Comparative composition of bacteria in the human intestinal microflora during remission and active ulcerative colitis

Ulcerative colitis is a severe, relapsing and remitting disease of the human large intestine characterised by inflammation of the mucosa and submucosa. The main site of disease is the sigmoid/rectal region of the large bowel but the aetiology remains unknown. There is considerable evidence to indicate that the components of the resident colonic microflora can play an important role in initiation of the disease. The present study was aimed at characterising the faecal microflora of ulcerative colitis patients in remission and active phases to determine profile differences. Faecal samples were obtained from 12 patients, 6 with active colitis and 6 in remission. The samples were analysed for populations of lactobacilli, bifidobacteria, clostridia, bacteroides, sulphate-reducing bacteria (SRB) and total bacteria using culture independent fluorescence in situ hybridisation (FISH). Lactobacillus-specific denaturing gradient gel electrophoresis (DGGE) was then performed to compare the species present. Numbers of lactobacilli were significantly lower (p<0.05) during the active phase of the disease but the other populations tested did not differ. DGGE analysis revealed that Lactobacillus salivarus, Lactobacillus manihotivorans and Pediococcus acidilactici were present in remission, but not during active inflammation. These results imply that a reduction in intestinal Lactobacillus species may be important in the initiation of ulcerative colitis.

A novel galactooligosaccharide mixture increases the bifidobacterial population numbers in a continuous in vitro fermentation system and in the proximal colonic contents of pigs in vivo

Prebiotics are nondigestible food ingredients that encourage proliferation of selected groups of the colonic microflora, thereby altering the composition toward a more beneficial community. In the present study, the prebiotic potential of a novel galactooligosaccharide (GOS) mixture, produced by the activity of galactosyltransferases from Bifidobacterium bifidum 41171 on lactose, was assessed in vitro and in a parallel continuous randomized pig trial. In situ fluorescent hybridization with 16S rRNA-targeted probes was used to investigate changes in total bacteria, bifidobacteria, lactobacilli, bacteroides, and Clostridium histolyticum group in response to supplementing the novel GOS mixture. In a 3-stage continuous culture system, the bifidobacterial numbers for the first 2 vessels, which represented the proximal and traverse colon, increased (P < 0.05) after the addition of the oligosaccharide mixture. In addition, the oligosaccharide mixture strongly inhibited the attachment of enterohepatic Escherichia coli (P < 0.01) and Salmonella enterica serotype Typhimurium (P < 0.01) to HT29 cells. Addition of the novel mixture at 4% (wt:wt) to a commercial diet increased the density of bificlobacteria (P < 0.001) and the acetate concentration (P < 0.001), and decreased the pH (P < 0.001) compared with the control diet and the control diet supplemented with inulin, suggesting a great prebiotic potential for the novel oligosaccharide mixture. J. Nutr. 135: 1726-1731, 2005.

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

Aims: To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. Materials and Methods: Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. Results: Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. Conclusions: Arabino-oligosaccharides can be considered as potential prebiotics. Significance and Impact of the Study: Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.

Effects of a novel galactooligosaccharide on the faecal microbiota of healthy and inflammatory bowel disease cats during a randomized, double-blind, cross-over feeding study

Inflammatory bowel disease (IBD) is a common gastrointestinal disorder of cats with no known aetiological agent. Previous work has suggested that the faecal microbiota of IBD cats is significantly different from that of healthy cats, including significantly lower bifidobacteria, bacteroides and total counts in IBD cats and significantly lower levels of sulfate-reducing bacteria in healthy cats. Prebiotics, including galactooligosaccharides (GOS), have been shown to elicit a bifidogenic effect in humans and other animals. The purpose of the current study was to examine the impact of a novel GOS supplementation on the faecal microbiota of healthy and IBD cats during a randomized, double-blind, cross-over feeding study. Eight oligonucleotide probes targeting specific bacterial populations and DAPI stain (total bacteria) were used to monitor the feline faecal microbiota. Overall, inter-animal variation was high; while a trend of increased bifidobacterial levels was seen with GOS supplementation it was not statistically significant in either healthy or IBD cats. No significant differences were observed in the faecal microbiota of IBD cats and healthy cats fed the same diet. Members of the family Coriobacteriaceae (Atopobium cluster) were found to be the most abundant bacteria in the feline microbiota.

Prebiotic effect of fruit and vegetable shots containing Jerusalem artichoke inulin : a human intervention study

The present study aimed to determine the prebiotic effect of fruit and vegetable shots containing inulin derived from Jerusalem artichoke (JA). A three-arm parallel, placebo-controlled, double-blind study was carried out with sixty-six healthy human volunteers (thirty-three men and thirty-three women, age range: 18–50 years). Subjects were randomised into three groups (n 22) assigned to consume either the test shots, pear-carrot-sea buckthorn (PCS) or plum-pear-beetroot (PPB), containing JA inulin (5 g/d) or the placebo. Fluorescent in situ hybridisation was used to monitor populations of total bacteria, bacteroides, bifidobacteria, Clostridium perfringens/histolyticum subgroup, Eubacterium rectale/Clostridium coccoides group, Lactobacillus/Enterococcus spp., Atopobium spp., Faecalibacterium prausnitzii and propionibacteria. Bifidobacteria levels were significantly higher on consumption of both the PCS and PPB shots (10·0 (sd 0·24) and 9·8 (sd 0·22) log10 cells/g faeces, respectively) compared with placebo (9·3 (sd 0·42) log10 cells/g faeces) (P < 0·0001). A small though significant increase in Lactobacillus/Enterococcus group was also observed for both the PCS and PPB shots (8·3 (sd 0·49) and 8·3 (sd 0·36) log10 cells/g faeces, respectively) compared with placebo (8·1 (sd 0·37) log10 cells/g faeces) (P = 0·042). Other bacterial groups and faecal SCFA concentrations remained unaffected. No extremities were seen in the adverse events, medication or bowel habits. A slight significant increase in flatulence was reported in the subjects consuming the PCS and PPB shots compared with placebo, but overall flatulence levels remained mild. A very high level of compliance (>90 %) to the product was observed. The present study confirms the prebiotic efficacy of fruit and vegetable shots containing JA inulin.

The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota.

We have examined the gut bacterial metabolism of pomegranate by-product (POMx) and major pomegranate polyphenols, punicalagins, using pH-controlled, stirred, batch culture fermentation systems reflective of the distal region of the human large intestine. Incubation of POMx or punicalagins with faecal bacteria resulted in formation of the dibenzopyranone-type urolithins. The time course profile confirmed the tetrahydroxylated urolithin D as the first product of microbial transformation, followed by compounds with decreasing number of phenolic hydroxy groups: the trihydroxy analogue urolithin C and dihydroxylated urolithin A. POMx exposure enhanced the growth of total bacteria, Bifidobacterium spp. and Lactobacillus spp., without influencing the Clostridium coccoides–Eubacterium rectale group and the C. histolyticum group. In addition, POMx increased concentrations of short chain fatty acids (SCFA) viz. acetate, propionate and butyrate in the fermentation medium. Punicalagins did not affect the growth of bacteria or production of SCFA. The results suggest that POMx oligomers, composed of gallic acid, ellagic acid and glucose units, may account for the enhanced growth of probiotic bacteria.

Changes in the intestinal microbiota after a short period of dietary over-indulgence, representative of a holiday or festival season

The effects on the intestinal microbiota of a short period of marginal over-eating, characteristic of holiday or festival periods, were investigated in a pilot study. Fourteen healthy male subjects consumed a diet rich in animal protein and fat for seven days. During this period, the subjects significantly increased their dietary energy, protein, carbohydrate and fat intakes by 56, 59, 53 and 58%, respectively (all P < 0.05). The mean weight gain of 0.27 kg was less than the expected 1 kg, but this was consistent with a degree of under-reporting on the baseline diet. Fluorescence in situ hybridisation analysis confirmed the relative stability of each individual’s faecal microbiota but showed considerable variations between them. The diet was associated with a significant increase in numbers of total faecal bacteria and the bacteroides group, as detected by the universal bacterial probe (DAPI) and Bacteroides probe (Bac 303), respectively. Overall, there was a decrease in numbers of the Lactobacillus/Enterococcus group (Lab 158 probe; 2.8 ± 3.0% to 1.8 ± 1.8%) and the Bifidobacterium group (Bif 164 probe; 3.0 ± 3.7% to 1.7 ± 1.2%), although there was considerable inter-individual variation. Analysis of the relative proportions of each bacterial group as a percentage of the subject’s total bacteria showed a trend for a change in the intestinal microbiota that might be considered potentially unhealthy.

Investigation of the faecal microbiota of geriatric cats

Aims: To investigate the faecal microbiota of geriatric cats, as aging affects the nutrient digestibility and metabolic function of the feline intestine. Methods and results: 20 geriatric cats were randomly assigned to two groups that were fed different foods. Coriobacteriaceae, Clostridium cluster XIV, bifidobacteria, and lactic acid bacteria were the dominant faecal bacterial groups, accounting for ∼40% of total bacteria. Clostridium cluster IX was less predominant (0.5% of total bacteria), while the remaining bacterial populations enumerated only accounted for 0.2% of total bacteria. Highly diverse microbial profiles were demonstrated for geriatric cats with denaturing gradient gel electrophoresis, although a few common bands were evident. Some differences were seen in the feline faecal microbiota between animal groups at the same time or over time for individual animals. However, no obvious clustering based on animal group or sample time was indicated. Conclusions: geriatric cats harboured a complex faecal microbiota and ∼41% of total bacteria have been detected with the probes employed. Significance and impact of study: First molecular-based study examining faecal microbiota of geriatric felines. Knowledge of the microbiota associated with ageing in cats may allow improved development of foods specific for the needs of senior cats.

Investigation of the faecal microbiota of kittens: monitoring bacterial succession and effect of diet

Weaning is a stressful process for kittens, and is often associated with diarrhoea and the onset of infectious diseases. The gastrointestinal microbiota plays an essential role in host well-being, including improving homeostasis. Composition of the gastrointestinal microbiota of young cats is poorly understood, and the impact of diet on the kitten microbiota unknown. The aims of this study were to monitor the faecal microbiota of kittens and determine the effect(s) of diet on its composition. Bacterial succession was monitored in two groups of kittens (at 4 and 6 weeks, and 4 and 9 months of age) fed different foods. Age-related microbial changes revealed significantly different counts of total bacteria, lactic acid bacteria, Desulfovibrionales, Clostridium cluster IX and Bacteroidetes between 4-week- and 9-month-old kittens. Diet-associated differences in the faecal microbiota of the two feeding groups were evident. In general, fluorescence in situ hybridization analysis demonstrated bifidobacteria, Atopobium group, Clostridium cluster XIV and lactic acid bacteria were dominant in kittens. Denaturing gradient gel electrophoresis profiling showed highly complex and diverse faecal microbiotas for kittens, with age- and/or food-related changes seen in relation to species richness and similarity indices. Four-week-old kittens harboured more diverse and variable profiles than those of weaned kittens.

Determination of the in vivo prebiotic potential of a maize based wholegrain breakfast cereal: a human feeding study

Epidemiological studies have shown an inverse relationship between risk of CVD and intake of whole grain (WG)-rich food. Regular consumption of breakfast cereals can provide not only an increase in dietary WG but also improvements to cardiovascular health. Various mechanisms have been proposed, including prebiotic modulation of the colonic microbiota. In the present study, the prebiotic activity of a maize-derived WG cereal (WGM) was evaluated in a double-blind, placebo-controlled human feeding study (n 32). For a period of 21 d, healthy men and women, mean age 32 (sd 8) years and BMI 23·3 (sd 0·58) kg/m2, consumed either 48 g/d WG cereal (WGM) or 48 g placebo cereal (non-whole grain (NWG)) in a crossover fashion. Faecal samples were collected at five points during the study on days 0, 21, 42, 63 and 84 (representing at baseline, after both treatments and both wash-out periods). Faecal bacteriology was assessed using fluorescence in situ hybridisation with 16S rRNA oligonucleotide probes specific for Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum/perfringens subgroup, Lactobacillus–Enterococcus subgroup and total bacteria. After 21 d consumption of WGM, mean group levels of faecal bifidobacteria increased significantly compared with the control cereal (P = 0·001). After a 3-week wash-out period, bifidobacterial levels returned to pre-intervention levels. No statistically significant changes were observed in serum lipids, glucose or measures of faecal output. In conclusion, this WG maize-enriched breakfast cereal mediated a bifidogenic modulation of the gut microbiota, indicating a possible prebiotic mode of action

In Vitro Fermentation of NUTRIOSE® FB06, a wheat dextrin soluble fibre, in a continuous culture human colonic model system

Wheat dextrin soluble fibre may have metabolic and health benefits, potentially acting via mechanisms governed by the selective modulation of the human gut microbiota. Our aim was to examine the impact of wheat dextrin on the composition and metabolic activity of the gut microbiota. We used a validated in vitro three-stage continuous culture human colonic model (gut model) system comprised of vessels simulating anatomical regions of the human colon. To mimic human ingestion, 7 g of wheat dextrin (NUTRIOSE® FB06) was administered to three gut models, twice daily at 10.00 and 15.00, for a total of 18 days. Samples were collected and analysed for microbial composition and organic acid concentrations by 16S rRNA-based fluorescence in situ hybridisation and gas chromatography approaches, respectively. Wheat dextrin mediated a significant increase in total bacteria in vessels simulating the transverse and distal colon, and a significant increase in key butyrate-producing bacteria Clostridium cluster XIVa and Roseburia genus in all vessels of the gut model. The production of principal short-chain fatty acids, acetate, propionate and butyrate, which have been purported to have protective, trophic and metabolic host benefits, were increased. Specifically, wheat dextrin fermentation had a significant butyrogenic effect in all vessels of the gut model and significantly increased production of acetate (vessels 2 and 3) and propionate (vessel 3), simulating the transverse and distal regions of the human colon, respectively. In conclusion, wheat dextrin NUTRIOSE® FB06 is selectively fermented in vitro by Clostridium cluster XIVa and Roseburia genus and beneficially alters the metabolic profile of the human gut microbiota.

An assessment of the prebiotic potential of single and blended substrates in anaerobic in vitro batch culture fermentations using canine faecal samples as inocula

Previously, using an in vitro static batch culture system, it was found that rice bran (RB), inulin, fibersol, mannanoligosaccharides (MOS), larch arabinogalactan and citrus pectin elicited prebiotic effects (in terms of increased numbers of bifidobacteria and lactic acid bacteria) on the faecal microbiota of a dog. The aim of the present study was to confirm the prebiotic potential of each individual substrate using multiple faecal donors, as well as assessing the prebiotic potential of 15 substrate blends made from them. Anaerobic static and stirred, pH-controlled batch culture systems inoculated with faecal samples from healthy dogs were used for this purpose. Fluorescence in situ hybridization (FISH) analysis using seven oligonucleotide probes targeting selected bacterial groups and DAPI (total bacteria) was used to monitor bacterial populations during fermentation runs. High-performance liquid chromatography was used to measure butyrate produced as a result of bacterial fermentation of the substrates. RB and a MOS/RB blend (1:1, w/w) were shown to elicit prebiotic and butyrogenic effects on the canine microbiota in static batch culture fermentations. Further testing of these substrates in stirred, pH-controlled batch culture fermentation systems confirmed the prebiotic and butyrogenic effects of MOS/RB, with no enhancement of Clostridium clusters I and II and Escherichia coli populations.

Dynamics and diversity of the 'Atopobium cluster' in the human faecal microbiota, and phenotypic characterization of 'Atopobium cluster' isolates

This study monitored the dynamics and diversity of the human faecal 'Atopobium cluster' over a 3-month period using a polyphasic approach. Fresh faecal samples were collected fortnightly from 13 healthy donors (6 males and 7 females) aged between 26 and 61 years. Fluorescence in situ hybridization was used to enumerate total (EUB338mix) and 'Atopobium cluster' (ATO291) bacteria, with counts ranging between 1.12 × 1011 and 9.95 × 1011, and 1.03 × 109 and 1.16 × 1011 cells (g dry weight faeces)-1, respectively. The 'Atopobium cluster' population represented 0.2-22 % of the total bacteria, with proportions donor-dependent. Denaturing gradient gel electrophoresis (DGGE) using 'Atopobium cluster'-specific primers demonstrated faecal populations of these bacteria were relatively stable, with bands identified as Collinsella aerofaciens, Collinsella intestinalis/Collinsella stercoris, Collinsella tanakaei, Coriobacteriaceae sp. PEAV3-3, Eggerthella lenta, Gordonibacter pamelaeae, Olsenella profusa, Olsenella uli and Paraeggerthella hongkongensis in the DGGE profiles of individuals. Colony PCR was used to identify 'Atopobium cluster' bacteria isolated from faeces (n = 224 isolates). 16S rRNA gene sequence analysis of isolates demonstrated Collinsella aerofaciens represented the predominant (88 % of isolates) member of the 'Atopobium cluster' found in human faeces, being found in nine individuals. Eggerthella lenta was identified in three individuals (3.6 % of isolates). Isolates of Collinsella tanakaei, an 'Enorma' sp. and representatives of novel species belonging to the 'Atopobium cluster' were also identified in the study. Phenotypic characterization of the isolates demonstrated their highly saccharolytic nature and heterogeneous phenotypic profiles, and 97 % of the isolates displayed lipase activity.

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.