[Why could gut microbiota become a medication?]. / Pourquoi la flore intestinale a-t-elle vocation à devenir médicament ?

The gut microbiota (or gut flora) is a set of bacteria living in symbiosis with the host. Strictly associated with the intestinal tract and interacting with it, the gut microbiota is not a tissue nor an organ, but a supra-organism. A disruption of dialogue between bacteria and human cells is a risk factor or a possible cause of various diseases. The restoration of this dialogue, thanks to the transfer of the gut microbiota of a healthy individual to a patient whose balance of gut flora has been broken, is a new therapeutic approach. If its exact effect still eludes scientific understanding, its clinical benefit is well established for an indication, and is recently being tested for many others. The proven contribution of gut microbiota in the human physiological balance calls for intensifying research throughout the world about the state of knowledge and technologies, as well as on the legal and ethical dimension of fecal microbiota transfer. This didactic paper updates the questions in relation with this therapeutic act.

[A new era in gut research concerning interactions between microbiota and human health]. / Une ère nouvelle dans le domaine des interactionsentre le microbiote et la santé humaine.

The scope of this introduction is to make a small review of the texts presented here and to emphasize some points that are not developed but that concern human microbiota functions.

[The human intestinal microbiota]. / Le microbiote intestinal humain.

The human intestinal microbiota constitutes a complex ecosystem which is now well recognized for its impact on human health and well-being. It contributes to maturation of the immune system and provides a direct barrier against colonization by pathogens. Its possible implication in diseases of modern societies, currently increasing in prevalence, has been reported. These include allergies, inflammatory bowel diseases and possibly metabolic and degenerative disorders. The analysis of the molecular composition of the human intestinal microbiota indicates marked inter-individual variations which may seem paradoxical considering the high degree of conservation of major functions of the intestinal microbiota such as anaerobic digestion of alimentary fibres. We have characterized a phylogenetic core within the human intestinal microbiota, in terms of composition, i.e., a set of conserved species that could be responsible for major conserved functions. Based on culture-independent molecular assessments, current knowledge enables a definition of criteria qualifying the normal state of the human intestinal microbiota that we call normobiosis. This further enables the identification of specific deviations from normobiosis, i.e., dysbiosis in immune, metabolic or degenerative diseases. Notably, Crohn's disease, an inflammatory bowel disease of yet unknown aetiology, is associated with intestinal dysbiosis with a lower representation of the Clostridium leptum group among the Firmicutes phylum. We further showed that the bacterial species Faecalibacterium prausnitzii exerts anti-inflammatory properties in vitro and in animal models; this could explain its ability, when detected in the mucosa-associated microbiota of patients in vivo, to protect patients from post-operative recurrence of endoscopic signs of inflammation 6 months after surgical resection of the ileocecal region of the gut. By confirming the major role of the microbiota in bowel-related disorders, which are especially associated with a disruption of homeostasis, we are currently applying high throughput functional metagenomic screens in order to identify signal molecules and mechanisms of bacteria-host cross-talk. Together with the high resolution description of the human intestinal metagenome, as well as explorations of environmental proteins and metabolites, these observations will further our understanding of the functional roles bacteria play in the maintenance of health and well-being in humans. It will open new perspectives for the monitoring and design of strategies for modulating the microbiota for health.

The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age.

BACKGROUND: In humans, the intestinal microbiota plays an important role in the maintenance of host health by providing energy, nutrients, and immunological protection. Applying current molecular methods is necessary to surmount the limitations of classical culturing techniques in order to obtain an accurate description of the microbiota composition. RESULTS: Here we report on the comparative assessment of human fecal microbiota from three age-groups: infants, adults and the elderly. We demonstrate that the human intestinal microbiota undergoes maturation from birth to adulthood and is further altered with ageing. The counts of major bacterial groups Clostridium leptum, Clostridium coccoides, Bacteroidetes, Bifidobacterium, Lactobacillus and Escherichia coli were assessed by quantitative PCR (qPCR). By comparing species diversity profiles, we observed age-related changes in the human fecal microbiota. The microbiota of infants was generally characterized by low levels of total bacteria. C. leptum and C. coccoides species were highly represented in the microbiota of infants, while elderly subjects exhibited high levels of E. coli and Bacteroidetes. We observed that the ratio of Firmicutes to Bacteroidetes evolves during different life stages. For infants, adults and elderly individuals we measured ratios of 0.4, 10.9 and 0.6, respectively. CONCLUSION: In this work we have confirmed that qPCR is a powerful technique in studying the diverse and complex fecal microbiota. Our work demonstrates that the fecal microbiota composition evolves throughout life, from early childhood to old age.

Allergic sensitization to bovine beta-lactoglobulin: comparison between germ-free and conventional BALB/c mice.

BACKGROUND: The 'hygiene hypothesis' suggests that high hygienic standards met in western countries lead to a lack of microbial exposure, thus promoting the development of atopy by preventing the proper maturation of the immune system. Germ-free animals are deprived of the immune stimulation that occurs during postnatal gut colonization by commensal bacteria. Germ-free mice could thereby provide an attractive model for studying the impact of gut microbiota on the development of Th2-mediated disorders such as allergy. METHODS: Germ-free and conventional BALB/c mice were sensitized to beta-lactoglobulin (BLG), a major cow's milk allergen, by means of intraperitoneal injections in the presence of incomplete Freund's adjuvant. Time courses of serum and fecal BLG-specific antibody responses were monitored and cytokine production was assayed in BLG-reactivated splenocytes. RESULTS: Serum BLG-specific IgG1 and IgE concentrations were significantly higher in germ-free mice during the primary immune response and IgE production persisted longer in germ-free mice. Furthermore, secretion of BLG-specific IgA was evidenced only in feces from germ-free mice while, in contrast, fecal IgG1 concentrations were at least 3-fold higher in conventional mice than in germ-free mice. Production of IL-5, IL-10 and IFN-gamma was 3-fold enhanced in BLG-reactivated splenocytes from germ-free mice. CONCLUSION: The absence of gut microbiota significantly affects the BLG-specific immune response in BALB/c mice, thus suggesting that this model might be of interest for further studies exploring the influence of gut colonization by different bacterial strains on the development of an allergic-type sensitization.

In vivo transfer of plasmid from food-grade transiting lactococci to murine epithelial cells.

We recently demonstrated that noninvasive food-grade Lactococcus lactis (L. lactis) can deliver eukaryotic expression plasmid in mammalian cells in vitro. Here, we evaluated, in vivo, whether a eukaryotic expression plasmid carried by lactococci can translocate to the epithelial cells of the intestinal membrane. The strain LL(pLIG:BLG1) carrying one plasmid containing a eukaryotic expression cassette encoding beta-lactoglobulin (BLG), a major allergen of cow's milk, was orally administered by gavage to mice. BLG cDNA was detected in the epithelial membrane of the small intestine of 40% of the mice and BLG was produced in 53% of the mice. Oral administration of LL(pLIG:BLG1) induced a low and transitory Th1-type immune response counteracting a Th2 response in case of further sensitization. We demonstrated for the first time the transfer of a functional plasmid to the epithelial membrane of the small intestine in mice by noninvasive food-grade lactococci.

Prevalence and pathogenicity of Clostridium difficile in hospitalized patients. A French multicenter study.

BACKGROUND: Although Clostridium difficile is the main agent responsible for nosocomial diarrhea in adults, its prevalence in stool cultures sent to hospital microbiology laboratories is not clearly established. OBJECTIVES: To determine the prevalence of C difficile in inpatient stools sent to hospital microbiology laboratories and to assess the relationship between serotypes and toxigenicity of the strains isolated and the clinical data. METHODS: From January 18, 1993, to July 31, 1993, the presence of C difficile was systematically investigated in a case-control study on 3921 stool samples sent for stool culture to 11 French hospital microbiology laboratories. The prevalence of C difficile in this population (cases) was compared with that of a group of 229 random hospital controls matched for age, department, and length of stay (controls). Stool culture from controls was requested by the laboratory although not prescribed by the clinical staff. Serotype and toxigenesis of the strains isolated were compared. RESULTS: The overall prevalence of C difficile in the cases was twice the prevalence in the controls (9.7% vs 4.8%; P < .001) and was approximately 4 times as high in diarrheal stools (ie, soft or liquid) as in normally formed stools from controls (11.5% vs 3.3%; P < .001). The strains isolated from diarrheal stools were more frequently toxigenic than those isolated from normally formed stools. Serogroup D was never toxigenic, and its proportion was statistically greater in the controls than in the cases (45% vs 18%; chi 2 = 5.2; P < .05). Conversely, serogroup C was isolated only from the cases. Clostridium difficile was mainly found in older patients ( > 65 years), suffering from a severe disabling disease, who had been treated with antibiotics and hospitalized for more than 1 week in long-stay wards or in intensive care. CONCLUSIONS: This multicenter period prevalence study clearly supports the hypothesis of a common role of C difficile in infectious diarrhea in hospitalized patients. Disease associated with C difficile should therefore be systematically evaluated in diarrheal stools from inpatients.

Plasmid vectors for gram-positive bacteria switching from high to low copy number.

A set of vectors for Gram-positive bacteria was constructed with a new feature which enables the switching down of their copy number per cell. These vectors carry the replication region of pAM beta 1, containing a gene essential for replication, repE, and its regulator, copF. The latter gene was inactivated by inserting a linker into its unique KpnI site. Since copF downregulates the expression of repE, its inactivation leads to an increase in the plasmid copy number per cell. The original low copy state can be restored by removal of the linker via KpnI cleavage and ligation. The new replicon was used to build (i) vectors for studying gene regulation by transcriptional or translational fusion with the bacterial luciferase gene, (ii) vectors for gene expression, and (iii) cassettes of the replicon with different multiple cloning sites, which would facilitate construction of vectors for novel purposes.

A recombinant vaccinia virus expressing the major capsid protein of Simian rotavirus-induced anti-rotavirus antibodies.

cDNA molecules encoding the major structural protein (VP6) of the Simian rotavirus SA11 were inserted under the control of the vaccinia virus 7.5 kDa promoter into the thymidine kinase gene. Synthesis of VP6 was demonstrated by immunoprecipitation of recombinant virus-infected cell. Mice inoculated via several routes with this recombinant vaccinia produce high titers of antirotavirus antibodies lacking neutralizing activity.

Kinetics of appearance of intestinal lesions in mice mono-associated with a lethal or non-lethal strain of Clostridium difficile.

The kinetics of the appearance of intestinal lesions induced by orogastric inoculation of gnotobiotic mice with a lethal strain of Clostridium difficile (VPI) that produced toxins A and B in vivo and in vitro was studied and compared with the lesions induced by non-lethal C. difficile strain 786 that produced toxins A and B in vitro but only toxin B in measurable amounts in vivo. Different portions of the intestine were removed 12, 20, 26 and 30 h after inoculation and studied by scanning electronmicroscopy. The remaining portions were homogenised for enumeration of C. difficile and quantification of toxin A by enzyme immunoassay and toxin B by cytotoxicity. The results showed that, following inoculation: (i) measurable amounts of both toxins were necessary to produce lesions; (ii) with strain VPI, the caecum and the colon were rapidly impaired and completely destroyed after 1 day, whereas the small intestine was damaged to a lesser extent; (iii) C. difficile strain 786 did not cause mucosal damage but induced mucus-like or serum-like secretion and morphological changes in the caecal enterocytes only.

Serogroup F strains of Clostridium difficile produce toxin B but not toxin A.

Most toxigenic strains of Clostridium difficile produce two toxins: an enterotoxin (toxin A) and a cytotoxin (toxin B). Only one strain (strain 8864) has been reported to produce toxin B but no toxin A. Serogroup F strains (44) of C. difficile, often isolated from asymptomatic infants, have been examined for toxin production. These strains, which were from distinct geographical and clinical sources, did not produce any detectable toxin A in vitro when examined in three distinct immunoassays. Nevertheless, all the strain produced a cytotoxin. Immunological differences between the cytotoxin of the serogroup F strains and that produced by C. difficile strain VPI 10463 (serogroup G) were demonstrated with monoclonal antibodies specific for either the toxin B produced by C. difficile strain VPI 10463 or C. sordellii lethal toxin (LT). Polymerase chain reaction amplification with primers derived from C. difficile strain VPI 10463 toxin A and B genes showed that serogroup F strains seem to possess a toxin B gene homologous with that of strain VPI 10463 and at least fragments of the toxin A gene. When axenic mice were inoculated with serogroup F strains, the animals survived; they did not develop diarrhoea and no toxin A could be detected in their faeces. However, cytotoxin was detected. Furthermore, these mice were protected against subsequent challenge with the otherwise lethally toxigenic C. difficile strain VPI 10463. The serogroup F strains appeared to be homogeneous and distinct from other C. difficile strains with regard to toxin production.

Detection of rotavirus immune complexes: relationship between rotavirus antibodies and rotavirus antigens in faeces.

An ELISA was developed for the identification of rotavirus immune complexes in pig faeces. The relationship between rotavirus antigens, rotavirus immune complexes and rotavirus antibodies of IgA class was examined.

Effect of oral Saccharomyces boulardii treatment on the activity of Clostridium difficile toxins in mouse digestive tract.

Human antibiotic-associated diarrhoea and pseudomembranous colitis are partly due to toxin production by Clostridium difficile. It is now well documented that Saccharomyces boulardii protects against C. difficile induced diseases. In an attempt to understand better the mechanism of this protective effect, the action of S. boulardii on a crude toxin preparation was studied in vitro and in vivo. The results showed that the yeast had no effect on the toxins in vitro but was able to protect mice inoculated with these toxins. Furthermore, the observation by scanning electron microscopy that the mucosa of S. boulardii protected mice was not damaged suggest that the yeast mainly acts on the intestinal mucosa.

Production of monoclonal antibodies against Clostridium difficile cytotoxin using immunosorbent binding bioassay procedure.

In the following study, a novel screening approach was used to develop monoclonal antibodies specific for toxin B of Clostridium difficile. The approach, which consisted of an immunosorbent binding bioassay (ISBBA), is based on antigen immunocapture by monoclonal antibodies and detection of biological activity. Our results showed ISBBA, which uses unpurified antigen, to be more sensitive than the neutralization assay and ELISA for the detection of toxin B antibody.

Design and evaluation of a 16S rRNA-targeted oligonucleotide probe for specific detection and quantitation of human faecal Bacteroides populations.

Colonic Bacteroides include several species which, by their population level and activities, are significant contributers to the metabolic activity and health of man and animals. Yet, the understanding of their ecology has been hampered by the lack of highly specific and reliable enumeration techniques. Based on 16S rRNA sequence comparisons within the available database, we have designed an 18-mer oligonucleotide that targets a region common to-and specific for the Bacteroides-Porphyromonas-Prevotella group. We have tested the specificity of the probe and its usefulness for studies of human faecal samples. Under experimentally optimized hybridization conditions, the probe was shown to similarly recognize the rDNA obtained from 40 strains representing 8 species of the Bacteroides-Porphyromonas-Prevotella group. Importantly, it did not recognize 31 strains of microorganisms representing 8 genera of the dominant human faecal microbiota. Among selected colonies of dominant microorganisms of the faecal flora of two human individuals, strains identified as B. vulgatus by immunoblots using a species-specific monoclonal antibody were all detected by the probe. Colony hybridization was used to enumerate total Bacteroides-group microorganisms in faecal specimen from children and adults. The probe described therein was further used in quantitative RNA blots to monitor fluctuations of the Bacteroides-group versus Bifidobacterium genus in frozen faecal samples from a child between 85 and 125 days of age. It will be applicable to similar investigations of other anaerobic environments.

The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.

Cellular and humoral immune response in pigs given vaccinal and chronic hog cholera viruses.

Humoral immune response (seroneutralization) and cellular immune response (lymphocyte-stimulation test) were analyzed in pigs inoculated with low virulent strains of hog cholera virus or vaccinated with a live-virus vaccine. Vaccinated animals exhibited an intense neutralizing antibody production, but cellular immune response was not detected. Neutralizing antibodies were not found in infected pigs, but a brief cellular immune response (18 days after inoculation) was observed.

Detection of Clostridium difficile toxins in stools. Comparison between a new enzyme immunoassay for toxin A and other routine tests.

An enzyme-linked immunosorbent assay (ELISA) for detection of Clostridium difficile toxin A (enterotoxin) using a monoclonal antibody is described. No cross-reaction was observed with any of the Clostridium species tested except for toxigenic Clostridium difficile. One hundred and eight stool specimens from hospitalized patients harbouring C. difficile in their intestine and 43 samples negative for C. difficile isolation were studied to compare this test with a cytotoxicity assay, the isolation of toxigenic C. difficile and the commercial EIA Premier test (Meridian). As compared with the cytotoxicity assay and EIA, specificity was 91 and 100%, while sensitivity 83 and 74% respectively. This ELISA technique could be used as a routine test for toxin A detection in stools.