Philippe Pochart

Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients

A decrease in the abundance and biodiversity of intestinal bacteria within the dominant phylum Firmicutes has been observed repeatedly in Crohn disease (CD) patients. In this study, we determined the composition of the mucosa-associated microbiota of CD patients at the time of surgical resection and 6 months later using FISH analysis. We found that a reduction of a major member of Firmicutes, Faecalibacterium prausnitzii, is associated with a higher risk of postoperative recurrence of ileal CD. A lower proportion of F. prausnitzii on resected ileal Crohn mucosa also was associated with endoscopic recurrence at 6 months. To evaluate the immunomodulatory properties of F. prausnitzii we analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro (cellular models) and in vivo [2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced] colitis in mice. In Caco-2 cells transfected with a reporter gene for NF-κB activity, F. prausnitzii had no effect on IL-1β-induced NF-κB activity, whereas the supernatant abolished it. In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii led to significantly lower IL-12 and IFN-γ production levels and higher secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis, as demonstrated by real-time quantitative PCR (qPCR) analysis. F. prausnitzii exhibits anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-κB activation and IL-8 production. These results suggest that counterbalancing dysbiosis using F. prausnitzii as a probiotic is a promising strategy in CD treatment.

Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization

ABSTRACT To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroidesgroup-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%.Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while Bifidobacterium andLactobacillus groups made up less than 2%.

Comparative study of bacterial groups within the human cecal and fecal microbiota.

ABSTRACT The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, andBacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.

Gut flora and inflammatory bowel disease

The pathogenesis of inflammatory bowel disease involves interactions between the host susceptibility, mucosal immunity and intestinal microflora. There is therefore great interest in the changes in the endogenous flora in inflammatory bowel disease patients and in the establishment of potential genetic variations in host responses to endogenous bacteria. In this review, we summarize the modifications in the various regional ecosystems in the gastrointestinal tract during inflammatory bowel disease (luminal bacteria in faeces or inside the gastrointestinal tract, bacteria in mucus and bacteria directly attached to the mucosa). Results were obtained following a ‘candidate microorganism strategy’ and, as is occurring increasingly frequently, following a ‘full description strategy’, which has progressed largely due to the development of culture‐independent techniques. The possibility of modifying the ecosystem using prebiotics or probiotics offers hope for new treatment developments, particularly in the prevention of relapse.

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.

Effect of feeding yogurt versus milk in children with persistent diarrhea.

Although the pathophysiology of persistent diarrhea in children remains unclear, it has been suggested that it may be related to the composition of the food ingested. Since lactase deficiency and cows milk protein intolerance are often identified in children with persistent diarrhea, replacement of milk with yogurt should be beneficial. We, therefore, compared the clinical outcome of children (aged 3–36 months) with persistent diarrhea randomly assigned to receive either milk or yogurt for 5 days. Preliminary results on 45 of the 100 children indicated clinical failure, which was determined after a 5% loss of body weight per day or the persistence of diarrhea after 5 days, in only 14% of the children fed yogurt compared to 42% of those fed milk (p < 0.05). These preliminary results strongly suggest a clinical advantage of feeding yogurt rather than milk in children with persistent diarrhea.

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.

Patchy distribution of mucosal lesions in ileal Crohn's disease is not linked to differences in the dominant mucosa‐associated bacteria: A study using fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis

Background The mucosa‐associated bacteria (MAB) are suspected of being involved in the pathogenesis of Crohns disease. We analyzed and compared the MAB in noninflamed and inflamed ileal mucosa of Crohns disease patients (n = 22). Methods Tissue samples from the inflamed ileal mucosa and from the adjacent noninflamed ileal mucosa were taken from surgical resection specimens. The MAB were investigated using fluorescence in situ hybridization with 7 group‐specific probes and temporal temperature gradient gel electrophoresis (TTGE). Results Samples from both noninflamed and inflamed mucosa were obtained from 15 patients. The distribution of the bacterial populations was not different between noninflamed and inflamed mucosa. The Bacteroidetes phylum was dominant and accounted for 29% of MAB (0%–74%) in noninflamed tissues and 32% (0%–70%) in inflamed areas. The &ggr; Proteobacteria represented 12% (0%–70%) of MAB both in noninflamed and inflamed areas. The Clostridium coccoides group (Firmicutes phylum) represented 15% of MAB in noninflamed tissues versus 7% in inflamed areas. For most of the patients the similarity index between TTGE paired profiles was very high. Conclusion The dominant MAB do not differ between noninflamed and inflamed ileal mucosa in Crohns disease. This argues against a localized dysbiosis to explain the patchy distribution of mucosal lesions. (Inflamm Bowel Dis 2007)

Pyxigraphic sampling to enumerate methanogens and anaerobes in the right colon of healthy humans

BACKGROUND A major limitation in studying the proximal human colonic flora is the lack of suitable sampling methods. The aim of this study was (1) to describe a prototype technique, pyxigraphy, which uses swallowed capsules containing a mechanism allowing the remote control of sampling the gastrointestinal tract contents, and (2) to use this sampling method to examine the distribution of methanogens in the colon of methane (CH4) excretors and non-CH4 excretors. METHODS In six CH4 excretors and four non-CH4 excretors, samples of the right colonic contents were obtained by means of the pyxigraphic sampling method. Methanogens and total anaerobes were enumerated in both the right colonic and fecal contents. RESULTS In CH4 excretors, the concentration of methanogens was higher in the feces than in the right colonic contents, representing 12% and 0.003%, respectively, of the total anacrobes (P < 0.02). In non-CH4 excretors, no difference was observed, methanogens representing < 0.003% of the total anaerobes in both the right colonic and fecal contents. CONCLUSIONS Pyxigraphy is a noninvasive, simple, and safe sampling method that allows to study the microbial populations of the proximal colon. The results obtained showed that methanogens preferentially colonize the distal part of the colon in CH4 excretors.