Fabien Magne

Conditions of bifidobacterial colonization in preterm infants : A prospective analysis

Background: Premature birth results in a delayed and abnormal qualitative pattern of gut colonization. This abnormal pattern is thought to affect intestinal development and contribute to a higher risk of gastrointestinal infectious diseases such as neonatal necrotizing enterocolitis (NEC). In particular, bifidobacteria are thought to play a major role. We therefore studied bifidobacterial colonization in preterm infants during the first month of life. Patients and Methods: Fecal samples were prospectively analyzed in 52 infants born at a gestational age ranging from 30 to 35 weeks fed with a preterm formula alone and, in 18, with their mothers milk. Fecal samples were collected twice per week during the hospital stay. Bifidobacterial colonization was analyzed with culture and a molecular method. Results: Bifidobacterial colonization occurred in 18 infants at a median age of 11 days, always greater than the corrected mean gestational age of 35.4 weeks (SD, 0.9) and greater than 34 weeks for 16 of 18. Colonization by bifidobacteria was affected by neither birthweight nor mode of delivery nor antibiotics given to the mother or infant. In contrast, birth gestational age had a significant impact on colonization by bifidobacteria (P < 0.05), which always occurred in children born at a birth gestational age greater than 32.9 weeks (P < 0.05). Conclusions: Birth gestational age seems to act as a major determinant of bifidobacterial colonization in the premature infant, suggesting the role of gut maturation, a finding that should probably be taken into account in manipulations of the gut flora aimed at reducing NEC.

PCR-TTGE Analysis of 16S rRNA from Rainbow Trout (Oncorhynchus mykiss) Gut Microbiota Reveals Host-Specific Communities of Active Bacteria

This study assessed the relative contributions of host genetics and diet in shaping the gut microbiota of rainbow trout. Full sibling fish from four unrelated families, each consisting of individuals derived from the mating of one male and one female belonging to a breeding program, were fed diets containing either vegetable proteins or vegetable oils for two months in comparison to a control diet consisting of only fish protein and fish oil. Two parallel approaches were applied on the same samples: transcriptionally active bacterial populations were examined based on RNA analysis and were compared with bacterial populations obtained from DNA analysis. Comparison of temporal temperature gradient gel electrophoresis (TTGE) profiles from DNA and RNA showed important differences, indicating that active bacterial populations were better described by RNA analysis. Results showed that some bacterial groups were significantly (P<0.05) associated with specific families, indicating that microbiota composition may be influenced by the host. In addition, the effect of diet on microbiota composition was dependent on the trout family.

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss).

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpoB gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpoB-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpoB-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus, and Shewanella marinus. In contrast to 16S rRNA gene-TTGE, rpoB-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpoB leads to a single band in TTGE, the rpoB gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.

Fecal microbial community in preterm infants

The gastrointestinal tract of a healthy fetus is sterile. In full term infants, the bacterial colonization of the gastrointestinal tract has been extensively studied if not entirely understood. During the birth process and rapidly thereafter, microbes from the mother and the surrounding environment colonize the gastrointestinal tract until a dense and complex bacterial community is established. In vaginally delivered neonates, bacteria appear in the stools during the first day of life, with usually Escherichia coli and Enterococcus spp., among the first, followed within the first 5 days by Bifidobacterium spp. Because, at this stage, the composition of the gut bacterial community is strongly influenced by the diet, a shift in the bacterial composition can be observed. By 10 days of age, most healthy full term neonates are colonized with a heterogeneous bacterial flora, with bifidobacteria dominant in breast-fed infants and a more diversified flora in formula-fed infants (1). A dynamic balance exists between the bacterial community, the host physiology, and the diet: all of them influence initial acquisition, subsequent development and eventual stability of the gut ecosystem (2). In contrast, in preterm infants, especially extremely low birth weight preterm infants (weighting less than 1,000 g at birth), bacterial colonization and its consequences on health have not been extensively studied. Many factors influence the biodiversity of the intestinal flora and may increase the risk of gastrointestinal disease such as necrotizing enterocolitis (NEC): immaturity of the main vital functions, the characteristics of the medical environment, from delivery to hospital discharge, the developmental stage of gastrointestinal and immune functions, the mode and environment of delivery, the feeding regimen, the kind of drug therapy (such as antibiotics, corticoids, etc.), or of other therapy (such as oxygenation). In adults, the human cecal flora differs quantitatively and qualitatively from the fecal flora. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces (3). So, the biodiversity of the bacterial community should be studied at these different levels of the gastrointestinal tract. For obvious reasons, most of the studies were performed on stool samples. In recent years, the use of ribosomal RNA, in particular, sequences of the 16S rRNA genes, has greatly facilitated the study of gastrointestinal tract ecology because it allows a culture-independent analysis of the fecal microbial community. In adult fecal samples, molecular tools have indicated that 60% to 80% of the total human microflora has not been cultivated (4). Techniques such as polymerase chain reaction, gene sequencing, in situ hybridization, and denaturing gradient gel electrophoresis are routinely used to study gut microbial ecology and have been recently reviewed by Suau (5). So far, only three studies inspected the microbial composition of premature infants using molecular methods (6–8). Thus, the aim of the present review is to describe the composition of fecal microbial community in preterm infants from the colonization of sterile gut, to present the different factors that contribute to its alterations, and to link the gut microflora to diseases such as NEC. This review is essentially based on data collected using traditional bacterial culture from fecal samples.

Effects on faecal microbiota of dietary and acidic oligosaccharides in children during partial formula feeding.

Objective: To test the safety and effect on faecal microbiota of a formula with prebiotic oligosaccharides alone or in combination with acidic oligosaccharides in infants at the age of partial formula feeding. Patients and Methods: The study was a double-blind, placebo-controlled, randomised intervention trial in which 82 healthy, full-term, partially breast-fed children, from 1 week to 3 months old, were given 1 of the following formulae: whey-based formula (control group), whey-based formula with galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS group), or whey-based formula with galacto- and long-chain fructo-oligosaccharides added with pectin-derived acidic oligosaccharides (scGOS/lcFOS/pAOS group). Children were studied for the duration of the partial formula feeding period and every 2 weeks for 2 months after breast-feeding cessation. The total bacteria count and the proportion of 7 bacterial families were determined using in situ hybridisation coupled to flow cytometry. Results: The total bacterial count did not alter with time or type of feeding (9.9 ± 0.1 log10 cells per gram wet weight). Compared with the control group, there was an increase of the Bifidobacterium genus (P = 0.0001), and a decrease of proportions for the Bacteroides group (P = 0.02) and the Clostridium coccoides group (P = 0.01) in both oligosaccharide groups. The proportion of bifidobacteria was significantly higher in the scGOS/lcFOS/pAOS compared with the scGOS/lcFOS group (P < 0.01). Conclusions: Infant formulae appear to be clinically safe and effective on infant microbiota. They minimize the alteration of faecal microbiota after cessation of breast-feeding and promote bifidobacteria proportions, with a stronger effect when acidic oligosaccharides are present.

A fermented formula in pre-term infants: clinical tolerance, gut microbiota, down-regulation of faecal calprotectin and up-regulation of faecal secretory IgA

Intestinal bacterial colonisation in pre-term infants is delayed compared with full-term infants, leading to an increased risk of gastrointestinal disease. Modulation of colonisation through dietary supplementation with probiotics or prebiotics could decrease such a risk. The present study evaluated clinical tolerance, the effects on gut microbiota, and inflammatory and immunological mucosal responses to an infant formula adapted for pre-term infants that included in its manufacturing process a fermentation step with two probiotic strains, Bifidobacterium breve C50 and Streptococcus thermophilus 065, inactivated by heat at the end of the process. A total of fifty-eight infants (gestational age: 30-35 weeks), fed either the fermented pre-term formula or a standard pre-term formula, were followed up during their hospital stay. Clinical tolerance, faecal microbiota using a culture and a culture-independent method (temporal temperature gel electrophoresis), faecal calprotectin and secretory IgA were analysed weekly. No difference was observed regarding anthropometric data and digestive tolerance, except for abdominal distension, the incidence of which was lower in infants fed the fermented formula for 2 weeks. Bacterial colonisation was not modified by the type of feeding, particularly for bifidobacteria. Faecal calprotectin was significantly lower in infants fed the fermented formula for 2 weeks, and secretory IgA increased with both mothers milk and the fermented formula. The fermented formula was well tolerated and did not significantly modulate the bacterial colonisation but had benefits on inflammatory and immune markers, which might be related to some features of gastrointestinal tolerance.

Metronidazole effects on microbiota and mucus layer thickness in the rat gut

Both mucus and mucosa-associated bacteria form a specific environment in the gut; their disruption may play a crucial role in the development of intestinal bowel disease (IBD). Metronidazole, an antibiotic used in the treatment of IBD, alters gut microbiota and reduces basal oxidative stress to proteins in colonic tissue of healthy rats. The aim of this study was to evaluate the impact of the altered microbiota due to the metronidazole on the thickness of the mucus layer. This study was performed in healthy untreated rats (control group) or rats treated by metronidazole (metronidazole-treated rats, 1 mg mL(-1) in drinking water for 7 days). Both PCR-temporal temperature gradient gel electrophoresis and quantitative PCR (qPCR) revealed an altered microbiota with an increase in bifidobacteria and enterobacteria in metronidazole-treated rats compared with control rats. Moreover, a dominant bifidobacterial species, Bifidobacterium pseudolongum, was detected. Using qPCR and FISH, we showed that bifidobacteria were also increased in the microbiota-associated mucosa. At the same time, the mucus layer thickness was increased approximately twofold. These results could explain the benefits of metronidazole treatment and warrant further investigations to define the role of bifidobacteria in the colonic mucosa.

Debaryomyces hansenii and Rhodotorula mucilaginosa comprised the yeast core gut microbiota of wild and reared carnivorous salmonids, croaker and yellowtail.

This is the first study using molecular and culture-based methods aimed at investigating the composition of the intestinal yeast microbiota of wild and reared carnivorous salmonids, croaker and yellowtail, to characterize their cores and to evaluate the enzymatic activities of the cultivated yeast. Among 103 samples from salmonids, croaker and yellowtail, yeast were detected in 85.4%, with 43 species identified. The core of reared fish was composed of eight species, in contrast to the wild fish core, which consisted of two species: Debaryomyces hansenii and Rhodotorula mucilaginosa. Despite the smaller diversity of the wild fish core, similar enzymatic profiles were detected for the species from the wild and reared cores. For principal component analysis, samples grouped together independently of host species, domestication status and location. A high proportion of yeast produced aminopeptidases and lipases, which may be explained by the high proportion of protein and lipids in the carnivorous diet. This study reveals the presence of a yeast community in the fish gut that appears to be strongly shaped by a carnivorous diet. Yeast in the gut increases the repertoire of microorganisms interacting with the host intestine, which could influence health and disease.

Differential Effects of Bifidobacterium pseudolongum Strain Patronus and Metronidazole in the Rat Gut

ABSTRACT In the luminal contents of metronidazole-treated rats, there was a dominant Bifidobacterium species. A strain has been isolated, its 16S rRNA gene has been sequenced, and the strain has been named Bifidobacterium pseudolongum strain Patronus. In this study, using an experimental model of healthy rats, the effects of metronidazole treatment and B. pseudolongum strain Patronus administration on the luminal and mucosa-associated microbiota and on gut oxidation processes were investigated. Metronidazole treatment and the daily gavage of rats with B. pseudolongum strain Patronus increased the numbers of bifidobacteria in cecal contents and in cecal mucosa-associated microbiota compared with those in control rats. Metronidazole reduced the colonic oxidative damage to proteins. This is the first evidence that B. pseudolongum strain Patronus exerts an effect on a biomarker of oxidative damage by reducing the susceptibility to oxidation of proteins in the colon and the small bowel. Antioxidant effects of metronidazole could be linked to the bifidobacterial increase but also to other bacterial modifications.

The gut microbiota of healthy chilean subjects reveals a high abundance of the phylum verrucomicrobia

The gut microbiota is currently recognized as an important factor regulating the homeostasis of the gastrointestinal tract and influencing the energetic metabolism of the host as well as its immune and central nervous systems. Determining the gut microbiota composition of healthy subjects is therefore necessary to establish a baseline allowing the detection of microbiota alterations in pathologic conditions. Accordingly, the aim of this study was to characterize the gut microbiota of healthy Chilean subjects using 16S rRNA gene sequencing. Fecal samples were collected from 41 young, asymptomatic, normal weight volunteers (age: 25 ± 4 years; ♀:48.8%; BMI: 22.5 ± 1.6 kg/m2) with low levels of plasma (IL6 and hsCRP) and colonic (fecal calprotectin) inflammatory markers. The V3-V4 region of the 16S rRNA gene of bacterial DNA was amplified and sequenced using MiSeq Illumina system. 109,180 ± 13,148 sequences/sample were obtained, with an α-diversity of 3.86 ± 0.37. The dominant phyla were Firmicutes (43.6 ± 9.2%) and Bacteroidetes (41.6 ± 13.1%), followed by Verrucomicrobia (8.5 ± 10.4%), Proteobacteria (2.8 ± 4.8%), Actinobacteria (1.8 ± 3.9%) and Euryarchaeota (1.4 ± 2.7%). The core microbiota representing the genera present in all the subjects included Bacteroides, Prevotella, Parabacteroides (phylum Bacteroidetes), Phascolarctobacterium, Faecalibacterium, Ruminococcus, Lachnospira, Oscillospira, Blautia, Dorea, Roseburia, Coprococcus, Clostridium, Streptococcus (phylum Firmicutes), Akkermansia (phylum Verrucomicrobia), and Collinsella (phylum Actinobacteria). Butyrate-producing genera including Faecalibacterium, Roseburia, Coprococcus, and Oscillospira were detected. The family Methanobacteriaceae was reported in 83% of the subjects and Desulfovibrio, the most representative sulfate-reducing genus, in 76%. The microbiota of the Chilean individuals significantly differed from those of Papua New Guinea and the Matses ethnic group and was closer to that of the Argentinians and sub-populations from the United States. Interestingly, the microbiota of the Chilean subjects stands out for its richness in Verrucomicrobia; the mucus-degrading bacterium Akkermansia muciniphila is the only identified member of this phylum. This is an important finding considering that this microorganism has been recently proposed as a hallmark of healthy gut due to its anti-inflammatory and immunostimulant properties and its ability to improve gut barrier function, insulin sensitivity and endotoxinemia. These results constitute an important baseline that will facilitate the characterization of dysbiosis in the main diseases affecting the Chilean population.