Annick Bernalier-Donadille

Functional dysbiosis within the gut microbiota of patients with constipated-irritable bowel syndrome.

The role of the gut microbiota in patho‐physiology of irritable bowel syndrome (IBS) is suggested by several studies. However, standard cultural and molecular methods used to date have not revealed specific and consistent IBS‐related groups of microbes.

The cellulolytic microflora of the human colon: evidence of microcrystalline cellulose-degrading bacteria in methane-excreting subjects

Abstract In humans, plant cell wall polysaccharides (mainly cellulose and hemicelluloses) represent an important source of dietary fibres that are digested by the gut microflora. However, the fibrolytic micro-organisms involved in the breakdown of these substrates remain largely unknown. Our objective was to quantify the microcrystalline-cellulose-degrading and methanogenic microbial communities in faecal samples (n=34) from both methane- and non-methane-excreting individuals and to identify the predominant cellulolytic organisms in these two categories of subjects. Microcrystalline cellulose degraders could only be enumerated in faecal samples from methane excretors while this community remained undetectable in non-methane-excretors. The cellulolytic isolates corresponded to new Ruminococcus species and to Enterococcus sp. closely related to Enterococcus faecalis. The presence of such fibrolytic species seems to be linked to that of methanogenic archaea in the gut, the relationships between these two microbial communities needing further investigation. Our findings suggest that the structure and activity of the cellulolytic communities differ in methane- and non-methane-excreting individuals.

The hypersensitivity to colonic distension of IBS patients can be transferred to rats through their fecal microbiota

Alterations of intestinal microbiota and hypersensitivity to colonic distension are two features of the irritable bowel syndrome (IBS). However, the role of intestinal microbiota in visceral hypersensitivity of IBS patients is far to be established. The aim of our study was to determine whether the intestinal microbiota is involved in the visceral hypersensitivity in IBS.

Human Microbiota-Secreted Factors Inhibit Shiga Toxin Synthesis by Enterohemorrhagic Escherichia coli O157:H7

ABSTRACT Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx2 mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx2 repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.

Lactate has the potential to promote hydrogen sulphide formation in the human colon

High concentrations of sulphide are toxic for the gut epithelium and may contribute to bowel disease. Lactate is a favoured cosubstrate for the sulphate-reducing colonic bacterium Desulfovibrio piger, as shown here by the stimulation of sulphide formation by D. piger DSM749 by lactate in the presence of sulphate. Sulphide formation by D. piger was also stimulated in cocultures with the lactate-producing bacterium Bifidobacterium adolescentis L2-32. Other lactate-utilizing bacteria such as the butyrate-producing species Eubacterium hallii and Anaerostipes caccae are, however, expected to be in competition with the sulphate-reducing bacteria (SRB) for the lactate formed in the human colon. Strains of E. hallii and A. caccae produced 65% and 96% less butyrate from lactate, respectively, in a coculture with D. piger DSM749 than in a pure culture. In triculture experiments involving B. adolescentis L2-32, up to 50% inhibition of butyrate formation by E. hallii and A. caccae was observed in the presence of D. piger DSM749. On the other hand, sulphide formation by D. piger was unaffected by E. hallii or A. caccae in these cocultures and tricultures. These experiments strongly suggest that lactate can stimulate sulphide formation by SRB present in the colon, with possible consequences for conditions such as colitis.

Ruminococcus champanellensis sp. nov., a cellulose-degrading bacterium from human gut microbiota

A strictly anaerobic, cellulolytic strain, designated 18P13(T), was isolated from a human faecal sample. Cells were Gram-positive non-motile cocci. Strain 18P13(T) was able to degrade microcrystalline cellulose but the utilization of soluble sugars was restricted to cellobiose. Acetate and succinate were the major end products of cellulose and cellobiose fermentation. 16S rRNA gene sequence analysis revealed that the isolate belonged to the genus Ruminococcus of the family Ruminococcaceae. The closest phylogenetic relative was the ruminal cellulolytic strain Ruminococcus flavefaciens ATCC 19208(T) (<95% 16S rRNA gene sequence similarity). The DNA G+C content of strain 18P13(T) was 53.05±0.7 mol%. On the basis of phylogenetic analysis, and morphological and physiological data, strain 18P13(T) can be differentiated from other members of the genus Ruminococcus with validly published names. The name Ruminococcus champanellensis sp. nov. is proposed, with 18P13(T) (=DSM 18848(T)=JCM 17042(T)) as the type strain.

The cellulose-degrading microbial community of the human gut varies according to the presence or absence of methanogens

Cellulose-degrading microorganisms involved in the breakdown of plant cell wall material in the human gut remain rather unexplored despite their role in intestinal fermentation. Microcrystalline cellulose-degrading bacteria were previously identified in faeces of methane-excreting individuals, whereas these microorganisms were undetectable in faecal samples from non-methane excretors. This suggested that the structure and activity of the cellulose-degrading community differ in methane- and non-methane-excreting individuals. The purpose of this study was to characterize in depth this cellulose-degrading community in individuals of both CH(4) statuses using both culture-dependent and molecular methods. A new real-time PCR analysis was developed to enumerate microcrystalline cellulose-degrading ruminococci and used to confirm the predominance of these hydrolytic ruminococci in methane excretors. Culture-dependent methods using cell wall spinach (CWS) residue revealed the presence of CWS-degrading microorganisms in all individuals. Characterization of CWS-degrading isolates further showed that the main cellulose-degrading bacteria belong essentially to Bacteroidetes in non-methane-excreting subjects, while they are predominantly represented by Firmicutes in methane-excreting individuals. This taxonomic diversity was associated with functional diversity: the ability to degrade different types of cellulose and to produce H(2) from fermentation differed depending on the species. The structure of the cellulolytic community was shown to vary depending on the presence of methanogens in the human gut.

Bacteroides xylanisolvens sp. nov., a xylan-degrading bacterium isolated from human faeces.

During the course of a study on the xylan-degrading community from the human gut, six xylanolytic, Gram-negative, anaerobic rods were isolated from faecal samples. 16S rRNA gene sequence analysis showed that the isolates were closely related to each other (> or =99 % sequence similarity) and that they belonged to the genus Bacteroides. On the basis of 16S rRNA gene sequence similarity, representative strain XB1AT was most closely related to the type strains of Bacteroides ovatus (97.5 %), B. finegoldii (96.5 %) and B. thetaiotaomicron (95.5 %). DNA-DNA hybridization results revealed that strain XB1AT was distinct from its closest relative, B. ovatus. The DNA G+C content of strain XB1AT (42.8 mol%) and major fatty acid composition (anteiso-C15 : 0, 33.8 %) further supported its affiliation to the genus Bacteroides. The novel isolates degraded different types of xylan, and were also able to grow on a variety of carbohydrates. Unlike most other Bacteroides species isolated from the human gut, these isolates were not able to degrade starch. Other biochemical tests further demonstrated that strain XB1AT could be differentiated from the closest related Bacteroides species. Xylan and sugars were converted by strain XB1AT mainly into acetate, propionate and succinate. Based on physiological, phenotypic and phylogenetic data, the six novel strains are considered to represent a novel species of the genus Bacteroides, for which the name Bacteroides xylanisolvens sp. nov. is proposed. The type strain is XB1AT (=DSM 18836T =CCUG 53782T).

Assessment of metabolic diversity within the intestinal microbiota from healthy humans using combined molecular and cultural approaches

The human gut harbours a wide range of bacterial communities that play key roles in supplying nutrients and energy to the host through anaerobic fermentation of dietary components and host secretions. This fermentative process involves different functional groups of microorganisms linked in a trophic chain. Although the diversity of the intestinal microbiota has been studied extensively using molecular techniques, the functional aspects of this biodiversity remain mostly unexplored. The aim of the present work was to enumerate the principal metabolic groups of microorganisms involved in the fermentative process in the gut of healthy humans. These functional groups of microorganisms were quantified by a cultural approach, while the taxonomic composition of the microbiota was assessed by in situ hybridization on the same faecal samples. The functional groups of microorganisms that predominated in the gut were the polysaccharide-degrading populations involved in the breakdown of the most readily available exogenous and endogenous substrates and the predominant butyrate-producing species. Most of the functional groups of microorganisms studied appeared to be present at rather similar levels in all healthy volunteers, suggesting that optimal numbers of these various bacterial groups are crucial for efficient gut fermentation, as well as for host nutrition and health. Significant interindividual differences were, however, confirmed with respect to the numbers of methanogenic archaea, filter paper-degrading and acetogenic bacteria and the products formed by lactate-utilizing bacteria.

Efficacy and safety profile of LCR35 complete freeze-dried culture in irritable bowel syndrome: a randomized, double-blind study.

AIM To assess the effects and safety of Lactobacillus casei rhamnosus LCR35 complete freeze-dried culture (LCR35) in patients suffering from irritable bowel syndrome (IBS). METHODS A randomized, double-blind pilot study was performed in 50 patients complaining of IBS symptoms complying with Rome III criteria. Patients were allocated to receive either LCR35 (n = 25) at a minimum daily dose of 6 × 10(8) colony forming units or placebo (n = 25) for 4 wk. At inclusion, after treatment and 2 wk later, patients completed the IBS severity scale. Change from baseline in the IBS severity score at the end of treatment was the primary efficacy criterion. Changes were compared between groups in the whole population and in IBS subtypes (IBS with predominance of constipation, IBS with predominance of diarrhoea, mixed IBS, unsubtyped IBS). The presence of lactobacillus casei rhamnosus in stools was investigated at inclusion and at the end of treatment. The gastrointestinal quality of life questionnaire and the hospital anxiety and depression (HAD) scale were also completed. RESULTS Both groups were balanced for baseline characteristics. In 85% of patients, stool analyses showed that lactobacillus casei rhamnosus able to survive in the digestive tract. In the whole population, improvements in the IBS severity score did not differ significantly between treatments with a 25% decrease after 4-wk treatment, and a 15% decrease from baseline 2 wk later in both groups. In IBS subgroups, statistical analysis could not be performed due to small sample size, but a clinical response in favour of LCR35 was observed in IBS patients with predominance of diarrhoea: no change in the symptom severity score was seen with the placebo after 4 wk treatment, whereas a clinically relevant decrease occurred with LCR35 (-37% vs -3%). Furthermore, in spite of an increase in symptom intensity, the IBS severity score was maintained below the baseline value 2 wk later with LCR35 (-19% from baseline), whilst a slight 5% increase from baseline was observed with placebo. In the IBS subgroup with predominance of diarrhoea only, a clinically relevant decrease in abdominal pain severity score (-36%) was observed with LCR35, whereas no change occurred with placebo. In mixed IBS patients, the 20% and 30% decreases in the IBS severity score observed after treatment with LCR35 and placebo, respectively, were maintained 2 wk later in both groups. A clinical response slightly in favour of placebo was observed at the end of the treatment period in IBS patients with predominance of constipation (-41% vs -20%) and unsubtyped IBS patients (-47% vs -17%), with the same value maintained 2 wk later. In both groups, no clinically relevant changes were observed either for the gastrointestinal quality of life index or HAD score. Thus, these results suggest that sub-grouping of IBS patients may be important for optimizing treatment responses by the physician. CONCLUSION This pilot study suggests that LCR35 could have some efficacy in IBS patients complaining of diarrhoea. These preliminary results need to be confirmed in larger studies.