Gérard Corthier

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.

Low Counts of Faecalibacterium prausnitzii in Colitis Microbiota

Background: The intestinal microbiota is suspected to play a role in colitis and particularly in inflammatory bowel disease (IBD) pathogenesis. The aim was to compare the fecal microbiota composition of patients with colitis to that of healthy subjects (HS). Methods: fecal samples from 22 active Crohns disease (A‐CD) patients, 10 CD patients in remission (R‐CD), 13 active ulcerative colitis (A‐UC) patients, 4 UC patients in remission (R‐UC), 8 infectious colitis (IC) patients, and 27 HS were analyzed by quantitative real‐time polymerase chain reaction (PCR) targeting the 16S rRNA gene. Bacterial counts were transformed to logarithms (Log10 CFU) for statistical analysis. Results: Bacteria of the phylum Firmicutes (Clostridium leptum and Clostridium coccoides groups) were less represented in A‐IBD patients (9.7; P = 0.004) and IC (9.4; P = 0.02), compared to HS (10.8). Faecalibacterium prausnitzii species (a major representative of the C. leptum group) had lower counts in A‐IBD and IC patients compared to HS (8.8 and 8.3 versus 10.4; P = 0.0004 and P = 0.003). The Firmicutes/Bacteroidetes ratio was lower in A‐IBD (1.3; P = 0.0001) and IC patients (0.4; P = 0.002). Compared to HS, Bifidobacteria were less represented in A‐IBD and IC (7.9 and 7.7 versus 9.2; P = 0.001 and P = 0.01). Conclusions: The fecal microbiota of patients with IBD differs from that of HS. The phylum Firmicutes and particularly the species F. prausnitzii, are underrepresented in A‐IBD patients as well as in IC patients. These bacteria could be crucial to gut homeostasis since lower counts of F. prausnitzii are consistently associated with a reduced protection of the gut mucosa. (Inflamm Bowel Dis 2009)

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

BackgroundIn 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.ResultsHere 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.ConclusionIn 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.

Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding

DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254–767 bp) and of a shorter fragment of this intron (the P6 loop, 10–143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trn L (UAA) intron (254-767 bp) and of a shorter fragment of this intron (the P6 loop, 10-143 bp) amplified with highly conserved primers. The main limitation of the whole trn L intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trn L intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.

Microbial dysbiosis in colorectal cancer (CRC) patients.

The composition of the human intestinal microbiota is linked to health status. The aim was to analyze the microbiota of normal and colon cancer patients in order to establish cancer-related dysbiosis. Patients and Methods Stool bacterial DNA was extracted prior to colonoscopy from 179 patients: 60 with colorectal cancer, and 119 with normal colonoscopy. Bacterial genes obtained by pyrosequencing of 12 stool samples (6 Normal and 6 Cancer) were subjected to a validated Principal Component Analysis (PCA) test. The dominant and subdominant bacterial population (C. leptum, C. coccoides, Bacteroides/Prevotella, Lactobacillus/Leuconostoc/Pediococcus groups, Bifidobacterium genus, and E. coli, and Faecalibacterium prausnitzii species) were quantified in all individuals using qPCR and specific IL17 producer cells in the intestinal mucosa were characterized using immunohistochemistry. Findings Pyrosequencing (Minimal sequence 200 nucleotide reads) revealed 80% of all sequences could be assigned to a total of 819 taxa based on default parameter of Classifier software. The phylogenetic core in Cancer individuals was different from that in Normal individuals according to the PCA analysis, with trends towards differences in the dominant and subdominant families of bacteria. Consequently, All-bacteria [log10 (bacteria/g of stool)] in Normal, and Cancer individuals were similar [11.88±0.35, and 11.80±0.56, respectively, (P = 0.16)], according to qPCR values whereas among all dominant and subdominant species only those of Bacteroides/Prevotella were higher (All bacteria-specific bacterium; P = 0.009) in Cancer (-1.04±0.55) than in Normal (-1.40±0.83) individuals. IL17 immunoreactive cells were significantly expressed more in the normal mucosa of cancer patients than in those with normal colonoscopy. Conclusion This is the first large series to demonstrate a composition change in the microbiota of colon cancer patients with possible impact on mucosal immune response. These data open new filed for mass screening and pathophysiology investigations.

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.

Should yoghurt cultures be considered probiotic

Probiotics are live micro-organisms that when administered in adequate amounts confer a health benefit on the host. Consumption of yoghurt has been shown to induce measurable health benefits linked to the presence of live bacteria. A number of human studies have clearly demonstrated that yoghurt containing viable bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii sp. bulgaricus) improves lactose digestion and eliminates symptoms of lactose intolerance. Thus, these cultures clearly fulfil the current concept of probiotics.

Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR

Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan probes were defined for the qPCR technique used for dominant microbiota. Human faecal microbiota was compared with that of farm animals using faecal samples collected from rabbits, goats, horses, pigs, sheep and cows. Three dominant bacterial groups (Bacteroides/Prevotella, Clostridium coccoides and Bifidobacterium) of the human microbiota showed differential population levels in animal species. The Clostridium leptum group showed the lowest differences among human and farm animal species. Human subdominant bacterial groups were highly variable in animal species. Partial least squares regression indicated that the human microbiota could be distinguished from all farm animals studied. This culture-independent comparative assessment of the faecal microbiota between humans and farm animals will prove useful in identifying biomarkers of human and animal faecal contaminations that can be applied to microbial source tracking methods.

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.

A Novel Mucosal Vaccine Based on Live Lactococci Expressing E7 Antigen and IL-12 Induces Systemic and Mucosal Immune Responses and Protects Mice against Human Papillomavirus Type 16-Induced Tumors

Current strategies to prevent or treat human papillomavirus type 16 (HPV-16) infection are promising, but remain costly. More economical but efficient vaccines are thus needed. In this study, we evaluated the protective effects of mucosally coadministered live Lactococcus lactis strains expressing cell wall-anchored E7 Ag and a secreted form of IL-12 to treat HPV-16-induced tumors in a murine model. When challenged with lethal levels of tumor cell line TC-1 expressing E7, immunized mice showed full prevention of TC-1-induced tumors, even after a second challenge, suggesting that this prophylactic immunization can provide long-lasting immunity. Therapeutic immunization with L. lactis recombinant strains, i.e., 7 days after TC-1 injection, induced regression of palpable tumors in treated mice. The antitumor effects of vaccination occurred through a CTL response, which is CD4+ and CD8+ dependent. Furthermore, immunized mice developed an E7-specific mucosal immune response. These preclinical results suggest the feasibility of the low-cost mucosal vaccination and/or immunotherapy strategies against HPV-related cervical cancer in humans.