Hugues Piloquet

Early Intestinal Bacterial Colonization and Necrotizing Enterocolitis in Premature Infants: The Putative Role of Clostridium

Necrotizing enterocolitis (NEC) is among the most severe conditions that can affect preterm infants. Although the etiology of NEC remains unknown, initial bacterial colonization could play a pivotal role in the development of NEC. To further explore the putative relationship between pathogen microorganisms and NEC, we conducted a prospective case-control study in 12 preterm infants with a new approach based on molecular techniques. Over an inclusion period of 24 mo, 12 neonates of <34 wk gestational age admitted to the neonatal unit were enrolled. The group included three cases of NEC, and nine control infants without evidence of NEC who were matched for gestational age and birth weight. Stool samples were collected at weekly intervals from all infants. PCR and temporal temperature gradient gel electrophoresis of 16S ribosomal DNA were used to detect the establishment of bacterial communities in the digestive tract. A salient feature of the bacteriological pattern was observed only in the three infants who later developed NEC: A band corresponding to the Clostridium perfringens subgroup could be detected in early samples, before diagnosis. There was no evidence for this specific band in any of the nine controls. To our knowledge, the current report is the first to demonstrate that the use of molecular techniques based on the study of bacterial 16S rRNA genes allowed the recognition of C. perfringens species in the first 2 wk of life of three infants who later displayed symptoms of NEC. A significant temporal relationship was thus established between early colonization by Clostridium and the later development of NEC. Compared with conventional bacteriological culturing methods, the use of this new molecular approach to analyze the gastrointestinal ecosystem should therefore allow a more complete and rapid assessment of intestinal flora. Although the current data do not constitute definitive proof that the identified bacterial species was a causative agent in the development of NEC, they outline the promise of this new technique based on molecular biology, and suggest that large-scale studies on a much wider population at high risk for NEC may be warranted.

Oral supplementation with probiotics in very-low-birth-weight preterm infants: a randomized, double-blind, placebo-controlled trial

BACKGROUND Although recent reports suggest that supplementation with probiotics may enhance intestinal function in premature infants, the mechanisms are unclear, and questions remain regarding the safety and efficacy of probiotics in extremely low-birth-weight infants. OBJECTIVE The objective was to evaluate the efficacy of probiotics on the digestive tolerance to enteral feeding in preterm infants born with a very low or extremely low birth weight. DESIGN In a bicentric, double-blind, randomized controlled clinical trial that was stratified for center and birth weight, 45 infants received enteral probiotics (Bifidobacterium longum BB536 and Lactobacillus rhamnosus GG; BB536-LGG) and 49 received placebo. The primary endpoint was the percentage of infants receiving >50% of their nutritional needs via enteral feeding on the 14th day of life. A triangular test was used to perform sequential analysis. RESULTS The trial was discontinued after the fourth sequential analysis concluded a lack of effect. The primary endpoint was not significantly different between the probiotic (57.8%) and placebo (57.1%) groups (P = 0.95). However, in infants who weighed >1000 g, probiotic supplementation was associated with a shortening in the time to reach full enteral feeding (P = 0.04). Other than colonization by the probiotic strains, no alteration in the composition of intestinal microbiota or changes in the fecal excretion of calprotectin was observed. No colonization by probiotic strains was detected in infants who weighed < or =1000 g, presumably because of more frequent suspensions of enteral feeding, more courses of antibiotic treatment, or both. CONCLUSIONS Supplementation with BB536-LGG may not improve the gastrointestinal tolerance to enteral feeding in very-low-birth-weight infants but may improve gastrointestinal tolerance in infants weighing >1000 g. This trial was registered at clinicaltrials.gov as NCT 00290576.

Acute Effects of Intravenous Glutamine Supplementation on Protein Metabolism in Very Low Birth Weight Infants: A Stable Isotope Study

Although very low birth weight infants are subjected to severe stress and glutamine is now considered a conditionally essential amino acid that may attenuate stress-induced protein wasting in adults, current amino acid solutions designed for neonatal parenteral nutrition do not contain glutamine. To determine whether a short-term supplementation with i.v. glutamine would affect protein metabolism in very low birth weight infants, 13 preterm neonates (gestational age, 28–30 wk; birth weight, 820–1610 g) receiving parenteral nutrition supplying 1.5 g · kg−-1 · d−1 amino acids and approximately 60 nonprotein kcal · kg−1 · d−1 were randomized to receive an i.v. supplement made of either 1) natural l-glutamine (0.5 g · kg−1 · d−1; glutamine group), or 2) an isonitrogenous glutamine-free amino acid mixture (control group), for 24 h starting on the third day of life. On the fourth day of life, they received a 2-h infusion of NaH13CO3 to assess the recovery of 13C in breath, immediately followed by a 3-h l-[1-13C]leucine infusion. Plasma ammonia did not differ between the groups. Glutamine supplementation was associated with 1) higher plasma glutamine (629 ± 94 versus 503 ± 83 μM, mean ± SD;p < 0.05, one-tailed unpaired t test), 2) lower rates of leucine release from protein breakdown (−16%, p < 0.05) and leucine oxidation (−35%, p < 0.05), 3) a lower rate of nonoxidative leucine disposal, an index of protein synthesis (−20%, p < 0.05), and 4) no change in protein balance (nonoxidative leucine disposal − leucine release from protein breakdown, NS). We conclude that although parenteral glutamine failed to enhance rates of protein synthesis, glutamine may have an acute protein-sparing effect, as it suppressed leucine oxidation and protein breakdown, in parenterally fed very low birth weight infants.

Dépister la dénutrition de l’enfant en pratique courante

Protein energy malnutrition (PEM) occurs when energy and protein intake do not meet requirements. It has a functional and structural impact and increases both morbidity and mortality of a given disease. The Nutrition Committee of the French Pediatric Society recommends weighing and measuring any child when hospitalized or seen in consultation. The body mass index (BMI) must be calculated and analyzed according to references any time growth kinetics cannot be analyzed. Any child with a BMI below the third centile or -2 standard deviations for age and sex needs to be examined looking for clinical signs of malnutrition and signs orienting toward an etiology and requires having his BMI and height dynamics plotted on a chart. PEM warrants drawing up a nutritional strategy along with the overall care plan. A target weight needs to be determined as well as the quantitative and qualitative nutritional care including its implementation. This plan must be evaluated afterwards in order to adapt the nutritional therapy.

Impact of Prophylactic Fundoplication on Survival without Growth Disorder in Left Congenital Diaphragmatic Hernia Requiring a Patch Repair

Growth impairment is frequent in surviving newborns with congenital diaphragmatic hernia requiring a patch repair. This multicenter retrospective study included 57 newborns and showed a significant relationship between prophylactic fundoplication performed during initial diaphragmatic repair and survival without disordered growth, after adjustment for propensity score (adjusted OR 4.7 [1.2-18.5]; P=.03).

Role of glucose and glutamine synthesis in the differential recovery of 13CO2 from infused [2-13C] versus [1-13C] acetate

Carbon exchange in the Krebs cycle may result in underestimation of substrate oxidation measured with 13C-labeled substrates, since carbon labeled in position 2 of acetyl-coenzyme A (CoA) could be incorporated into glucose (via gluconeogenesis) and glutamine. Five healthy volunteers were therefore infused with [1-13C] and [2-13C] acetate at a rate of 0.5 micromol x kg(-1) x min(-1) for 165 minutes on two different occasions in randomized order. Whole body acetate turnover did not differ between the two tracers: 7.9+/-0.3 and 7.5+/-0.6 micromol x kg(-1) x min(-1) (nonsignificant [NS]) for [1-13C] and [2-13C] acetate, respectively. Isotopic 13C enrichment was higher in expired CO2 (0.177+/-0.021 v 0.089+/-0.009 atom percent excess [APE], P < .01) and lower in glucose (0.074+/-0.017 v0.291+/-0.061 mole percent excess [MPE], P < .01) for [1-13C] acetate compared with [2-13C] acetate, respectively, at the end of the infusions. Glutamine isotopic enrichment was slightly but not significantly higher when infusing [1-13C] acetate versus [2-13C] acetate (0.348+/-0.038 v0.495+/-0.069 MPE, NS, respectively). At the end of the experiment, the recovery of 13CO2 from [1-13C] acetate was 44.8%+/-2.7%, and from [2-13C] acetate, 22.6%+/-1.3%. A significant correlation was observed between the differences in 13C enrichment of CO2 for the two tracers and glucose (deltaCO2=0.424 x deltaglucose + 0.001, R2=.9856, P=.0007) or glutamine (deltaCO2=0.621 x deltaglutamine + 0.004, R2=.9573, P=.0038) during the infusion. These results suggest that (1) although gluconeogenesis appears to be more responsible than glutamine for the differential recovery of [2-13C] versus [1-13C] acetate, other secondary pathways are probably also implicated; and (2) different recovery correction factors should be applied when measuring substrate oxidation with a stable isotope tracer depending on the expected position of 13C in acetyl-CoA.

Preweaning modulation of intestinal microbiota by oligosaccharides or amoxicillin can contribute to programming of adult microbiota in rats

OBJECTIVE Increasing evidence suggests that early nutrition has programming effects on adult health. Identifying mechanisms underlying nutritional programming would aid in the design of new disease prevention strategies. The intestinal microbiota could be a key player in this programming because it affects host metabolic homeostasis, postnatal gut colonization is sensitive to early nutrition, and initial microbial set-up is thought to shape microbiota composition for life. The aim of this study was to determine whether early manipulation of intestinal microbiota actually programs adult microbiota in rats. METHODS Suckling rats pups were supplemented with fructo-oligosaccharides, galacto-oligosaccharides/long-chain fructan mix (GOS/lcF, 9/1), acidic oligosaccharides, amoxicillin, or vehicle from the fifth to the fourteenth day of life, and weaned to standard chow at day 21. Ceco-colonic microbiota was characterized at 14 and 131 d by real-time polymerase chain reaction analysis. RESULTS At day 14, all treatments affected microbiota. Amoxicillin had the most significant effect. All oligosaccharides decreased Firmicutes levels, whereas only fructo-oligosaccharides and GOS/lcF increased bifidobacteria. At day 131, most of these effects had faded away but a significant, albeit minor, adult microbiota programming was observed for rats that received GOS/lcF mix before weaning, regarding Roseburia intestinalis cluster, one subdivision of the Erysipelotrichaceae family as well as butyrate kinase gene. CONCLUSIONS As revealed by a targeted quantitative polymerase chain reaction approach, programming of adult intestinal microbiota seems to vary according to the nature of the preweaning microbiotal modulator. This suggests that intestinal microbiota may, only under specific circumstances, serve as a relay of neonatal nutrition and thus potentially contribute to nutritional programming of host physiology.

Recovery of Exfoliated Cells From the Gastrointestinal Tract of Premature Infants: A New Tool to Perform “Noninvasive Biopsies?”

To gain insight into specific gene expression in the gastrointestinal (GI) tract of preterm infants, we adapted a method to isolate exfoliated epithelial cells. Gastric residual fluid aspirates (n = 89) or stool samples (n = 10) were collected from 96 neonates (gestational age, 24–36 wk). Cells were characterized by microscopic observation, cytokeratin-18 immunodetection, and expression of transcripts. The human origin of cellular DNA was confirmed by amplification of specific X and Y chromosome sequences. Isolation yielded 100–500 cells per sample for gastric aspirates (n = 8) and 10–20 cells for fecal samples (n = 5). Epithelial origin was confirmed by immunodetection of cytokeratin 18. Analyses of reverse transcribed products, using two independent methods, from 15 gastric fluid and two stool samples showed that 18S-rRNA and transcripts of beta-actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), and period1 were in quantities corresponding to at least 10 cells. On 59 aspirates, we found beta-actin transcripts (all but one), cytokeratin 18 (eight positive of eight samples), SLC26-A7-1 (13 positive of 19 samples), period2 (17 positive of 17 samples), and clock (25 positive of 26 samples). Exfoliated cells can be recovered from gastric aspirates and fecal samples and serve as a tool to investigate the impact of therapeutic and nutritional regimens on the maturation of GI functions.

Can Antibiotic Treatment in Preweaning Rats Alter Body Composition in Adulthood

Background: It is suggested that antibiotherapy in infancy might program adult body composition and thus could be a determinant of obesity risk. Although not convincingly substantiated by existing literature, this assumption is plausible since antibiotics affect intestinal microbiota, whose composition in adulthood is potentially programmable during infancy and which is able to interact with both fat development and central control of appetite. Objectives: In order to substantiate the link between antibiotherapy and programming of adult body composition, the present study investigated the impact of a course of amoxicillin treatment in neonatal period on subsequent growth and body composition in rats. Methods: Suckling rat pups were treated by oral gavage with an amoxicillin solution (150 mg·kg–1) or vehicle from postnatal day (PND)5 to PND15. All animals were fully weaned at PND21 then fed a standard diet until PND130. Animal growth and food intake were followed up until PND130, when body composition and plasma leptin were measured. Faecal microbiota was typified at regular intervals using real-time quantitative polymerase chain reaction. Results: Preweaning amoxicillin treatment affected the composition of the faecal microbiota of pups at PND21 but this impact did not sustain long beyond the antibiotic supplementation. Immediately after weaning, a transient increase in food intake (+11%) was noticed in amoxicillin-treated animals. However, no significant impact on either growth or body composition at adulthood was observed. Conclusions: In a neonatal animal model there is no evidence of a programming of adult body weight and composition by wide-spectrum antibiotic treatment in early life.

Role of Lactoferrin in Neonates and Infants: An Update

Abstract Lactoferrin is one of the most represented and important bioactive proteins in human and mammal milk. In humans, lactoferrin is responsible for several actions targeting anti‐infective, immunological, and gastrointestinal domains in neonates, infants, and young children. Evidence‐based data vouch for the ability of supplemented lactoferrin to prevent sepsis and necrotizing enterocolitis in preterm infants and to reduce the burden of morbidity related to gastrointestinal and respiratory pathogens in young children. However, several issues remain pending regarding answers and clarification related to quality control, correct intakes, optimal schedules and schemes of supplementations, interactions with probiotics, and different types of milk and formulas. This review summarizes the current evidence regarding lactoferrin and discusses the areas in need of further guidance prior to the adoption of strategies that include a routine use of lactoferrin in neonates and young children.