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International audience; Aim: Very preterm birth is associated with a high risk of enteropathies. Diagnosis is challenging, especially in mild forms, leading to unnecessary periods of cessation of enteral feeding. This study aimed at establishing a prognosis score of enteropathy combining clinical parameters and faecal calprotectin concentration.Methods: This prospective multicentric study included preterm neonates born at a gestational age of 33 weeks or less. Stools were collected weekly until hospital discharge, and daily in case of digestive events for calprotectin measurement (ELISA and immunochromatography) and microbiota analyses (16S rRNA gene sequencing).Results: Among the 121 neonates included, 21 experienced at least one episode of enteropathy, mainly mild forms. By ELISA testing, median faecal calprotectin was 88 (8-798) µg/g faeces. No statistically significant association was found between the outset of enteropathy and maternal and neonatal characteristics, and calprotectin levels. The agreement between ELISA and immunochromatography assay was moderate (intra-class correlation coefficient 0.58, 95%CI [0.47-0.66]). Comparison of species diversity and relative bacterial abundance profiles between infants with or without enteropathy revealed no specific alterations associated with enteropathy.Conclusion: The study failed to propose a prognostic score of enteropathy, probably due the large inter- and intra-individual variability of faecal calprotectin in very preterm neonates.

Key Points Question What are the long-term outcomes associated with dysbiosis of gut microbiota in very preterm newborns? Findings In this cohort study of 577 very preterm newborns across 24 neonatal intensive care units from a French nationwide cohort, gut microbiota at week 4 after birth showed 6 bacterial patterns that varied according to gestational age, perinatal characteristics, individual treatments, and neonatal intensive care unit strategies. Three clusters were associated with 2-year outcomes after adjustment for these confounders. Meaning Modifying strategies associated with alterations in microbiota, such as promoting enteral nutrition, reducing sedation use, promoting early extubation, or skin-to-skin practice, may be correlated with outcomes in preterm newborns., Importance In very preterm newborns, gut microbiota is highly variable with major dysbiosis. Its association with short-term health is widely studied, but the association with long-term outcomes remains unknown. Objective To investigate in preterm newborns the associations among practice strategies in neonatal intensive care units (NICUs), gut microbiota, and outcomes at 2 years. Design, Setting, and Participants EPIFLORE is a prospective observational cohort study that includes a stool sample collection during the fourth week after birth. Preterm newborns of less than 32 weeks of gestational age (GA) born in 2011 were included from 24 NICUs as part of the French nationwide population-based cohort, EPIPAGE 2. Data were collected from May 2011 to December 2011 and analyzed from September 2016 to December 2018. Exposures Eight NICU strategies concerning sedation, ventilation, skin-to-skin practice, antibiotherapy, ductus arteriosus, and breastfeeding were assessed. A NICU was considered favorable to a practice if the percentage of that practice in the NICU was more than the expected percentage. Main Outcomes and Measures Gut microbiota was analyzed by 16S ribosomal RNA gene sequencing and characterized by a clustering-based method. The 2-year outcome was defined by death or neurodevelopmental delay using a Global Ages and Stages questionnaire score. Results Of 577 newborns included in the study, the mean (SD) GA was 28.3 (2.0) weeks, and 303 (52.5%) were male. Collected gut microbiota was grouped into 5 discrete clusters. A sixth cluster included nonamplifiable samples owing to low bacterial load. Cluster 4 (driven by Enterococcus [n = 63]), cluster 5 (driven by Staphylococcus [n = 52]), and cluster 6 (n = 93) were significantly associated with lower mean (SD) GA (26.7 [1.8] weeks and 26.8 [1.9] weeks, respectively) and cluster 3 (driven by Escherichia/Shigella [n = 61]) with higher mean (SD) GA (29.4 [1.6] weeks; P = .001). Cluster 3 was considered the reference. After adjustment for confounders, no assisted ventilation at day 1 was associated with a decreased risk of belonging to cluster 5 or cluster 6 (adjusted odds ratio [AOR], 0.21 [95% CI, 0.06-0.78] and 0.19 [95% CI, 0.06-0.62], respectively) when sedation (AOR, 10.55 [95% CI, 2.28-48.87] and 4.62 [1.32-16.18], respectively) and low volume of enteral nutrition (AOR, 10.48 [95% CI, 2.48-44.29] and 7.28 [95% CI, 2.03-26.18], respectively) was associated with an increased risk. Skin-to-skin practice was associated with a decreased risk of being in cluster 5 (AOR, 0.14 [95% CI, 0.04-0.48]). Moreover, clusters 4, 5, 6 were significantly associated with 2-year nonoptimal outcome (AOR, 6.17 [95% CI, 1.46-26.0]; AOR, 4.53 [95% CI, 1.02-20.1]; and AOR, 5.42 [95% CI, 1.36-21.6], respectively). Conclusions and Relevance Gut microbiota of very preterm newborns at week 4 is associated with NICU practices and 2-year outcomes. Microbiota could be a noninvasive biomarker of immaturity., This cohort study of 577 very preterm newborns across 24 neonatal intensive care units from a French nationwide cohort investigates associations among practice strategies, gut microbiota, and outcomes at 2 years.

International audience; Until recently the in utero environment of pregnant women was considered sterile. Recent high-sensitivity molecular techniques and high-throughput sequencing lead to some evidence for a low-biomass microbiome associated with the healthy placenta. Other studies failed to reveal evidence for a consistent presence of bacteria using either culture or molecular based techniques. Comparing conflicting "placental microbiome" studies is complicated by the use of varied and inconsistent protocols. Given this situation, we undertook an evaluation of the in utero environment sterility using several controlled methods, in the same study, to evaluate the presence or absence of bacteria and to explain contradictions present in the literature. Healthy pregnant women (n = 38) were recruited in three maternity wards. Placenta were collected after cesarean section with or without Alexis ® and vaginal delivery births. For this study we sampled fetal membranes, umbilical cord and chorionic villi. Bacterial presence was analyzed using bacterial culture and qPCR on 34 fetal membranes, umbilical cord and chorionic villi samples. Shotgun metagenomics was performed on seven chorionic villi samples. We showed that the isolation of meaningful quantities of viable bacteria or bacterial DNA was possible only outside the placenta (fetal membranes and umbilical cords) highlighting the importance of sampling methods in studying the in utero environment. Bacterial communities described by metagenomics analysis were similar in chorionic villi samples and in negative controls and were dependent on the database chosen for the analysis. We conclude that the placenta does not harbor a specific, consistent and functional microbiota.

Acute graft-versus-host disease (aGVHD) is the main complication of hematopoietic stem cell transplantation (HSCT). Changes in gut microbiota composition have been associated with subsequent aGVHD, and reconstitution of healthy microbiota is currently being explored as a therapeutic approach. However, the specific actors in the intestinal ecosystem involved in the pathologic process at the time of aGVHD onset are not yet fully known. We prospectively collected stool samples from patients who underwent allogeneic HSCT. Patients sampled at aGVHD onset were compared with non-GVHD patients. To identify phylogenetic and functional signatures of the disease process, we determined fecal short-chain fatty acid (SFCA) profiles and used high-throughput DNA sequencing and real-time quantitative polymerase chain reaction to assess the microbiota composition. Microbiota alterations were highly specific of gastrointestinal (GI) aGVHD severity. Bacterial biomass and a-diversity were lower in severe aGVHD. We identified several bacterial signatures associated with severe aGVHD at disease onset; a negative correlation was observed with anaerobic bacteria of the Lachnospiraceae, especially the Blautia genus, and Ruminococcaceae families. In parallel, in severe aGVHD patients, we showed a dramatic decrease in the levels of the main SFCAs: acetate (75.8%), propionate (95.8%), and butyrate (94.6%). Mild aGVHD patients were characterized by conserved levels of propionate and Blautia propionate producers. Butyrate was significantly decreased in all GI aGVHD stages, representing a potential diagnostic marker of the disease. Specific microbiota and metabolic alterations were thus associated with aGVHD severity and may be useful for diagnostic and pathophysiologic purposes., Stem-cell Transplantation; Diversity

Certaines pathologies semblent avoir une origine développementale et, aujourd’hui, le microbiote apparaît comme un déterminant de l’état de santé de l’homme et son établissement, une étape importante chez le nouveau-né. Des variations dans sa constitution, incluant un déséquilibre (ou dysbiose), ont ainsi été associées à de nombreuses pathologies. De récents travaux suggèrent qu’une colonisation bactérienne de l’utérus, du liquide amniotique ou encore du placenta, des sites auparavant pensés stériles, existerait. Durant les phases de son développement, le foetus pourrait ainsi rencontrer des bactériesin utero. Elles contribueraient à l’établissement de son microbiote avant même l’accouchement et donc avant la rencontre avec les microorganismes des microbiotes vaginal, fécal et cutané, ceux-ci variant selon les modes d’accouchement (voie basse ou césarienne). Les premières études sur l’existence d’un microbiotein utero, qui se caractérise par une faible biomasse, sont cependant controversées.

The developmental origin of health and disease highlights the importance of the period of the first 1000 days (from the conception to the 2 years of life). The process of the gut microbiota establishment is included in this time window. Various perinatal determinants, such as cesarean section delivery, type of feeding, antibiotics treatment, gestational age or environment, can affect the pattern of bacterial colonization and result in dysbiosis. The alteration of the early bacterial gut pattern can persist over several months and may have long-lasting functional effects with an impact on disease risk later in life. As for example, early gut dysbiosis has been involved in allergic diseases and obesity occurrence. Besides, while it was thought that the fetus developed under sterile conditions, recent data suggested the presence of a microbiotain utero, particularly in the placenta. Even if the origin of this microbiota and its eventual transfer to the infant are nowadays unknown, this placental microbiota could trigger immune responses in the fetus and would program the infant’s immune development during fetal life, earlier than previously considered. Moreover, several studies demonstrated a link between the composition of placental microbiota and some pathological conditions of the pregnancy. All these data show the evidence of relationships between the neonatal gut establishment and future health outcomes. Hence, the use of pre- and/or probiotics to prevent or repair any early dysbiosis is increasingly attractive to avoid long-term health consequences.

Aim This Lebanese study tested the hypothesis that differences would exist in the gut microbiota of preterm infants with and without necrotising enterocolitis (NEC), as reported in Western countries. Methods This study compared 11 infants with NEC and 11 controls, all born at 27-35 weeks, in three neonatal intensive care units between January 2013 and March 2015. Faecal samples were collected at key time points, and microbiota was analysed by culture, quantitative PCR (qPCR) and temperature temporal gel electrophoresis (TTGE). Results The cultures revealed that all preterm infants were poorly colonised and harboured no more than seven species. Prior to NEC diagnosis, significant differences were observed by qPCR with a higher colonisation by staphylococci (p = 0.034) and lower colonisations by enterococci (p = 0.039) and lactobacilli (p = 0.048) in the NEC group compared to the healthy controls. Throughout the study, virtually all of the infants were colonised by Enterobacteriaceae at high levels. TTGE analysis revealed no particular clusterisation, showing high interindividual variability. Conclusion The NEC infants were poorly colonised with no more than seven species, and the controls had a more diversified and balanced gut microbiota. Understanding NEC aetiology better could lead to more effective prophylactic interventions and a reduced incidence.

International audience

International audience; Background: The pathophysiology of necrotizing enterocolitis (NEC) remains poorly understood. Objective: We assessed the relation between feeding strategies, intestinal microbiota composition, and the development of NEC. Design: We performed a prospective nationwide population-based study, EPIPAGE 2 (Etude Epidemiologique sur les Petits Ages Ges-tationnels), including preterm infants born at,32 wk of gestation in France in 2011. From individual characteristics observed during the first week of life, we calculated a propensity score for the risk of NEC (Bell's stage 2 or 3) after day 7 of life. We analyzed the relation between neonatal intensive care unit (NICU) strategies concerning the rate of progression of enteral feeding, the direct-breastfeeding policy, and the onset of NEC using general linear mixed models to account for clustering by the NICU. An ancillary propensitymatched case-control study, EPIFLORE (Etude Epidemiologique de la flore), in 20 of the 64 NICUs, analyzed the intestinal microbiota by culture and 16S ribosomal RNA gene sequencing. Results: Among the 3161 enrolled preterm infants, 106 (3.4%; 95% CI: 2.8%, 4.0%) developed NEC. Individual characteristics were significantly associated with NEC. Slower and intermediate rates of progression of enteral feeding strategies were associated with a higher risk of NEC, with an adjusted OR of 2.3 (95% CI: 1.2, 4.5; P = 0.01) and 2.0 (95% CI: 1.1, 3.5; P = 0.02), respectively. Less favorable and intermediate direct-breastfeeding policies were associated with higher NEC risk as well, with an adjusted OR of 2.5 (95% CI: 1.1, 5.8; P = 0.03) and 2.3 (95% CI: 1.1, 4.8; P = 0.02), respectively. Microbiota analysis performed in 16 cases and 78 controls showed an association between Clostridium neonatale and Staphylococcus aureus with NEC (P = 0.001 and P = 0.002). Conclusions: A slow rate of progression of enteral feeding and a less favorable direct-breastfeeding policy are associated with an increased risk of developing NEC. For a given level of risk assessed by propensity score, colonization by C. neonatale and/or S. aureus is significantly associated with NEC. This trial (EPIFLORE study) was registered at clinicaltrials.gov as NCT01127698.

International audience; In the last years there has been a growing interest in the use of genetically modified bacteria to deliver molecules of therapeutic interest at mucosal surfaces. Due to the well-recognized probiotic properties of some strains, bifidobacteria represent excellent candidates for the development of live vehicles to produce and deliver heterologous proteins at mucosal surfaces. However, very few studies have considered this genus because of its complexity to be genetically manipulated. In this work, we report the development of a new Bifidobacteria Expression SysTem (BEST) allowing the production of heterologous proteins in Bifidobacterium bifidum. This system is based on: i) the broad host range plasmid pWV01, ii) a stress-inducible promoter, and iii) two different signal peptides (SPs) one issued from Lactococcus lactis (SPExp4) and issued from Bifidobacterium longum (SPBL1181). The functionality of BEST system was validated by cloning murine interleukin-10 (IL-10) and establishing the resulting plasmids (i.e., pBEST(Exp4):IL-10 and pBEST(BL1181):IL-10) in the strain of B. bifidum BS42. We then demonstrated in vitro that recombinant B. bifidum BS42 harboring pBEST(BL1181):IL-10 plasmid efficiently secreted IL-10 and that this secretion was significantly higher (sevenfold) than its counterpart B. bifidum BS42 harboring pBEST(Exp4):IL-10 plasmid. Finally, we validated in vivo that recombinant B. bifidum strains producing IL-10 using BEST system efficiently delivered this cytokine at mucosal surfaces and exhibit beneficial effects in a murine model of low-grade intestinal inflammation.

Some diseases seem to have a developmental origin. Today, the microbiota is recognized as a determinant in health and diseases and one important step is its establishment in the neonate. Some variations in its composition including an imbalance (also called dysbiosis) have been associated to several pathologies. Recent studies suggest a bacterial colonization in the non-pregnant uterus, in the amniotic fluid and in the placenta, which were previously thought sterile. So, during deve-lopmental phases, the fetus could have encounter bacteria in utero. These bacteria could contribute to its microbiota establishment before parturition and therefore before the encounter with all microorganisms from vaginal, fecal and cutaneous microbiotas according to the delivery mode. However, studies stating the existence of such in utero microbiota, characterized by a low biomass, are somewhat disputed.

Food allergies can have significant effects on morbidity and on quality of life. Therefore, the development of efficient approaches to reduce the risk of developing food allergies is of considerable interest. The aim of this study was to identify and select probiotic strains with preventive properties against allergies using a combination of in vitro and in vivo approaches. To that end, 31 strains of bifidobacteria and lactic acid bacteria were screened for their immunomodulatory properties in two cellular models, namely, human peripheral blood mononuclear cells (PBMCs) and T helper 2 (Th2)-skewed murine splenocytes. Six strains inducing a high interleukin-10 (IL-10)/IL-12p70 ratio and a low secretion of IL-4 on the two cellular models were selected, and their protective impact was tested in vivo in a murine model of food allergy to β-lactoglobulin. Three strains showed a protective impact on sensitization, with a decrease in allergen-specific IgE, and on allergy, with a decrease in mast cell degranulation. Analysis of the impact of these three strains on the T helper balance revealed different mechanisms of action. The Lactobacillus salivarius LA307 strain proved to block Th1 and Th2 responses, while the Bifidobacterium longum subsp. infantis LA308 strain induced a pro-Th1 profile and the Lactobacillus rhamnosus LA305 strain induced pro-Th1 and regulatory responses. These results demonstrate that a combination of in vitro and in vivo screening is effective in probiotic strain selection and allowed identification of three novel probiotic strains that are active against sensitization in mice.

Clostridium butyricum is a Gram-positive bacterium involved in the development of necrotizing enterocolitis (NEC) in preterm infants. To colonize the digestive tract, components of the cell wall of C. butyricum must interact with the intestinal mucosa. The D-alanylation of cell wall components such as teichoic acids results in a net positive charge on the cell wall, which is important for many functions of Gram-positive bacteria. Notably, D-alanylation mediates resistance to antimicrobial peptides and antibiotics. Here, we show that the dlt operon of C. butyricum encodes the enzymes responsible for the D-alanylation of cell wall components and influences the surface properties of the cell wall. We show that the D-alanylation of cell wall components controls the septation of C. butyricum, which is an essential mechanism during vegetative growth. Furthermore, we find that D-alanylation is involved in the resistance of C. butyricum to some cationic antimicrobial peptides (CAMPs) and lysozyme. Finally, we show that the D-alanylation of cell wall components influences vancomycin-induced lysis.

Amino acid-based formulas (AAFs) are recommended for children with cow's-milk allergy (CMA) failing to respond to extensively hydrolysed formulas (eHFs). We evaluated the effects of a new thickened AAF (TAAF, Novalac), containing a pectin-based thickener, and a reference AAF (RAAF, Neocate) on allergy symptoms and safety, through blood biochemistry analysis and growth.Infants (ages18 months) with CMA symptoms failing to respond to eHFs were randomised in a double-blind manner to receive TAAF or RAAF for 3 months. All of the infants were then fed TAAF for 3 additional months. Paediatric visits occurred at 1, 3, and 6 months. Blood samples were collected at inclusion and 3 months.Results at 1 month were previously described. The 75 infants with proven CMA and eHF intolerance tolerated their allocated formula. At 3 months, the dominant allergic symptom had disappeared in 76.2% of the infants with TAAF and in 51.5% of the infants with RAAF (P = 0.026). The Scoring Atopic Dermatitis Index significantly improved more with TAAF than with RAAF (-27.3 ± 2.3 vs -20.8 ± 2.2, P = 0.048). Of the infants, 92.9% had normal stools (soft or formed consistency) with TAAF vs 75.8% with RAAF (P = 0.051). More infants in TAAF group had better quality of nighttime sleep (P = 0.036) and low frequency of irritability signs (P 0.001). With both formulas, all of the biochemical parameters were within normal ranges. There were no differences between the 2 groups in any of the anthropometric z scores.The new TAAF was tolerated by all of the infants with CMA and intolerance to eHFs. Anthropometric and clinical data showed that both formulas were safe.

International audience; Objectives: Head injury (HI) induces a hypercatabolic state, dysimmunity, and septic complications that increase morbidity and mortality. Although compromised immune function is usually incriminated in infection occurrence, gut dysbiosis could also be involved in this phenomenon and, to our knowledge, has never been considered. To assess if HI could affect microbiota, we explored the impact of HI on intestinal microbiota in a rodent model of fluid percussion.Methods: Nineteen rats were randomly assigned to two groups: Healthy rats fed ad libitum (n ¼ 7) and HI rats (n ¼ 12), which received standard enteral nutrition for 4 d. Four days after HI, rats were euthanized and cecal contents were sampled. Cecal microbiota was assessed using real-timequantitative polymerase chain reaction.Results: HI significantly decreased the cecal content of strict anaerobic groups, Bacteroides/Prevotella group (HI 8.9 versus healthy controls 9.3 median log10 colony forming units [CFU]/g, P ¼ 0.007), Clostridium cluster XIVab (HI 7.9 versus healthy controls 8.9 median log10 CFU/g, P ¼ 0.002), Lactobacillus/Leuconostoc group (HI 8.5 versus healthy controls 9.4 median log10 CFU/g, P ¼ 0.044), and Bifidobacterium sp. (HI 3.0 versus healthy controls 8.2 median log10 CFU/g, P < 0.001). In contrast, colonization by Escherichia coli was dramatically increased (HI 10.5 versus healthy controls 7.0 median log10 CFU/g, P < 0.001).Conclusions: HI profoundly modified the gut microbiota homeostasis and thus could contribute to infection in head trauma patients. These preliminary results open a new field of research in the management of patients with HI.

We aimed at identifying potential bacterial factors linking clostridia with necrotizing enterocolitis (NEC). We compared the phenotypic traits, stress responses, cellular cytotoxicity, and inflammatory capabilities of the largest collection of Clostridium butyricum and Clostridium neonatale strains isolated from fecal samples of NEC preterm neonates (PN) and control PNs. When strain characteristics were used as explanatory variables, a statistical discriminant analysis allowed the separation of NEC and control strains into separate groups. Strains isolated from NEC PN were characterized by a higher viability at 30°C ( P = 0.03) and higher aerotolerance ( P = 0.01), suggesting that NEC strains may have a competitive and/or survival advantage in the environmental gastrointestinal tract conditions of NEC PN. Heat-treated NEC bacteria induced higher production of interleukin-8 in Caco-2 cells ( P = 0.03), suggesting proinflammatory activity. In vitro , bacteria, bacterial components, and fecal filtrates showed variable cytotoxic effects affecting the cellular network and/or cell viability, without specific association with NEC or control samples. Altogether, our data support the existence of a specific clostridial strain signature associated with NEC. IMPORTANCE Clostridia are part of the commensal microbiota in preterm neonates (PN). However, microbiota analyses by culture and metagenomics have linked necrotizing enterocolitis (NEC) and intestinal colonization with clostridial species. Nevertheless, little is known about the specific characteristics that may be shared by clostridia associated with NEC compared to commensal clostridia. Therefore, our goal was to identify specific bacterial factors linking clostridial strains with NEC. We report the existence of a specific bacterial signature associated with NEC and propose that activation of the innate immune response may be a unifying causative mechanism for the development of NEC independent of a specific pathogenic organism. The present study provides new insights into NEC pathophysiology that are needed for better diagnostics and strategies for implementing prevention of the disease.

Introduction L’allergie aux proteines de lait de vache (APLV) est l’allergie la plus frequente dans la petite enfance. Ici, nous rapportons les effets du transfert de microbiote fecal (TMF) d’un nourrisson controle sain (CS) et d’un nourrisson atteints d’APLV dans un modele murin d’allergie. Methodes Des nourrissons (6 CS, 5 APLV ; 5 a 16 mois) ont ete recrutes respectivement en ville et au Great Ormond Street Hospital (Londres, Royaume-Uni) (Comite d’ethique ref. 14/LO/0364). Le microbiote fecal a ete analyse par FISH. Des souris axeniques âgees de trois semaines ont ete gavees avec une selle soit d’un nourrisson CS (TMF-CS) soit d’un nourrisson APLV (TMF-APLV). Douze jours apres, les souris ont ete soit sensibilisees une fois par semaine pendant 5 semaines avec des proteines du lactoserum et la toxine cholerique (S), soit ont recu uniquement la toxine cholerique (NS). Apres un test de provocation orale avec de la β-lactoglobuline (BLG), les reponses allergiques et la sensibilisation ont ete mesurees (scores cliniques, mMCP1, immunoglobulines totales et specifiques de l’allergene : IgE IgG1, IgG2a). Resultats La caracterisation des selles de nourrissons sains et allergiques apparies sur l’âge, le sexe et le mode d’accouchement, nous a permis de selectionner un echantillon representatif de chaque groupe, avec dans les selles de nourrissons APLV, un moindre niveau de Bifidobacterium spp. et un niveau plus eleve du groupe Eubacterium rectale/Clostridium coccoides. Chez les souris gnotobiotiques, la reponse clinique allergique a ete augmentee dans le groupe TMF-APLV-S par rapport au groupe TMF-CS-S (p Conclusion Le microbiote intestinal associe a l’APLV influence la reponse clinique et oriente le systeme immunitaire vers un terrain atopique dont le mecanisme suggere est non IgE medie.

Introduction L’allergie aux proteines de lait de vache (APLV) touche jusqu’a 5 % des enfants. De nombreuses donnees suggerent qu’un microbiote intestinal desequilibre peut contribuer au developpement d’allergies alimentaires, validant l’utilisation de probiotiques a titre preventif ou curatif. Methodes Les proprietes curatives de trois souches probiotiques (Lactobacillus rhamnosus LA305, L. salivarius LA307, Bifidobacterium longum subsp. infantis LA308), ont ete evaluees chez des souris Balb/c sensibilisees per os avec des proteines de lactoserum et de la toxine cholerique, une fois par semaine pendant 5 semaines. Un test de provocation orale a la β-lactoglobuline (BLG) a ensuite ete realise avant l’administration orale de souches probiotiques pendant 3 semaines. Apres un second test de provocation a la BLG, les reponses immunitaires ont ete evaluees par dosage serique de marqueurs de sensibilisation et de degranulation des mastocytes, metabolome serique, dosage de cytokines secretees pas les splenocytes re-stimules par la BLG et mesure de l’expression des genes ileaux associes aux T-helper. Resultats Le traitement probiotique n’a pas eu d’impact sur la degranulation des mastocytes et la production d’immunoglobulines specifiques anti-BLG. Cependant, la secretion de cytokines IL-2, Th2 (IL-4, IL-5) et Th1 (IFNg) par les splenocytes de souris traitees a ete diminuee avec un profil souche dependant. L’expression ileale des genes foxp3, tbet, gata3 et rorgt a confirme cette regulation immunitaire. Les 3 souches ont aussi induit une diminution des kynurenines seriques. Discussion Si les capacites de regulation des reponses immunitaire et inflammatoire dans un modele murin d’APLV sont souche-dependantes, la diminution des kynurenines seriques montrent que les trois souches ont un effet anti-inflammatoire. Conclusion Ces resultats confirment le potentiel therapeutique de ces probiotiques base notamment sur une reduction de la reponse inflammatoire au cours de la reaction allergique.

International audience; Adequate nutritional support is a major challenge in brain injury patients, because malnutrition cannot be reversed by standard enteral nutrition. We hypothesized that an oligomeric formula could improve nutritional status by restoring intestinal trophicity. Eighteen male Sprague-Dawley rats (300-330 g) underwent gastrostomy on day-7 (D-7) and traumatic brain injury (TBI) by hydraulic percussion (D0) and were then fed for 4 days with either a polymeric formula (Sondalis® HP, TBIP, n = 9), or an oligomeric formula (Peptamen® HN, TBIO, n = 9). In addition, a control group of healthy gastrostomized rats was fed the polymeric diet (control, n = 8). All rats were weighed daily. On D+4, the rats were euthanized. Blood was collected for plasma amino acid determination. Organs were removed and weighed. Intestinal morphometry was studied. Protein content was assessed on intestine and muscles. Enterobacterial translocation and dissemination were evaluated. Results were expressed as means ± SEM and compared using analysis of variance+Newman-Keuls test. TBI induced a significant decrease in whole body weight (TBIP vs. control, p < 0.05) that was totally blunted by the oligomeric diet (TBIP vs. TBIO, p < 0.01). Thymus weight significantly decreased after TBI (TBIP vs. control, p < 0.05) and was restored by the oligomeric formula (TBIO vs. TBIP, p < 0.05). Glutamine (GLN) concentration was improved by the oligomeric diet in both plasma (TBIO: 688 ± 19 vs. control: 591 ± 45 and TBIP: 615 ± 42 μmol/L, p < 0.05) and soleus muscle. These results show that the use of an oligomeric diet may limit response to injury after brain injury and could be a simple nutritional strategy in this setting.

Bifidobacteria are part of the human gastrointestinal microbiota and are used as probiotics in functional food products because of their health promoting properties. However, only few data are available on the phenotypic characteristics displayed by human bifidobacteria strain populations. In this study we compared the in vitro tolerance to acid, bile and oxygen of the largest number of independent human intestinal strains. Bile and acid tolerance varied among species and independent strains within a species: B. adolescentis strains were the most tolerant to bile followed by Bifidobacterium longum and B. breve; B. longum, B. breve and B. dentium showed the highest viability levels after exposure to acid pH. Oxygen tolerance was largely distributed among intestinal bifidobacteria: B. longum, B. breve and B. bifidum showed the highest oxygen tolerance. B. adolescentis showed the highest susceptibility to acid and oxygen stresses. The present study gave us the opportunity to update our knowledge about the phenotypic characteristics of human intestinal bifidobacteria. B. longum and B. breve harboured the best tolerance to oxygen, bile and acid stresses. Based on such biological characters, B. longum and B. breve species showed the highest interest in terms of potential selection of human probiotics.

Linezolid (LZD) has arisen as an alternative treatment in diabetic foot osteitis due to staphylococci. LZD resistance selection is difficult and involved various molecular mechanisms. As a better knowledge of those mechanisms could be beneficial for pathogenic strains' screening, we simulated in vitro the spontaneous mutagenesis process that leads to LZD-resistant strains from two Staphylococcus epidermidis strains responsible for monomicrobial diabetic foot osteitis. LZD high resistance was selected for both strains, with the same timeline of mutation appearance. Mutation in L3 protein (G152D) occurred first and quickly, but did not cause phenotypically detectable resistance or fitness cost. It was later followed by different 23S rRNA mutations (G2505A, G2447T), leading this time to detectable resistance (minimum inhibitory concentration [MIC] ≥8 mg/L). This phenomenon underlies the difficulty of resistance selection in coagulase-negative staphylococci (CoNS). This study is the first description of G2505A mutation in CoNS. Various phenotypical impacts were observed depending on strain and mutation: (i) fitness cost of G2505A and G2447T mutations; (ii) loss of erythromycin resistance concomitantly with L3 mutation selection; (iii) correlation between number of mutated rrl copies and LZD resistance level for G2447T. In conclusion, the risk of selection of high-level LZD-resistant S. epidermidis strains is weak, but does exist. It could probably appear in case of long-term treatment and be favored in the case of a pre-existing mutation in L3 ribosomal protein. Thus, broad screening conditions for pathogenic strains should probably be considered.

The establishment and development of the intestinal microbiota is known to be associated with profound short- and long-term effects on the health of full-term infants (FTI), but studies are just starting for preterm infants (PTI). The data also mostly come from western countries and little information is available for the Middle East. Here, we determined the composition and dynamics of the intestinal microbiota during the first month of life for PTI (n = 66) and FTI (n = 17) in Lebanon. Fecal samples were collected weekly and analyzed by quantitative PCR (q-PCR) and temporal temperature gradient gel electrophoresis (TTGE). We observed differences in the establishment and composition of the intestinal microbiota between the two groups. q-PCR showed that PTI were more highly colonized by Staphylococcus than FTI in the first three weeks of life; whereas FTI were more highly colonized by Clostridium clusters I and XI. At one month of life, PTI were mainly colonized by facultative anaerobes and a few strict anaerobes, such as Clostridium cluster I and Bifidobacterium. The type of feeding and antibiotic treatments significantly affected intestinal colonization. TTGE revealed low species diversity in both groups and high inter-individual variability in PTI. Our findings show that PTI had altered intestinal colonization with a higher occurrence of potential pathogens (Enterobacter, Clostridium sp) than FTI. This suggests the need for intervention strategies for PTI to modulate their intestinal microbiota and promote their health.

PURPOSE OF REVIEW The gut microbiota is a very complex ecosystem which interacts extensively with the host, influencing multiple metabolic and physiological functions. Several diseases have been shown to be associated with specific alterations in gut microbiota. It is more and more underscored as playing a major role in the development of insulin resistance and inflammation associated with excess weight gain. RECENT FINDINGS Recent studies in obese patients have shown perturbations in gut microbiota with a weight gain-associated increase in the Firmicutes/Bacteroidetes ratio ameliorated by various attempts at inducing weight loss. SUMMARY Intestinal microbiota may contribute to the development of inflammation and insulin resistance by two main mechanisms. First, gut microbiota might facilitate energy harvest from the gut leading via perturbation in energy homeostasis to fat deposition and increased adipokine production and plasma free fatty acid levels both contributing to insulin resistance and inflammation. Alternatively, it can initiate an inflammatory process either originating from the intestine or generated at the peripheral level via endotoxin leakage into the blood from the intestine, both leading secondarily to insulin resistance.

Some clinical studies have suggested a relationship between allergic diseases and gut microbiota. We aimed to study bifidobacterial colonization at species and strain levels in ten allergic French infants included at their first clinical consultation and 20 controls matching for age at sampling, mode of delivery, per partum antibiotics, type of feeding and antibiotics in the first weeks of life. The faecal microbiota was analyzed by culture methods and TTGE. Bifidobacterial species and strains were identified using multiplex PCR and Box-PCR fingerprinting. No differences were observed between groups in the number of colonized infants or in the levels of colonization by the main aerobic and anaerobic genera. All infants were colonized with high levels of Bifidobacterium except for one in each group. One to 5 Bifidobacterium species and 1 to 7 strains were observed per subject independently of allergic status and age at sampling. Our study showed the infants to be colonized by several species and strains, including several strains from the same species. This diversity in Bifidobacterium colonization was not related with the allergic status and showed that the link between Bifidobacterium colonization and allergic diseases is complex and cannot be restricted to the role attributed to Bifidobacterium species.

The various bacterial communities associated with humans have many functions and the gut microbiota has a major role in the host. Bacterial imbalance in the gut, known as dysbiosis, has therefore been linked to several diseases. Probiotics, that is, microbial strains that have beneficial effects on the host, are thought to benefit this intestinal ecosystem. Hence, knowledge of the gut microbiota composition and an understanding of its functionalities are of interest. Recently, efforts have focused on developing new high-throughput techniques for studying microbial cells and complex communities. Among them, proteomics is increasingly being used. The purpose of this article is to focus on the recent development of this technology and its usefulness in analyzing the human gut ecosystem and probiotic strains.

L’intestin constitue un écosystème extrêmement complexe etune communauté microbienne dynamique où coexistent bactéries,archées, virus et eucaryotes (Weinstock 2012). Ce microbiote esten contact permanent avec les nutriments et les cellules de l’hôte,responsable d’interactions fortes. Les relations micro-organismeshôte ont cependant longtemps été regardées essentiellement sousl’angle de la pathogénicité, mais aujourd’hui le microbiote commensal est reconnu pour ses interactions bénéfiques l’organismehumain en faisant un véritable partenaire de l’hôte. Ce partenariatdébute à la naissance, la colonisation bactérienne commençantdès la rupture des membranes fœtales. Les bactéries, dont lenombre tout d’abord considéré comme 10 fois supérieur auxcellules eucaryotes, ont été récemment réévaluées à 3,8 1013, soitdans un rapport similaire à celui des eucaryotes de l’organismehumain (Sender et al. 2016). La majorité du microbiote résidedans l’intestin, essentiellement le côlon, où il est caractérisé parson haut niveau et sa large diversité. Le nombre d’espèces bactériennes a été évalué par les techniques de culture classique à 400 à500 espèces par individu. Par des techniques indépendantes de laculture ce nombre est évalué à plus de 1000 espèces par individu(Tap et al. 2009), soit environ 25 fois le génome humain (Qinet al. 2010). Le microbiote intestinal peut ainsi être considérécomme un véritable organe, ouvert, métaboliquement adaptableet rapidement renouvelable.

Depuis plusieurs decennies, l’incidence de l’allergie augmente dramatiquement dans les pays industrialises. Cette augmentation est actuellement reliee, entre autres facteurs, a un defaut de maturation du systeme immunitaire par les bacteries commensales, a une hygiene accrue et a l’utilisation frequente d’antibiotiques, responsables d’une modification d’etablissement du microbiote intestinal au cours des premiers mois de vie. Cette theorie est a l’origine de l’utilisation des probiotiques et des prebiotiques, modulateurs du microbiote, dans la prevention et le traitement de l’allergie, strategie qui suscite beaucoup d’interet. Cette revue fait le point sur les etudes cliniques randomisees, realisees en double insu, avec administration de pro- et/ou prebiotiques dans le traitement ou la prevention de l’allergie. Certaines etudes ont montre des resultats prometteurs. Meme si les connaissances actuelles ne permettent pas de recommander l’utilisation de probiotiques dans le traitement ni dans la prevention des allergies, cette approche est seduisante et merite d’etre approfondie.

Studies suggesting that the development of atopy is linked to gut microbiota composition are inconclusive on whether dysbiosis precedes or arises from allergic symptoms. Using a mouse model of cow's milk allergy, we aimed at investigating the link between the intestinal microbiota, allergic sensitization, and the severity of symptoms. Germ-free and conventional mice were orally sensitized with whey proteins and cholera toxin, and then orally challenged with β-lactoglobulin (BLG). Allergic responses were monitored with clinical symptoms, plasma markers of sensitization, and the T-helper Th1/Th2/regulatory-T-cell balance. Microbiota compositions were analysed using denaturing gradient gel electrophoresis and culture methods. Germ-free mice were found to be more responsive than conventional mice to sensitization, displaying a greater reduction of rectal temperature upon challenge, higher levels of blood mouse mast cell protease-1 (mMCP-1) and BLG-specific immunoglobulin G1 (IgG1), and a systemic Th2-skewed response. This may be explained by a high susceptibility to release mMCP-1 even in the presence of low levels of IgE. Sensitization did not alter the microbiota composition. However, the absence of or low Staphylococcus colonization in the caecum was associated with high allergic manifestations. This work demonstrates that intestinal colonization protects against oral sensitization and allergic response. This is the first study to show a relationship between alterations within the subdominant microbiota and severity of food allergy.

This study reports the antibiotic susceptibility and genetic resistance determinants of 39 Clostridium butyricum strains isolated from the faeces of preterm infants as well as one reference strain. Results showed that all the strains were susceptible to cefoxitin, imipenem, vancomycin, tigecycline, metronidazole, chloramphenicol and linezolid. Resistance was observed to clindamycin (100%), penicillin G, amoxicillin and piperacillin (15%), tetracycline (7.5%) and erythromycin (5%). Investigation of the genetic basis of the observed resistance phenotypes showed that resistance to penicillin was due to β-lactamase activity and that resistance to tetracycline involved tet(O) or tet(O/32/O) homologue genes. Clindamycin and erythromycin resistance may involve another genetic determinant, different from those commonly described for clostridia.

Immune-enhancing diets (IEDs) contain a mixture of nutrients claimed to have immunological properties. Therefore, it seemed relevant to determine the effect of each of their components. The aim of this study was to examine the role of arginine (Arg) and ω3 polyunsaturated fatty acids (ω3 PUFAs) in the effect of an IED (Crucial(®)) in a validated rat model of inflammation induced by turpentine (TI).Forty-two rats were randomized into five groups: AL (ad libitum), TI-EN (TI+standard enteral nutrition (EN): Sondalis(®)HP), TI-EN-Arg (TI+standard EN+Arg in equimolar concentration to Arg in the IED), TI-M-IED (TI+modified IED containing the same ω6/ω3 ratio as in standard EN) and TI-IED (TI+Crucial(®)). Blood was sampled to determine CD25 receptor density on lymphocytes. TNF-α, IL-6 and NO (production and expression) were evaluated on isolated macrophages. Mesenteric lymph nodes, spleen and liver were cultured for analysis of enterobacterial translocation and dissemination.CD25 density was decreased after TI and was corrected in the TI-EN-Arg, TI-M-IED and TI-IED groups (p0.05). TI induced an alteration of macrophage mRNA expression of IL-6, TNF-α and iNOS, corrected in the TI-EN-Arg and TI-M-IED groups (p0.05), but not by the IED. Enterobacterial translocation was observed in all treated groups, nevertheless the amount tended (p=0.054) to be lower in the TI-EN-Arg group.Arg and ω3 PUFAs make a major contribution to IED effects, but our study shows interaction between them on macrophage reactivity. This indicates that the individual properties of each pharmaconutrient are not additive in IEDs.

The beneficial effects ofBifidobacteriumare partly due to its immunostimulatory properties. These immunostimulatory properties may be linked to the presence of unmethylated CpG motifs specific to bacterial DNA, which may induce a TH1 response by activating Toll-like receptors (TLR). Usingin silicoanalyses, PCR amplification, and dot blotting, we characterized the CpG content of various bifidobacterial strains and evaluated the immunostimulatory properties and genomic heterogeneity of these motifs in the genus. Ourin silicostudy, based on entire genome sequences from five bifidobacterial strains, showed thatBifidobacteriumgenomes contain numerous CpG motifs, including 5′-purine-purine-CG-pyrimidine-pyrimidine-3′ and 5′-purine-TCG-pyrimidine-pyrimidine-3′ motifs, and biologically active sequences previously identified in lactic acid bacteria. We identified four CpG-rich sequences withBifidobacterium longumNCC2705. Two sequences with a percent G+C of about 68% included 14 and 16 CpG motifs. Two sequences with a percent G+C of about 60% included 16 and 6 CpG motifs. These sequences induce the production of monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor alpha (TNF-α) through a pattern of TLR9 stimulation on RAW 264.7 macrophages. No link could be established between their immunostimulatory properties, the number of CpG motifs, and percent G+C. We investigated inter- and intraspecies heterogeneity in 71 strains of various origins. These sequences were highly conserved in the genus. No link was found between the presence of the CpG-rich sequence and the origin of the strains (healthy, allergic, or preterm infants). The high frequency of CpG motifs in the DNA ofBifidobacteriummay play an important role in the immunostimulatory properties of commensal or probiotic bifidobacterial strains.

The genus Bifidobacterium comprises several species that are important contributors to the gut microbiome, with some strains having beneficial health effects. Understanding the evolutionary emergence of advantageous biological properties requires knowledge of the genetic diversity and clonal structure of species. We sequenced seven housekeeping genes in 119 Bifidobacterium strains of Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum. Phylogenetic analysis of concatenated sequences delineated sequence clusters that correspond to previously named taxa, and suggested that B. longum subsp. infantis is a nascent lineage emerging from within B. longum subsp. longum. Clear traces of recombination among distant bifidobacterial species indicate leaky species borders and warn against the practice of single gene-based identification. Multilocus sequence typing achieved precise strain genotyping, with discrimination indices above 99% in B. bifidum, B. breve and B. longum, providing a powerful tool for strain traceability, colonization dynamics and ecological studies. Frequent homologous recombination accelerates clonal diversification and may facilitate the transfer of biological properties among bifidobacterial strains.

Background: Newborns display high intestinal permeability and a naive adaptive immune system, but infections are rare, indicating strong innate defense mechanisms. Objective: To measure the kinetics of fecal β-defensin-2 (HBD2), an inducible endogenous antimicrobial peptide produced by intestinal epithelial cells, in full-term and preterm infants. Methods: As a first step of this bicentric study, we enrolled 30 healthy full-term infants and 20 healthy preterm infants, with fecal samples collected at days 3, 7 12 and 30 in full-term infants and at days 15, 30 and 60 in preterm infants. As a second step, we enrolled 10 preterm infants with intestinal distress, either necrotizing enterocolitis (NEC) Bell’s stage III (n = 3) or isolated rectal bleeding (n = 7) and 20 controls, cross-matched for gestational age and age at sampling. Results: HBD2 decreased significantly from day 3 to day 7 (227 ng/g; 14–440 vs. 117 ng/g; 30–470, p = 0.01) then moderately until day 30 (84 ng/g; 10–500) in healthy full-term infants. Healthy preterm infants showed similar high levels between days 15 and 60 (82 ng/g; 30–154 and 85 ng/g; 26–390, respectively). No significant variation of fecal HBD2 levels was observed between infants with clinical features of intestinal distress (77 ng/g, 2–1,271) and cross-matched controls (56 ng/g, 31–164). However, 2/3 infants with NEC and 1/7 infants with isolated rectal bleeding had HBD2 levels above the maximal level observed in controls. Conclusions: The kinetics of fecal HBD2 in the neonatal period indicate that this inducible defensin can be detected at high level in the feces of full-term and preterm infants, independently of gestational age or mode of feeding. The potential role of fecal HBD2 in detecting NEC is suggested.

The intestinal microbiota is a complex ecosystem harbouring about 10(14) micro-organisms composed of nearly 400 hundred species. It plays various important functions in the gut, including metabolic, flora, barrier and stimulation of the intestinal immune system. Most studies have been based on culture, but more recent molecular biology techniques have provided complementary information. The formation of this ecosystem begins rapidly in the newborn; it is sterile at birth and is based on contact with the maternal flora and the surrounding environment. Although little is known about the factors leading to the development of bacteria, numerous external factors will affect the microbial succession: mode of delivery, environmental conditions, type of feeding, gestational age, and antibiotics. Recent data report a delay in intestinal colonization especially of enteric maternal bacteria. Which may be due to strict hygiene measures during birth. The clinical impact of these variations is not known but they could lead to lack of barrier flora or poor immune system stimulation in the gut. Modulation of gut microbiota in neonates with infant formulas containing either probiotics, prebiotics or non viable bacterias and their metabolites, or nucleotides is discussed.

The benefit of immune-enhancing diets (IEDs) in the intensive care unit remains controversial. Considering their complexity, the role of each component, in particular arginine (Arg), in their properties is largely unknown. The aim of this study was to determine the role of arginine in the immunomodulatory effects of an IED (Crucial®) in head-injured rats. Thirty-four rats were randomized into five groups: AL (ad libitum), HI (head-injured), HI-STD (HI + standard enteral nutrition, EN), HI-STD-Arg (HI + standard EN + Arg in equimolar concentration to Arg in IED), and HI-IED (HI + IED). These isocaloric and isonitrogenous diets were administered over 4 days. After death, the thymus was removed and weighed. The density of CD25, CD4 and CD8 on lymphocytes from blood and from Peyer patches was evaluated. Mesenteric lymph nodes, liver and spleen were cultured for analysis of enterobacterial translocation and dissemination. HI induced an atrophy of the thymus which was not corrected by the standard diet (HI 0.27 ± 0.03, HI-STD 0.35 ± 0.03 vs. AL 0.49 ± 0.02 g; p