Christophe Lay

Specificities of the fecal microbiota in inflammatory bowel disease.

Background: Abnormalities have been described in the fecal microbiota of patients with IBD, but it is not known whether they are specific for inflammatory bowel disease (IBD) or to some extent common to other forms of colitis. The aim of this study was to compare the bacterial composition of the dominant fecal microbiota in patients with Crohns disease (CD), ulcerative colitis (UC), infectious colitis (IC), and in healthy subjects (HS). Methods: Fluorescent in situ hybridization adapted to flow cytometry was used to analyze the bacterial composition of fecal samples from 13 patients with active CD, 13 patients with active UC, 5 patients with IC, and 13 HS. We used 6 group‐specific probes targeting 16S rRNA and spanning the main phylogenetic groups of the fecal microbiota. Results: A significantly higher proportion of the total fecal bacteria were recognized by the 6 probes in HS (86.6% ± 12.7) and in IC (84.0% ± 11.7) than in patients with IBD (70.9% ± 15 in CD and 60.1% ± 25.7 in UC). The Clostridium coccoides group was reduced in UC (20.0% ± 13.3 versus 42.0% ± 12.0 in HS; P < .001), whereas the C leptum group was reduced in CD (13.1% ± 11.9 versus 25.2% ± 14.2 in HS; P = .002). The Bacteroides group was more abundant in IC (36.4% ± 22.9) than in the other 3 groups (13.8% ± 11.8 in CD, 11.7% ± 11.7 in UC, 12.1% ± 7.0 in HS; P < .001 for all 3 comparisons). Conclusions: In IBD the dominant fecal microbiota comprises unusual bacterial species. Moreover, CD and UC fecal microbiota harbor specific discrepancies and differ from that of IC and healthy subjects.

Colonic microbiota signatures across five northern European countries

ABSTRACT The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed. Clostridium coccoides and Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by the Bacteroides (8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed.

Analysis of bacterial bowel communities of IBD patients: what has it revealed?

The bacterial community, in whole or in part, resident in the bowel of humans is considered to fuel the chronic immune inflammatory conditions characteristic of Crohns disease and ulcerative colitis. Chronic or recurrent pouchitis in ulcerative colitis patients is responsive to antibiotic therapy, indicating that bacteria are the etiological agents. Microbiological investigations of the bacterial communities in stool or of biopsy‐associated bacteria have so far failed to reveal conclusively the existence of pathogens or bacterial communities of consistently altered composition in IBD patients relative to control subjects. Confounding factors need to be eliminated from future studies by using better‐defined patient populations of newly diagnosed and untreated individuals and by improved sampling procedures.

Supplementation of the Diet with High-Viscosity Beta-Glucan Results in Enrichment for Lactobacilli in the Rat Cecum

ABSTRACT BBn (BioBreeding) rats were fed casein-based diets supplemented with barley flour, oatmeal flour, cellulose, or barley β-glucans of high [HV] or low viscosity [LV] in order to measure the prebiotic effects of these different sources of dietary fiber. The dietary impact on the composition of the cecal microbiota was determined by the generation of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene sequences. The DGGE profiles produced from the cecal microbiota of rats within each dietary group were similar, but consensus profiles generated from pooled bacterial DNAs showed differences between rat groups. Animals fed HV glucans (HV-fed rats) had DGGE consensus profiles that were 30% dissimilar from those of the other rat groups. A 16S rRNA gene fragment that was more conspicuous in the profiles of HV-fed animals than in those of cellulose-fed rats had sequence identity with Lactobacillus acidophilus. Measurements of L. acidophilus rRNA abundance (DNA-RNA hybridization), the preparation of cloned 16S rRNA gene libraries, and the enumeration of Lactobacillus cells (fluorescent in situ hybridization) showed that lactobacilli formed a greater proportion of the cecal microbiota in HV-fed rats. In vitro experiments confirmed that some lactobacilli utilize oligosaccharides (degree of polymerization, 3 or 4) present in β-glucan hydrolysates. The results of this study have relevance to the use of purified β-glucan products as dietary supplements for human consumption.

Effect of a Milk Formula Containing Probiotics on the Fecal Microbiota of Asian Infants at Risk of Atopic Diseases

The fecal microbiota of 37 infants with (n = 20) or without (n = 17) probiotic administration was evaluated on D 3, and at 1, 3, and 12 mo by fluorescence in situ hybridization-flow cytometry (FISH-FC), PCR, and bacteriological culture methods. They represent consecutive subjects of an ongoing double-blind, placebo-controlled trial on a probiotic formula (LGG and Bifidobacterium longum) administered during the first 6 mo of life. Despite varying composition in each baby, there was a general bacterial colonization pattern in the first year. Bifidobacteria increased markedly (p = 0.0003) with a parallel decrease in Enterobacteriaceae (p < 0.001) and Bacteroides–Prevotella (p = 0.005) populations. Eubacterium rectale–Clostridium coccoides (p < 0.001) and Atopobium (p = 0.039) groups also gradually increased. This overall pattern was unaffected by probiotic administration (p > 0.05). B. longum (p = 0.005) and Lactobacillus rhamnosus (p < 0.001) were detected more frequently in probiotic group during supplementation, but no difference after supplementation had ceased (p > 0.05). Cultured lactic acid bacteria were also more numerous in the probiotic-administered babies during treatment period (log10 CFU/g 8.4 versus 7.4; p = 0.035). Our results indicate that supplemented strains could be detected but did not persist in the bowel once probiotic administration had ceased.

Comprehensive analysis of the bacterial content of stool from patients with chronic pouchitis, normal pouches, or familial adenomatous polyposis pouches.

Background: Chronic pouchitis is an important long‐term complication following ileal pouch–anal anastomosis for ulcerative colitis. Antibiotic administration reduces symptoms of pouchitis, indicating that bacteria have a role in pathogenesis. The aim of the research was to investigate the bacterial content of pouches using nucleic acid‐based methods. Methods: Stool microbiota of 17 patients with normal pouches (NP), 17 patients with pouchitis (CP) utilizing samples collected from each patient when antibiotic‐treated (CP‐on, asymptomatic) and when untreated (CP‐off, symptomatic), and 14 familial adenomatous polyposis (FAP) patients were analyzed by high‐throughput sequencing, fluorescence in situ hybridization technologies, and quantitative polymerase chain reaction (qPCR). Results: Fluorescence in situ hybridization analysis revealed an expanded phylogenetic gap in NP and CP‐off patients relative to FAP. Antibiotic treatment reduced the gap in CP stool. The phylogenetic gap of CP‐off patients was due to members of the bacterial families Caulobacteriaceae, Sphingomonadaceae, Comamonadaceae, Peptostreptococcaceae, and Clostridiaceae. There was a greater diversity of phylotypes of Clostridiaceae in CP‐off subjects. The phylogenetic gap of NP stool was enriched by Ruminococcaceae and Bifidobacteriaceae. CP stool microbiota had reduced diversity relative to NP and FAP stool due largely to a reduction in Lachnospiraceae/Insertae Sedis XIV/clostridial cluster IV groups. Conclusions: Bacterial groups within the expanded phylogenetic gap of pouch patients may have roles in the pathogenesis of pouchitis. Further research concerning the physiology of cultured members of these groups will be necessary to explain their specific roles. Members of the Lachnospiraceae, Incertae Sedis XIV, and clostridial cluster IV could be useful biomarkers of pouch health. (Inflamm Bowel Dis 2011;)

Influence of Camembert consumption on the composition and metabolism of intestinal microbiota: a study in human microbiota-associated rats

The objective of the present study was to evaluate the consequence of Camembert consumption on the composition and metabolism of human intestinal microbiota. Camembert cheese was compared with milk fermented by yoghurt starters and Lactobacillus casei as a probiotic reference. The experimental model was the human microbiota-associated (HM) rat. HM rats were fed a basal diet (HMB group), a diet containing Camembert made from pasteurised milk (HMCp group) or a diet containing fermented milk (HMfm group). The level of micro-organisms from dairy products was measured in faeces using cultures on a specific medium and PCR-temporal temperature gradient gel electrophoresis. The metabolic characteristics of the caecal microbiota were also studied: SCFA, NH3, glycosidase and reductase activities, and bile acid degradations. The results showed that micro-organisms from cheese comprised 10(5)-10(8) bacteria/g faecal sample in the HMCp group. Lactobacillus species from fermented milk were detected in HMfm rats. Consumption of cheese and fermented milk led to similar changes in bacterial metabolism: a decrease in azoreductase activity and NH3 concentration and an increase in mucolytic activities. However, specific changes were observed: in HMCp rats, the proportion of ursodeoxycholic resulting from chenodeoxycholic epimerisation was higher; in HMfm rats, alpha and beta-galactosidases were higher than in other groups and both azoreductases and nitrate reductases were lower. The results show that, as for fermented milk, Camembert consumption did not greatly modify the microbiota profile or its major metabolic activities. Ingested micro-organisms were able to survive in part during intestinal transit. These dairy products exert a potentially beneficial influence on intestinal metabolism.

Differentiation of Bifidobacterium longum subspecies longum and infantis by quantitative PCR using functional gene targets

Background Members of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy. Methods We developed a qPCR method targeting the sialidase gene (subsp. infantis) and sugar kinase gene (subsp. longum) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested by in silico, pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions. Results A pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp. infantis was detectable (0–32.4% prevalence) in the feces of Australian (n = 90), South-East Asian (n = 24), and Chinese babies (n = 91), but in all cases at low abundance (<0.01–4.6%) compared to subsp. longum (0.1–33.7% abundance; 21.4–100% prevalence). Discussion Our qPCR method differentiates B. longum subspecies longum and infantis using characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health.

Allergic diseases of the skin and drug allergies – 2013. Longitudinal analysis of fecal microbiota of infants followed-up for eczema till 2 years of age

Methods Children with eczema at 2 years old (n=26) and their matched (for gender, mode of delivery and feeding in first 6 months) healthy controls (n=26) were selected from the placebo group of a cohort of at-risk infants participating in an randomized double-blind placebo controlled trial on the protective effects of supplemental probiotics (first 6 months) on eczema and allergies. Children with eczema were subclassified into atopic eczema (n=12) and non-atopic eczema (n=14). Molecular evaluation of fecal microbiota were conducted using Fluorescence In Situ Hybridization-Flow Cytometry (FISH-FC) for fecal samples collected at 3 days, 1, 3, and 12 months. Probes were selected to target Eubacterium rectale-Clostridium coccoides group (Erec482), Clostridium leptum subgroup (Clep866 and the corresponding competitor probes), Bacteroides-Prevotella group (Bac303), Bifidobacterium genus (Bif164), Atopobium group (Ato291), LactobacilliEnterococci group (Lab158), Enterobacteriaceae family (Enter1432) and Clostridium perfringens (Cperf191). Linear mixed model was used to evaluate the longitudinal differences (i.e. 4 time points) of bacterial targets while adjusting for gender, mode of delivery, feeding up to 6 months, and allergic rhinitis and wheezing within the eczema group at 2 years of age. Results Longitudinal analyses over four time points showed that higher relative abundance of Enterobacteriaceae [coefficient (B): 1.104, 95% confidence interval (CI):0.175-2.033, adj p=0.022] in children with eczema by 2 years of age. Similar observations were made when eczema group was subanalyzed into non-atopic and atopic eczema, where higher relative abundance of Enterobacteriaceae [B:1.357, 95%CI; 0.382-2.332, adj p=0.008] and [B:1.165, 95%CI; 0.221-2.109, adj p=0.019] were observed respectively as compared to healthy controls. Relative abundance of Clostridium perfringens were also higher when subanalyzed for non-atopic [B:0.572, 95%CI; 0.0009-1.144, adj p=0.050] and atopic eczema [B:0.000451, 95%CI: 0.0001-0.0007, adj p=0.012] compared to healthy controls.

CR05 BACTERIA NOT COMMONLY ASSOCIATED WITH HUMAN FAECES IMPLICATED IN ETIOLOGY OF POUCHITIS

Purpose  Chronic pouchitis is an important long‐term complication following ileal pouch formation. Antibiotics reduce symptoms indicating bacteria play a role in pathogenesis. We analyzed and compared the bacterial content (microbiota) of stool collected from chronic pouchitis and familial adenomatous polyposis patients. Additionally, we compared the microbiota of chronic pouchitis patients on and off antibiotics. We aimed to determine which of the bacterial inhabitants of pouches had a role in the pathogenesis of pouchitis.