Michael Blaut

Differences in Fecal Microbiota in Different European Study Populations in Relation to Age, Gender, and Country: a Cross-Sectional Study

ABSTRACT A cross-sectional study on intestinal microbiota composition was performed on 230 healthy subjects at four European locations in France, Germany, Italy, and Sweden. The study participants were assigned to two age groups: 20 to 50 years (mean age, 35 years; n = 85) and >60 years (mean age, 75 years; n = 145). A set of 14 group- and species-specific 16S rRNA-targeted oligonucleotide probes was applied to the analysis of fecal samples by fluorescence in situ hybridization coupled with flow cytometry. Marked country-age interactions were observed for the German and Italian study groups. These interactions were inverse for the predominant bacterial groups Eubacterium rectale-Clostridium coccoides and Bacteroides-Prevotella. Differences between European populations were observed for the Bifidobacterium group only. Proportions of bifidobacteria were two- to threefold higher in the Italian study population than in any other study group, and this effect was independent of age. Higher proportions of enterobacteria were found in all elderly volunteers independent of the location. Gender effects were observed for the Bacteroides-Prevotella group, with higher levels in males than in females. In summary, age-related differences in the microbiota makeup were detected but differed between the study populations from the four countries, each showing a characteristic colonization pattern.

Oligofructose and long-chain inulin: influence on the gut microbial ecology of rats associated with a human faecal flora

Dietary incorporation of fermentable, indigestible fructans may be of benefit to gastrointestinal health by providing short-chain fatty acids, stimulating the proliferation of bifidobacteria or lactobacilli and suppressing potential pathogenic organisms in the gut. We tested the hypothesis that the effects of fructans on caecal, colonic and faecal short-chain fatty acid concentration and microflora composition depend on their chain length. Germ-free rats associated with a human faecal flora were randomly assigned to one of four treatments as follows: (1) commercial standard diet as a control (Con); (2) Con+50 g short-chain oligofructose/kg (OF); (3) C+50 g long-chain inulin/kg (lcIN); or (4) Con+50 g OF-lcIN/kg (Mix OF-lcIN). Changes in bacterial population groups in response to feeding these diets were investigated with 16S rRNA-targeted probes applied in in situ hybridization. Mix OF-lcIN- and lcIN-containing diets resulted in larger numbers of caecal, colonic and faecal bacteria of the Clostridium coccoides-Eubacterium rectale cluster than Con (10.6 and 10.3 v. 9.5 log10/g wet wt), whereas OF alone did not affect this bacterial group in caecum, colon or faeces. A bifidogenic effect was only observed in the colon and faeces of OF-treated rats. More lactobacilli were found in caecal and colonic contents of Mix OF-lcIN-fed rats and in faeces of OF-fed rats compared with Con. Mix OF-lcIN and OF led to significantly smaller numbers of caecal, colonic and faecal bacteria belonging to the Clostridium histolyticum and C. lituseburense groups than Con (6.8 and 6.9 v. 7.9 log10/g wet wt). Counts of total bacteria, Bacteroides-Prevotella and Enterobacteriaceae did not differ between the groups. OF and/or lcIN-containing diets significantly increased the caecal and colonic concentration of butyrate and its relative molar proportion. Only lcIN-containing diets resulted in a higher faecal concentration of butyrate than Con. Higher molar proportions of faecal butyrate were observed with all diets that had been supplemented with OF and/or lcIN. Stimulation of butyrate production could be of interest for the prevention of ulcerative colitis and colon cancer.

Effects of inulin on faecal bifidobacteria in human subjects.

A controlled study with eight healthy free-living subjects was carried out, in which energy intake was adjusted to the individual energy requirements. On administration of inulin, blood lipids, the faecal microflora, short-chain fatty acids and accompanying gastrointestinal symptoms were characterized in order to investigate the long-term effect of inulin. During the run-in phase (8 d), subjects received a typical Western diet providing 45% energy as fat and 40% energy as carbohydrate. Subsequently, the subjects consumed a fat-reduced diet which provided 30% energy as fat and 55% energy as carbohydrate for a period of 64 d using inulin as a fat replacer. The amounts of inulin consumed by the subjects (up to 34 g/d) were based on individual energy requirements with the aim to keep the diet isoenergetic with that used in the run-in period. To assess the effects of inulin administration, a control study (run-in and intervention) was carried out in which subjects consumed the same diet but devoid of inulin during the whole course of the study. To investigate the effect of inulin on faecal flora composition total bacteria and bifidobacteria in the faeces were enumerated by in situ hybridization with 16S rRNA targeted oligonucleotide probes. Inulin significantly increased bifidobacteria from 9.8 to 11.0 log10/g dry faeces and caused a moderate increase in gastrointestinal symptoms such as flatulence and bloatedness, whereas blood lipids and short-chain fatty acids remained essentially unaffected.

Absence of intestinal microbiota does not protect mice from diet-induced obesity.

The gut microbiota has been implicated in host nutrient absorption and energy homeostasis. We studied the influence of different diets on body composition in germ-free (GF) and conventional (CV) mice. GF and CV male adult C3H mice were fed ad libitum a semi-synthetic low-fat diet (LFD; carbohydrate-protein-fat ratio: 41:42:17; 19.8 kJ/g), a high-fat diet (HFD; 41:16:43; 21.4 kJ/g) or a commercial Western diet (WD; 41:19:41; 21.5 kJ/g). There was no difference in body weight gain between GF and CV mice on the LFD. On the HFD, GF mice gained more body weight and body fat than CV mice, and had lower energy expenditure. GF mice on the WD gained significantly less body fat than GF mice on the HFD. GF mice on both HFD and WD showed increased intestinal mRNA expression of fasting-induced adipose factor/angiopoietin-like protein 4 (Fiaf/Angptl4), but they showed no major changes in circulating Fiaf/Angptl4 compared with CV mice. The faecal microbiota composition of the CV mice differed between diets: the proportion of Firmicutes increased on both HFD and WD at the expense of the Bacteroidetes. This increase in the Firmicutes was mainly due to the proliferation of one family within this phylum: the Erysipelotrichaceae. We conclude that the absence of gut microbiota does not provide a general protection from diet-induced obesity, that intestinal production of Fiaf/Angptl4 does not play a causal role in gut microbiota-mediated effects on fat storage and that diet composition affects gut microbial composition to larger extent than previously thought.

Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls.

Background: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. Methods: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. Results: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls ( P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria , the Enterobacteriaceae , the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. Conclusion: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.

Metabolic Diversity of the Intestinal Microbiota: Implications for Health and Disease

The bacteria colonizing the human intestinal tract exhibit a high phylogenetic diversity that reflects their immense metabolic potential. By virtue of their catalytic activity, the human gut micro-organisms have an impact on gastrointestinal function and host health. All dietary components that escape digestion in the small intestine are potential substrates of the bacteria in the colon. The bacterial conversion of carbohydrates, proteins and nonnutritive compounds such as polyphenolic substances leads to the formation of a large number of compounds that may have beneficial or adverse effects on human health.

Development of the Intestinal Bacterial Composition in Hospitalized Preterm Infants in Comparison with Breast-Fed, Full-Term Infants

The establishment and succession of bacterial communities in hospitalized preterm infants has not been extensively studied. Because earlier studies depended on classical cultural techniques, their results were limited. This study monitored the establishment and succession of the neonatal microbiota in the first weeks of life by analyzing the 16S rDNA variety in fecal samples applying PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Fecal samples from 29 preterm infants hospitalized in a neonatal intensive care unit, including samples from antibiotic-treated infants and one with neonatal necrotizing enterocolitis, were subjected to PCR-DGGE analysis. Daily DGGE profiles from all preterm infants during the first 4 wk were obtained and analyzed. In addition, feces of 15 breast-fed, full-term infants and a variety of clinical bacterial isolates were examined and compared with the PCR-DGGE profiles of the preterm infants. During the first days of life, the DGGE profiles were rather simple but increased in their complexity over time. It became obvious that not only the intraindividual band-pattern similarity increased over time, but also the interindividual. During the observation period, similarity values (Cs) increased in each preterm infant from 0 to 80%, whereas interindividual Cs increased from 18.1 to 57.4%, revealing the acquisition of a highly similar bacterial community in these infants. In contrast, Cs-values obtained for breast-fed, full-term infants were rather low (11.2%). Escherichia coli, Enterococcus sp., and Klebsiella pneumoniae were the bacteria most commonly found in all preterm infants. The interindividual bacterial composition in hospitalized preterm infants is more similar in comparison with breast-fed, full-term infants and is not necessarily influenced by birth weight, diet, or antibiotic treatment.

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.

Detection of Diverse Bacterial Signatures in Atherosclerotic Lesions of Patients With Coronary Heart Disease

Background— Bacterial infection has been discussed as a potential etiologic factor in the pathophysiology of coronary heart disease (CHD). This study analyzes molecular phylogenies to systematically explore the presence, frequency, and diversity of bacteria in atherosclerotic lesions in patients with CHD. Methods and Results— We investigated 16S rDNA signatures in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients with CHD, control material from postmortem patients (n=15), and heart-beating organ donors (n=11) using clone libraries, denaturating gradient gel analysis, and fluorescence in situ hybridization. Bacterial DNA was found in all CHD patients by conserved PCR but not in control material or in any of the normal/unaffected coronary arteries. Presence of bacteria in atherosclerotic lesions was confirmed by fluorescence in situ hybridization. A high overall bacterial diversity of >50 different species, among them Staphylococcus species, Proteus vulgaris, Klebsiella pneumoniae, and Streptococcus species, was demonstrated in >1500 clones from a combined library and confirmed by denaturating gradient gel analysis. Mean bacterial diversity in atheromas was high, with a score of 12.33±3.81 (range, 5 to 22). A specific PCR detected Chlamydia species in 51.5% of CHD patients. Conclusions— Detection of a broad variety of molecular signatures in all CHD specimens suggests that diverse bacterial colonization may be more important than a single pathogen. Our observation does not allow us to conclude that bacteria are the causative agent in the etiopathogenesis of CHD. However, bacterial agents could have secondarily colonized atheromatous lesions and could act as an additional factor accelerating disease progression.

High-fat diet alters gut microbiota physiology in mice

The intestinal microbiota is known to regulate host energy homeostasis and can be influenced by high-calorie diets. However, changes affecting the ecosystem at the functional level are still not well characterized. We measured shifts in cecal bacterial communities in mice fed a carbohydrate or high-fat (HF) diet for 12 weeks at the level of the following: (i) diversity and taxa distribution by high-throughput 16S ribosomal RNA gene sequencing; (ii) bulk and single-cell chemical composition by Fourier-transform infrared- (FT-IR) and Raman micro-spectroscopy and (iii) metaproteome and metabolome via high-resolution mass spectrometry. High-fat diet caused shifts in the diversity of dominant gut bacteria and altered the proportion of Ruminococcaceae (decrease) and Rikenellaceae (increase). FT-IR spectroscopy revealed that the impact of the diet on cecal chemical fingerprints is greater than the impact of microbiota composition. Diet-driven changes in biochemical fingerprints of members of the Bacteroidales and Lachnospiraceae were also observed at the level of single cells, indicating that there were distinct differences in cellular composition of dominant phylotypes under different diets. Metaproteome and metabolome analyses based on the occurrence of 1760 bacterial proteins and 86 annotated metabolites revealed distinct HF diet-specific profiles. Alteration of hormonal and anti-microbial networks, bile acid and bilirubin metabolism and shifts towards amino acid and simple sugars metabolism were observed. We conclude that a HF diet markedly affects the gut bacterial ecosystem at the functional level.