal changes in microbiota composition and activity, reduced α-diversity of microbiota, and led to Lactobacillaceae becoming particularly more abundant after 60 days of privation. By inoculating GF mice with a simplified bacterial mixture, we show that the iron-depleted host favors the gut fitness of Bifidobacterium longum.

Faecalibacterium duncaniae A2-165 regulates the expression of butyrate synthesis, ferrous iron uptake, and stress-response genes based on acetate consumption.

The promising next-generation probiotic Faecalibacterium prausnitzii is one of the most abundant acetate-consuming, butyrate-producing bacteria in the healthy human gut. Yet, little is known about how acetate availability affects this bacterium's gene expression strategies. Here, we investigated the effect of acetate on temporal changes in the transcriptome of F. duncaniae A2-165 cultures using RNA sequencing. We compared gene expression patterns between two growth phases (early stationary vs. late exponential) and two acetate levels (low: 3 mM vs. high: 23 mM). Only in low-acetate conditions, a general stress response was activated. In high-acetate conditions, there was greater expression of genes related to butyrate synthesis and to the importation of B vitamins and iron. Specifically, expression was strongly activated in the case of the feoAABC operon, which encodes a FeoB ferrous iron transporter, but not in the case of the feoAB gene, which encodes a second putative FeoAB transporter. Moreover, excess ferrous iron repressed feoB expression but not feoAB. Lastly, FeoB but not FeoAB peptides from strain A2-165 were found in abundance in a healthy human fecal metaproteome. In conclusion, we characterized two early-stationary transcriptomes based on acetate consumption and this work highlights the regulation of feoB expression in F. duncaniae A2-165.

Near adult height and BMI changes in growth hormone treated short children with Noonan syndrome: the Belgian experience.

Introduction A variable near adult height (NAH) outcome after growth hormone (GH) therapy in Noonan syndrome (NS) patients with short stature has been reported. The main objective of this study was to evaluate NAH and body mass index (BMI) evolution in a large Belgian cohort of NS patients treated for short stature. The secondary objectives were to investigate whether sex, genotype, the presence of a thoracic deformity and/or a heart anomaly might affect NAH and to validate the recently developed NAH prediction model by Ranke et al. Methods Clinical and auxological data of GH treated short NS patients born before 2001 were extracted from the national Belgrow registry. NAH was available in 54 (35 male) genotyped NS using a gene panel of 9 genes, showing pathogenic variants in PTPN11 in 32 and in SOS1 in 5 patients, while in 17 patients gene panel analysis was inconclusive (no mutation group). Results After a median (P10; P90) duration of 5.4 (2.2-10.3) years of GH therapy with a median dose of 0.05 mg/kg/day NS patients reached a median NAH of -1.7 (-3.4; -0.8) SDS. Median total height gain was 1.1 (0.1; 2.3) SDS. Sex, genotype and the presence of a thoracic or cardiac malformation did not correlate with NAH or total height gain. Linear regression modelling revealed that height SDS at start (beta=0.90, p<0.001), mid-parental height SDS (beta =0.27; p=0.005), birth weight SDS (beta=0.15; p=0.051), age at start (beta=0.07; p=0032) were independently associated with NAH SDS. Median BMI SDS increased significantly (p<0.001) from -1.0 (-2.5; 0.0) at start to -0.2 (-1.5; 0.9) at NAH. The observed NAH in a subgroup of 44 patients with more than 3 years of GH treatment was not statistically different from the predicted NAH by the Noonan NAH prediction model of Ranke. Conclusion Long-term GH therapy at a dose of 0.05 mg/kg/day in short NS patients is effective in improving adult height and BMI, irrespective of the genotype and presence or absence of cardiac and or thoracic anomalies.

Pre-, pro-, syn-, and Postbiotics in Infant Formulas: What Are the Immune Benefits for Infants?

The first objective of infant formulas is to ensure the healthy growth of neonates and infants, as the sole complete food source during the first months of life when a child cannot be breastfed. Beyond this nutritional aspect, infant nutrition companies also try to mimic breast milk in its unique immuno-modulating properties. Numerous studies have demonstrated that the intestinal microbiota under the influence of diet shapes the maturation of the immune system and influences the risk of atopic diseases in infants. A new challenge for dairy industries is, therefore, to develop infant formulas inducing the maturation of immunity and the microbiota that can be observed in breastfed delivered vaginally, representing reference infants. Streptococcus thermophilus, Lactobacillus reuteri DSM 17938, Bifidobacterium breve (BC50), Bifidobacterium lactis Bb12, Lactobacillus fermentum (CECT5716), and Lactobacillus rhamnosus GG (LGG) are some of the probiotics added to infant formula, according to a literature review of the past 10 years. The most frequently used prebiotics in published clinical trials are fructo-oligosaccharides (FOSs), galacto-oligosaccharides (GOSs), and human milk oligosaccharides (HMOs). This review sums up the expected benefits and effects for infants of pre-, pro-, syn-, and postbiotics added to infant formula regarding the microbiota, immunity, and allergies.