Association between gut microbiota at 3.5 years of age and body mass index at 5 years: results from two French nationwide birth cohorts.

BACKGROUND/OBJECTIVES: The relationship between gut microbiota and changes in body mass index (BMI) or pediatric overweight in early life remains unclear, and information regarding the preterm population is scarce. This study aimed to investigate how the gut microbiota at 3.5 years of age is associated with (1) later BMI at 5 years, and (2) BMI z-score variations between 2 and 5 years in children from two French nationwide birth cohorts. SUBJECTS/METHODS: Bacterial 16S rRNA gene sequencing was performed to profile the gut microbiota at 3.5 years of age in preterm children (n = 143, EPIPAGE 2 cohort) and late preterm/full-term children (n = 369, ELFE cohort). The predicted abundances of metabolic functions were computed using PICRUSt2. Anthropometric measurements were collected at 2 and 5 years of age during medical examinations or retrieved from children's health records. Statistical analyses included multivariable linear and logistic regressions, random forest variable selection, and MiRKAT. RESULTS: The Firmicutes to Bacteroidetes (F/B) ratio at 3.5 years was positively associated with the BMI z-score at 5 years. Several genera were positively ([Eubacterium] hallii group, Fusicatenibacter, and [Eubacterium] ventriosum group) or negatively (Eggerthella, Colidextribacter, and Ruminococcaceae CAG-352) associated with the BMI z-scores at 5 years. Some genera were also associated with variations in the BMI z-scores between 2 and 5 years of age. Predicted metabolic functions, including steroid hormone biosynthesis, biotin metabolism, glycosaminoglycan degradation, and amino sugar and nucleotide sugar metabolism, were associated with lower BMI z-scores at 5 years. The unsaturated fatty acids biosynthesis pathway was associated with higher BMI z-scores. CONCLUSIONS: These findings indicate that the gut microbiota at 3.5 years is associated with later BMI during childhood, independent of preterm or term birth, suggesting that changes in the gut microbiota that may predispose to adult obesity begin in early childhood.

Faecal calprotectin and gut microbiota do not predict enteropathy in very preterm infants.

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.

Clostridial Strain-Specific Characteristics Associated with Necrotizing Enterocolitis.

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.

Preterm infants with necrotising enterocolitis demonstrate an unbalanced gut microbiota.

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.

Establishment and development of the intestinal microbiota of preterm infants in a Lebanese tertiary hospital.

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.

Three Novel Candidate Probiotic Strains with Prophylactic Properties in a Murine Model of Cow's Milk Allergy.

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 ß-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.

One-Step Multiplex PCR Assay for Differentiating Proposed New Species "Clostridium neonatale" from Closely Related Species.

"Clostridium neonatale" sp. nov., previously involved in an outbreak of neonatal necrotizing enterocolitis, was recently proposed as a new species of the Clostridium genus sensu stricto. We developed a one-step multiplex colony PCR for C. neonatale identification and investigated C. neonatale intestinal colonization frequency in healthy preterm neonates.

Safety of a New Amino Acid Formula in Infants Allergic to Cow's Milk and Intolerant to Hydrolysates.

OBJECTIVES: 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. METHODS: Infants (ages < 18 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: 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 ±â€Š2.3 vs -20.8 ±â€Š2.2, 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. CONCLUSIONS: 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.

Antibacterial Labdane Diterpenoids from Vitex vestita.

A large-scale in vitro screening of tropical plants using an antibacterial assay permitted the selection of several species with significant antibacterial activities. Bioassay-guided purification of the dichloromethane extract of the leaves of the Malaysian species Vitex vestita, led to the isolation of six new labdane-type diterpenoids, namely, 12-epivitexolide A (2), vitexolides B and C (3 and 4), vitexolide E (8), and vitexolins A and B (5 and 6), along with six known compounds, vitexolides A (1) and D (7), acuminolide (9), 3ß-hydroxyanticopalic acid (10), 8α-hydroxyanticopalic acid (11), and 6α-hydroxyanticopalic acid (12). Their structures were elucidated on the basis of 1D and 2D NMR analyses and HRMS experiments. Both variable-temperature NMR spectroscopic studies and chemical modifications were performed to investigate the dynamic epimerization of the γ-hydroxybutenolide moiety of compounds 1-4. Compounds were assayed against a panel of 46 Gram-positive strains. Vitexolide A (1) exhibited the most potent antibacterial activity with minimal inhibitory concentration values ranging from 6 to 96 µM, whereas compounds 2 and 6-9 showed moderate antibacterial activity. The presence of a ß-hydroxyalkyl-γ-hydroxybutenolide subunit contributed significantly to antibacterial activity. Compounds 1-4 and 6-9 showed cytotoxic activities against the HCT-116 cancer cell line (1 < IC50s < 10 µM) and human fetal lung fibroblast MRC5 cell line (1 < IC50s < 10 µM for compounds 1, 2, 7, 8, and 9).

The functional dlt operon of Clostridium butyricum controls the D-alanylation of cell wall components and influences cell septation and vancomycin-induced lysis.

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.

16S rRNA gene sequencing, multilocus sequence analysis, and mass spectrometry identification of the proposed new species "Clostridium neonatale".

In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, "Clostridium neonatale." To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies.

Bifidobacterium longum and Bifidobacterium breve isolates from preterm and full term neonates: comparison of cell surface properties.

We compared autoaggregation, surface hydrophobicity and Caco-2 cells adhesion capabilities of independent Bifidobacterium breve (n = 22) and Bifidobacterium longum (n = 25) strains isolated from preterm (n = 20) and full term neonates (n = 27). Concerning strains properties, a correlation between autoaggregation and surface hydrophobicity was found for B. longum (r = 0.40, p = 0.048), B. breve (r = 0.57, p = 0.005), and all strains independently of the species consideration (r = 0.46, p = 0.001). The absence of difference in adhesion capabilities between preterm and full term neonate strains suggests a strain-dependent property. However, B. longum strains from preterm neonates (n = 10) showed higher autoaggregation ability (p = 0.044). Additionally, independently of species consideration, preterm neonates strains showed lower surface hydrophobicity (p = 0.027). As far as species are considered, preterm neonate B. breve strains (n = 10) showed significantly lower surface hydrophobicity percentages (p = 0.043). Our results suggest the existence of variations in bifidobacteria membrane structure and/or composition that may reflect adaptation of these bacteria to the intestinal environment of either preterm or full term neonates. Such information is of interest when considering the use of bifidobacteria probiotic strains for modulation of preterm neonates gut microbiota.

Intestinal permeability and fecal eosinophil-derived neurotoxin are the best diagnosis tools for digestive non-IgE-mediated cow's milk allergy in toddlers.

BACKGROUND: Food allergy is a common problem in France involving 4%-6% of toddlers. As opposed to IgE-mediated cow's milk allergy (CMA), delayed-onset CMA, mostly, non-IgE-mediated, remains difficult to diagnose in toddlers. Our study assessed the diagnostic performances of intestinal permeability and of fecal markers, in comparison with the standard allergic work-up in children referred for CMA diagnosis. METHODS: Twenty-five consecutive children, mean age (standard deviation) 6.3 months (4.8) with digestive and/or extra-digestive manifestations suggesting CMA, were prospectively studied based on a standardized allergic work-up (specific cow's protein IgE and IgG, skin prick test, atopy patch test and oral open cow's milk challenge) and digestive work-up including fecal microbiota analysis, intestinal permeability determination (urinary lactitol/mannitol ratio) and fecal markers measurement, i.e., α(1)-antitrypsin, tumor necrosis factor-α, calprotectin, ß-defensin2, secretory IgA and eosinophil-derived neurotoxin (EDN). Receiver operating characteristic (ROC) curves were calculated for all markers in order to define cut-off levels. RESULTS: The cow's milk challenge was positive in 11 children and negative in 14. The global test performances, i.e., the number of true positive+negative cases/the total number of cases, were 76% for intestinal permeability; 72% for fecal EDN; contrasting with atopy patch test, 68%; IgE, 60%; skin prick test, 55% and IgG, 52%. CONCLUSIONS: In this routine diagnosis allergy work-up for CMA in toddlers, the best efficacy was seen for intestinal permeability compared to IgE, IgG, skin prick test and atopy patch test. Moreover, fecal EDN in a single spot sample displayed a similar performance.

Tolerance of Bifidobacterium human isolates to bile, acid and oxygen.

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.

Early Life Factors Influencing Children Gut Microbiota at 3.5 Years from Two French Birth Cohorts.

Early life gut microbiota-influencing factors may play an important role in programming individuals long-term health and substantial efforts have been devoted into studying the development of the gut microbiota in relation to early life events. This study aimed to examine in a single study, the persistence of associations between 20 factors occurring in the early life and the gut microbiota at 3.5 years of 798 children from two French nationwide birth cohorts, EPIPAGE 2 (very preterm children) and ELFE (late preterm and full-term children). Gut microbiota profiling was assessed using 16S rRNA gene sequencing-based method. Upon thorough adjustment of confounding factors, we demonstrated that gestational age was one of the factors most associated with gut microbiota differences with a noticeable imprint of prematurity at 3.5 years of age. Children born by cesarean section harbored lower richness and diversity and a different overall gut microbiota composition independently of preterm status. Children who had ever received human milk were associated with a Prevotella-driven enterotype (P_type) compared to those who had never received human milk. Living with a sibling was associated with higher diversity. Children with siblings and those attending daycare centers were associated with a P_type enterotype. Maternal factors including the country of birth and preconception maternal body mass index were associated with some microbiota characteristics: children born to overweight or obese mothers showed increased gut microbiota richness. This study reveals that multiple exposures operating from early life imprint the gut microbiota at 3.5 years that is a pivotal age when the gut microbiota acquires many of its adult characteristics.

Neonatal necrotizing enterocolitis: Clostridium butyricum and Clostridium neonatale fermentation metabolism and enteropathogenicity.

Bacterial colonization in the gut plays a pivotal role in neonatal necrotizing enterocolitis (NEC) development, but the relationship between bacteria and NEC remains unclear. In this study, we aimed to elucidate whether bacterial butyrate end-fermentation metabolites participate in the development of NEC lesions and confirm the enteropathogenicity of Clostridium butyricum and Clostridium neonatale in NEC. First, we produced C.butyricum and C.neonatale strains impaired in butyrate production by genetically inactivating the hbd gene encoding ß-hydroxybutyryl-CoA dehydrogenase that produces end-fermentation metabolites. Second, we evaluated the enteropathogenicty of the hbd-knockout strains in a gnotobiotic quail model of NEC. The analyses showed that animals harboring these strains had significantly fewer and less intense intestinal lesions than those harboring the respective wild-type strains. In the absence of specific biological markers of NEC, the data provide original and new mechanistic insights into the disease pathophysiology, a necessary step for developing potential novel therapies.

Occurrence of Neonatal Necrotizing Enterocolitis in Premature Neonates and Gut Microbiota: A Case-Control Prospective Multicenter Study.

BACKGROUND: Necrotizing enterocolitis (NEC) is still one of the leading causes of neonatal death. The present study reports the data from a French case-control prospective multicenter study. METHODS: A total of 146 preterm neonates (PNs) with or without NEC were included. Bacterial 16S rRNA gene sequencing was performed on stool samples (n = 103). Specific culture media were used to isolate Escherichia coli, Clostridium butyricum, and Clostridium neonatale, and strains were phenotypically characterized. RESULTS: The gut microbiota of PNs was dominated by Firmicutes and Proteobacteria, and five enterotypes were identified. The microbiota composition was similar between NEC cases and PN controls. However, differences were observed in the relative abundance of Lactobacillus genus, which was significantly lower in the NEC group, whereas that of the Clostridium cluster III was significantly higher (p < 0.05). Within enterotypes, several phylotypes were significantly more abundant in NEC cases (p < 0.05). Regarding perinatal factors, a statistical association was found between the gut microbiota and cesarean delivery and antifungal therapy. In NEC cases and PN controls, the carriage rates and virulence genes of uropathogenic E. coli were equivalent based on culture. No correlation was found between E. coli, C. butyricum, and C. neonatale carriages, beta-lactam resistance, and antibiotic treatment. CONCLUSIONS: At disease onset, our data support a microbiota dysbiosis between NEC and control infants at the genus level. In addition, it provides valuable information on bacterial antimicrobial susceptibility.

Arginine reduces bacterial invasion in rats with head injury: an in vivo evaluation by bioluminescence.

OBJECTIVES: The benefit of arginine in intensive care unit patients with severe sepsis is still controversial. An excessive supply of arginine could lead to an overproduction of nitric oxide and could be responsible for septic shock and multiorgan failure. However, this claim is not supported by any experimental or clinical data. We set out to determine whether an enteral supply of arginine would modulate bacterial invasion in rats with head injury. METHODS: Male Sprague-Dawley rats with head injury were randomized into two groups. Group 1 included rats with head injury fed a standard enteral nutrition (Sondalis HP, n = 10) and group 2 included rats with head injury fed the standard enteral nutrition plus arginine (4 g/kg/d, n = 11). Two days after head injury, the rats received a single enteral bolus of luminescent Escherichia coli Xen 14. Bacterial proliferation was evaluated in vivo at time + 2 hrs and time + 6 hrs after E. coli challenge. Four days after head injury, blood was sampled for arginine and fibrinogen assay. Muscles, intestine, spleen, and thymus were removed and weighed. RESULTS: There was no mortality in either group. The luminescence signal was similar in the two groups at time +2 hrs (group 1: 414 [5-823] vs. group 2: 496 [0.1-993] (median value[min-max]; not significant) and was significantly lower at time +6 hrs in group 2 (group 1: 71 [0-142] vs. group 2: 8.5 [0-17]; p = .026). Arginine treatment did not improve any nutritional parameters. CONCLUSIONS: Arginine was not responsible for mortality in rats with head injury with infectious complications and reduced the intensity of bacterial invasion.