Influence of the gut microbiota on blood acute-phase proteins.

Little is known about the bovine intestinal microbiota influence on systemic innate immune responses. The objective of the present study was to determine relationships between acute-phase proteins in blood serum of cows [C-reactive protein (CRP), LPS-binding protein (LBP) and haptoglobin (Hp)] and the faecal microbiota. Fifty-two healthy cows (2-8 years old) were investigated. Faecal bacteria were determent characterized by in situ hybridization with 16S/23S rRNA-targeted probes and by conventional culture methods. The population of Gram-negative faecal bacteria (Enterobacteriaceae) was correlated negatively with CRP and positively with LBP in blood plasma, independent of the method used. Similar results were observed with Clostridium perfringens. No correlation was found between the faecal population of intestinal bacteria and Hp levels in blood plasma. This datum indicates that intestinal bacteria, especially Enterobacteriaceae and C. perfringens, may influence the level of CRP and LBP in blood plasma. These findings can be very important for diagnostic evaluations of the intestinal microbiota and provide specific information about its regulation.

Bacterial and fungal microbiota in relation to probiotic therapy (VSL#3) in pouchitis.

BACKGROUND: The intestinal microbiota plays a critical role in the pathophysiology of pouchitis, a major complication after ileal pouch anal anastomosis in patients with ulcerative colitis. Recently, controlled trials have demonstrated that probiotics are effective in maintenance of remission in pouchitis patients. However, the mechanism by which therapy with probiotics works remains elusive. This study explores the role of the bacterial and fungal flora in a controlled trial for maintenance of remission in pouchitis patients with the probiotic VSL#3 compound. METHODS: The mucosa associated pouch microbiota was investigated before and after therapy with VSL#3 by analysis of endoscopic biopsies using ribosomal DNA/RNA based community fingerprint analysis, clone libraries, real time polymerase chain reaction (PCR), and fluorescence in situ hybridisation. Patients were recruited from a placebo controlled remission maintenance trial with VSL#3. RESULTS: Patients who developed pouchitis while treated with placebo had low bacterial and high fungal diversity. Bacterial diversity was increased and fungal diversity was reduced in patients in remission maintained with VSL#3 (p = 0.001). Real time PCR experiments demonstrated that VSL#3 increased the total number of bacterial cells (p = 0.002) and modified the spectrum of bacteria towards anaerobic species. Taxa specific clone libraries for Lactobacilli and Bifidobacteria showed that the richness and spectrum of these bacteria were altered under probiotic therapy. CONCLUSIONS: Probiotic therapy with VSL#3 increases the total number of intestinal bacterial cells as well as the richness and diversity of the bacterial microbiota, especially the anaerobic flora. The diversity of the fungal flora is repressed. Restoration of the integrity of a "protective" intestinal mucosa related microbiota could therefore be a potential mechanism of probiotic bacteria in inflammatory barrier diseases of the lower gastrointestinal tract.

Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers.

Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P<0.05). During the isomalt phase, the activity of bacterial beta-glucosidase decreased (P<0.05) whereas beta-glucuronidase, sulfatase, nitroreductase and urease remained unchanged. Faecal polyamines were not different between test periods with the exception of cadaverine, which showed a trend towards a lower concentration following isomalt (P=0.055). Faecal SCFA, lactate, bile acids, neutral sterols, N, NH3, phenol and p-cresol were not affected by isomalt consumption. In vitro, isomalt was metabolized in several bifidobacteria strains and yielded high butyrate concentrations. Isomalt, which is used widely as a low-glycaemic and low-energy sweetener, has to be considered a prebiotic carbohydrate that might contribute to a healthy luminal environment of the colonic mucosa.

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.

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.

Quantification of the flavonoid-degrading bacterium Eubacterium ramulus in human fecal samples with a species-specific oligonucleotide hybridization probe.

To investigate the occurrence of the flavonoid-degrading bacterium Eubacterium ramulus in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E. ram-0997-a-A-18 to fecal samples from 20 subjects indicated the presence of E. ramulus in each individual tested in numbers from 4.4 x 10(7) to 2.0 x 10(9) cells/g of fecal dry mass. Six fecal E. ramulus isolates were recognized by S-S-E.ram-0997-a-A-18 but exhibited different band patterns when analyzed by randomly amplified polymorphic DNA.

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.

Dietary guar gum and pectin stimulate intestinal microbial polyamine synthesis in rats.

The effects of two highly fermentable dietary fibers (guar gum and pectin) on the type and concentrations of cecal polyamines as affected by the intestinal microflora were studied in groups of germ-free (n = 10/group) and conventional rats (n = 6/group). Both germ-free and conventional rats were randomly assigned to one of three treatments as follows: 1) fiber-free control diet, 2) control diet + 10% guar gum and 3) control diet + 10% pectin. In germ-free rats, guar gum and pectin had no effect on cecal polyamine concentrations. Putrescine was confirmed to be the major endogenous polyamine within the gut lumen. In cecal contents of conventional rats, both guar gum and pectin led to the appearance of cadaverine and to elevated putrescine concentrations in comparison with the fiber-free control diet (1.35 +/- 0.15 and 2.27 +/- 0.32, respectively, vs. 0.20 +/- 0.03 micromol/g dry weight, P < 0.05). The cecal cadaverine concentration was higher in pectin- than in guar-fed rats (8.20 +/- 0.89 vs. 1.92 +/- 0.27 micromol/g dry weight, P < 0.05). Counts of total bacteria, bacteroides, fusobacteria and enterobacteria were higher (P < 0.05) in rats fed guar gum and pectin. Bifidobacteria were found exclusively in guar-fed rats. In vitro studies on selected species representing the numerically dominant population groups of the human gut flora (bacteroides, fusobacteria, anaerobic cocci and bifidobacteria) were examined for their ability to synthesize intracellular polyamines. These experiments demonstrated the ability of bacteroides, fusobacteria and anaerobic cocci to synthesize high amounts of putrescine and spermidine. Calculations based on these results suggest that the intestinal microflora are a major source of polyamines in the contents of the large intestine.

Feeding resistant starch affects fecal and cecal microflora and short-chain fatty acids in rats.

The effects of different forms of resistant potato starch (RS) on the major microbial population groups and short-chain fatty acids (SCFA) in the cecum and feces of rats were studied over a 5-mo feeding period. Thirty 8-wk-old male Wistar rats, averaging 210 g initial body weight, were adapted for 7 d to a balanced basal diet containing 60% waxy maize starch devoid of any RS. On d 8, three groups of 10 rats each were fed diets containing the following forms of starch: 1) rapidly digestible waxy maize starch (basal diet), 2) a mixture of 83.3% waxy maize starch and 16.7% native granular potato starch (RS 1), or 3) a mixture of 33.3% waxy maize starch and 66.7% modified potato starch (RS 2). The final RS content in RS 1 and RS 2 was 10%. Fecal samples were collected at d 8 and 1, 3, and 5 mo after the start of the experiment. Cecal contents were taken after 5 mo. The colony counts of microbial groups did not vary with time in the control or the RS 1 group (P > .05). Only the number of Bacteroides/fusobacteria decreased between mo 1 and 5 in rats fed RS 1 (P < .05). The RS 2 diet led to a significant increase in total culturable bacteria, lactobacilli, streptococci, and enterobacteria between mo 1 and 5. The RS 1 and RS 2 diets stimulated the growth of bifidobacteria. Cecal numbers of lactobacilli, streptococci, and enterobacteria were higher in rats fed RS 2 than in rats fed RS 1 or control diet (P < .05). Lactobacillus cellobiosus occurred only in rats fed RS 1 or RS 2. Acetate increased in mo 3 compared with d 8 in all groups (P < .05). The fecal and cecal SCFA displayed higher concentrations of acetate and propionate and a higher molar proportion of propionate in RS 2 than in RS 1 or control rats (P < .05). Stimulation of bifidobacteria, lactobacilli, and SCFA may be useful for the suppression of pathogenic organisms in the colon.

Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons.

Constipation is an ailment encountered often in elderly people. A study was initiated to test the effects of lactose or inulin on the bowel habits of constipated elderly patients and to correlate these effects with several variables measured in feces such as microflora composition, concentration of lactate and short-chain fatty acids (SCFAs), pH, and the activities of beta-glucosidase and beta-glucuronidase, Groups of 15 and 10 patients received lactose and inulin, respectively, for a period of 19 d. The dose, 20 g/d from days 1 to 8, was gradually increased to 40 g/d from days 9 to 11 and was kept at this dose from days 12 to 19. There was considerable interindividual variations with this kind of dietary intervention. Inulin increased bifidobacteria significantly from 7.9 to 9.2 log10/g dry feces, but decreased enterococci in number and enterobacteria in frequency. In individuals consuming lactose, a noticeable increase in fecal counts of enterococci and a decrease in lactobacilli and clostridia was detected. Total bacterial counts remained unchanged. No changes in the concentrations of fecal SCFAs and lactate were observed. SCFAs showed a slight trend toward higher molar ratios of acetate to butyrate in response to the intake of lactose or inulin. The fecal pH and the beta-glucosidase and beta-glucuronidase activities were not influenced by sugar intake. Inulin showed a better laxative effect than lactose and reduced functional constipation with only mild discomfort.

Influence of two infant formulas and human milk on the development of the faecal flora in newborn infants.

The establishment of the faecal flora of 39 full-term infants fed exclusively on breast milk (n = 20) or with two different modern adapted cow's milk formulas (n = 19) was studied during the first 3 months of life. One formula investigated was based on 100% bovine casein as the protein source whereas the other formula contained bovine milk proteins with a whey/casein ratio of 60:40. A faecal flora rich in bifidobacteria was found in all study groups; the growth of putrefactive bacteria (especially Bacteroides spp.), however, was limited. In formula-fed infants, significantly higher bacterial counts of enterococci and clostridia were detected compared to breast milk-fed infants. Similarities and differences due to the feeding regimen were particularly reflected in the pattern of the anaerobic bacterial species. Bifidobacterium bifidum, B. infantis and B. breve constituted the majority of the bifidobacterial flora independent of the type of milk feeding. Other bifidobacterial species such as B. longum, B. adolescentis, B. parabifidum and B. pseudo-catenulatum were detected in high numbers and at low frequencies in breastfed infants. The latter three were observed in infants fed the whey/casein formula as well. It seems that infants fed a casein formula develop a faecal flora more like that of breastfed infants concerning Lactobacillus spp. (especially L. fermentum and L. brevis).