Influence of isomalto-oligosaccharides on intestinal microbiota in rats

Aims: Isomalto‐oligosaccharides (IMO) with α(1→6) and α(1→4) glucosidic linkages are produced by enzymatic conversion of starch. IMO are only partially digestible but data on their influence on intestinal microbiota are limited. It was the aim of this study to investigate the effect of IMO diet on intestinal microbiota and short‐chain fatty acids production (SCFA) in rats. Methods and results: Three groups of F344 rats, each consisting of six animals, were fed IMO, inulin or a control diets for six weeks. A qualitative assessment of the intestinal microbiota was achieved by PCR‐denaturing gradient gel electrophoresis (DGGE). Major bacterial taxa were quantified by quantitative PCR (qPCR), and SCFA were measured using gas chromatography. Quantitative PCR demonstrated that lactobacilli were one of the dominant bacterial taxa in faecal samples from rats. IMO increased the number of lactobacilli and the total number of intestinal bacteria in rats fed IMO compared with animals receiving control and inulin diets. Furthermore, PCR‐DGGE with lactobacilli‐specific primers showed an altered biodiversity of lactobacilli in rats fed IMO compared with control diet. Conclusions: IMO selectively stimulates lactobacilli and increases their diversity in rats. Significance and impact of study: Isomalto‐oligosaccharides specifically stimulate growth of intestinal lactobacilli in a rat model system.     

Analysis of the large bowel microbiota of colitic mice using PCR/DGGE

AIM: To test combined polymerase chain reaction amplification of 16S rRNA gene sequences and denaturing gradient gel electrophoresis (PCR/DGGE) as an analytical method to investigate the composition of the large bowel microbiota of mice during the development of colitis. METHODS AND RESULTS: The colonic microbiota of formerly germfree interleukin 10 (IL-10)-deficient mice that had been exposed to the faecal microbiota of specific pathogen-free animals was screened using PCR/DGGE. The composition of the large bowel microbiota of IL-10-deficient mice changed as colitis progressed. DNA fragments originating from four bacterial populations ('Bacteroides sp.', Bifidobacterium animalis, Clostridium cocleatum, enterococci) were more apparent in PCR/DGGE profiles of colitic mice relative to non-colitic animals, whereas two populations were less apparent (Eubacterium ventriosum, Acidophilus group lactobacilli). Specific DNA:RNA dot blot analysis showed that bifidobacterial ribosomal RNA (rRNA) abundance increased as colitis developed. CONCLUSIONS: PCR/DGGE was shown to be an effective method to demonstrate changes in the composition of the large bowel microbiota of mice in relation to progression of inflammatory disease. The intensity of staining of DNA fragments in DGGE profiles reflected increased abundance of bifidobacterial rRNA in the microbiota of colitic animals. As bifidobacterial fragments in PCR/DGGE profiles generated from microbiota DNA showed increased intensity of fragment staining, an increase in bifidobacterial numbers in colitic mice was indicated. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR/DGGE analysis demonstrated an altered composition of the large bowel microbiota of colitic mice. This work will allow specific groups of bacteria to be targeted in future research concerning the pathogenesis of colitis.     

Supplementation of the diet with high-viscosity beta-glucan results in enrichment for lactobacilli in the rat cecum

BBn (BioBreeding) rats were fed casein-based diets supplemented with barley flour, oatmeal flour, cellulose, or barley beta-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 beta-glucan hydrolysates. The results of this study have relevance to the use of purified beta-glucan products as dietary supplements for human consumption.     

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

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.     

Intestinal Microbiota and Microbial Metabolites Are Changed in a Pig Model Fed a High-Fat/Low-Fiber or a Low-Fat/High-Fiber Diet

The intestinal microbiota and its metabolites appear to be an important factor for gastrointestinal function and health. However, research is still needed to further elaborate potential relationships between nutrition, gut microbiota and host’s health by means of a suitable animal model. The present study examined the effect of two different diets on microbial composition and activity by using the pig as a model for humans. Eight pigs were equally allotted to two treatments, either fed a low-fat/high-fiber (LF), or a high-fat/low-fiber (HF) diet for 7 weeks. Feces were sampled at day 7 of every experimental week. Diet effects on fecal microbiota were assessed using quantitative real-time PCR, DNA fingerprinting and metaproteomics. Furthermore, fecal short-chain fatty acid (SCFA) profiles and ammonia concentrations were determined. Gene copy numbers of lactobacilli, bifidobacteria (P<0.001) and Faecalibacterium prausnitzii (P<0.05) were higher in the LF pigs, while Enterobacteriaceae were more abundant in the HF pigs (P<0.001). Higher numbers of proteins affiliated to Enterobacteriaceae were also present in the HF samples. Proteins for polysaccharide breakdown did almost exclusively originate from Prevotellaceae. Total and individual fecal SCFA concentrations were higher for pigs of the LF treatment (P<0.05), whereas fecal ammonia concentrations did not differ between treatments (P>0.05). Results provide evidence that beginning from the start of the experiment, the LF diet stimulated beneficial bacteria and SCFA production, especially butyrate (P<0.05), while the HF diet fostered those bacterial groups which have been associated with a negative impact on health conditions. These findings correspond to results in humans and might strengthen the hypothesis that the response of the porcine gut microbiota to a specific dietary modulation is in support of using the pig as suitable animal model for humans to assess diet-gut-microbiota interactions.Data are available via ProteomeXchange with identifier PXD003447.     

Composition and temporal stability of gastrointestinal microbiota in irritable bowel syndrome--a longitudinal study in IBS and control subjects

Irritable bowel syndrome (IBS) is a common intestinal disorder that includes continuous or recurrent intestinal pain and discomfort and altered bowel habits. The pathophysiology of IBS is incompletely understood, but it may involve an altered intestinal microbiota. The aim of the present study was to compare the composition and temporal stability of faecal microbiota of IBS patients and healthy controls by applying culture-based techniques and PCR-DGGE analysis. No difference in the prevalence or mean culturable manners of bacteroides, bifidobacteria, spore-forming bacteria, lactobacilli, enterococci or yeasts were observed between the IBS and the control groups, whereas slightly higher numbers of coliforms as well as an increased aerobe:anaerobe ratio was observed in the IBS group. PCR-DGGE revealed more temporal instability in the predominant bacterial population of IBS subjects than in controls. In 9 out of 21 IBS subjects and 5 out of 17 controls the PCR-DGGE profiles obtained from the samples of the same individual on different occasions (sampling points 0, 3 and 6 months) were clearly different. However, the instability in some of the IBS subjects could partly be explained by the antibiotic consumption during the study. The present study suggests that instability of intestinal microbiota may be involved in IBS. However, further studies are needed to associate the instability with specific IBS symptoms or with specific bacterial groups and species.     

Effect of high fibre diets formulated with different fibrous ingredients on performance,nutrient digestibility and faecal microbiota of weaned piglets

The aim of the experiment on 180 weaned piglets (8.9 kg body weight) was to investigate the influence of high fibre diets formulated with different fibrous ingredients on performance, nutrient digestibility, diarrhoea incidence and numbers of faecal microbiota. The dietary treatments included a Control diet and five high fibre diets formulated with different fibre sources including wheat bran, soybean hulls, naked oat hulls, palm kernel expeller and bamboo fibre. The high fibre diets averaged 14.6% neutral detergent fibre with different non-starch polysaccharides (NSP) components and were fed ad libitum for 28 d. Faecal samples were collected during the last 3 d of the experiment and the apparent total tract digestibility of nutrients and fibre components were determined. Pigs fed the Control and wheat bran diets had a higher (p ≤ 0.05) average daily gain (ADG) than pigs fed the palm kernel expeller and bamboo meal diets. The reduced ADG for pigs appeared to be related to reductions in the digestibility of gross energy and dry matter, respectively. The feed-to-gain ratio was significantly higher (p ≤ 0.05) for pigs fed the fibre diets. The digestibility of NSP components was different among the treatments. The diarrhoea incidence was not affected by treatments. The abundance of faecal bifidobacteria was significantly higher (p ≤ 0.05) for pigs fed the wheat bran diet than for pigs fed the bamboo meal diet. It was concluded that the diets formulated with different fibre sources when fed to weaned piglets have different effects on pig performance, nutrient digestibility and numbers of faecal microbiota. The wheat bran diet rich in arabinoxylans enabled a better performance than the other tested diets with fibre addition.     

Effects of feeding finisher pigs with chicory or lupine feed for one week or two weeks before slaughter with respect to levels of Bifidobacteria and Campylobacter

This study aimed to assess whether inclusion of chicory or lupine (prebiotics) in the diet of pre-slaughter pigs for just 1 or 2 weeks could change the composition of their intestinal microbiota, stimulate the growth of bifidobacteria and help to lower the amount of thermoplilic Campylobacter spp. (mainly Campylobacter jejuni and Campylobacter coli), which are a major cause of food-borne infections in humans. A total of 48 pigs that had an initial live weight of 90 kg were fed with either a lupine (organic concentrate with 25% blue lupine seeds), chicory (organic concentrate with 10% dried chicory roots) or control (100% organic concentrate) diet for 1 week (24 pigs) or 2 weeks (24 pigs) before slaughter. The Campylobacter spp. level in rectal faecal samples after 0, 1 and 2 weeks of feeding and in the luminal content from ileum, caecum and colon at slaughter was determined by direct plating on modified charcoal-cefoperazone-deoxycholate agar plates. DNA extracted from the luminal content of distal ileum and caecum was used for terminal restriction fragment length polymorphism (T-RFLP) analysis of the composition of intestinal microbiota and for measuring the amount of bifidobacterial and total bacterial DNA by quantitative real-time PCR (qPCR). Campylobacter spp. were excreted by all pigs and present in the luminal content from distal ileum to midway colon with particularly high numbers in the caecum, but the excretion was reduced by 10-fold in pigs fed lupines for 1 week as compared with control- and chicory-fed pigs (mean log₁₀ 2.9 v. 4.1 CFU/g; P < 0.05). The qPCR analysis showed that feeding with lupines resulted in higher levels of bifidobacteria in caecum as compared with the other diets (P < 0.05). T-RFLP analysis showed that four of the most abundant bacteria with terminal restriction fragment values >5% relative to the intensity of total abundance differed between the feed treatments (P < 0.05). Therefore, this study showed that even a short-term alternative feeding strategy with prebiotics in the diet of pre-slaughter pigs elicited changes in the composition of the intestinal microbiota, where lupine increased the level of bifidobacteria in caecum and reduced the Campylobacter spp. excretion level after 1 week.     

Case study of the distribution of mucosa-associated Bifidobacterium species, Lactobacillus species, and other lactic acid bacteria in the human colon

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.     

Molecular monitoring of the fecal microbiota of healthy human subjects during administration of lactulose and Saccharomyces boulardii

Diet is a major factor in maintaining a healthy human gastrointestinal tract, and this has triggered the development of functional foods containing a probiotic and/or prebiotic component intended to improve the host's health via modulation of the intestinal microbiota. In this study, a long-term placebo-controlled crossover feeding study in which each subject received several treatments was performed to monitor the effect of a prebiotic substrate (i.e., lactulose), a probiotic organism (i.e., Saccharomyces boulardii), and their synbiotic combination on the fecal microbiota of three groups of 10 healthy human subjects differing in prebiotic dose and/or intake of placebo versus synbiotic. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was used to detect possible changes in the overall bacterial composition using the universal V(3) primer and to detect possible changes at the subpopulation level using group-specific primers targeting the Bacteroides fragilis subgroup, the genus Bifidobacterium, the Clostridium lituseburense group (cluster XI), and the Clostridium coccoides-Eubacterium rectale group (cluster XIVa). Although these populations remained fairly stable based on DGGE profiling, one pronounced change was observed in the universal fingerprint profiles after lactulose ingestion. Band position analysis and band sequencing revealed that a band appearing or intensifying following lactulose administration could be assigned to the species Bifidobacterium adolescentis. Subsequent analysis with real-time PCR (RT-PCR) indicated a statistically significant increase (P < 0.05) in total bifidobacteria in one of the three subject groups after lactulose administration, whereas a similar but nonsignificant trend was observed in the other two groups. Combined RT-PCR results from two subject groups indicated a borderline significant increase (P = 0.074) of B. adolescentis following lactulose intake. The probiotic yeast S. boulardii did not display any detectable universal changes in the DGGE profiles, nor did it influence the bifidobacterial levels. This study highlighted the capacity of an integrated approach consisting of DGGE analysis and RT-PCR to monitor and quantify pronounced changes in the fecal microbiota of healthy subjects upon functional food administration.     

Case Study of the Distribution of Mucosa-Associated Bifidobacterium Species, Lactobacillus Species, and Other Lactic Acid Bacteria in the Human Colon

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.     

Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota.

The porcine gastrointestinal tract (GIT) microbiota has been studied to increase production efficiency, improve product quality, and help attempt to reduce disease. During the developmental period from birth through weaning, the intestinal microbiota undergoes a rapid ecological succession. There is interest in developing a monitoring technique that allows for analysis of bacterial population levels and shifts within the pig intestine. The objective of this study was to determine if denaturant gradient gel electrophoresis (DGGE) could be effectively applied to measure changes in bacterial populations of the pig GIT, as influenced by age, diet or compartment. Bacterial genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (approximately 200 bp) obtained from primers specific for the domain Bacteria. Protocol development included optimization of: DNA extraction procedures, PCR amplification, removal of PCR artifacts, and optimization of gel preparation and image capture. DGGE analysis revealed diverse bacterial populations between pigs of different ages and among individual gut compartments. Comparison of fecal DNA from different aged pigs revealed several unique PCR product bands indicating the presence of unique bacterial populations. Comparison of different gut compartments demonstrated that bacterial populations were most similar (C, value > 50%) within a single compartment and between adjacent ones. Thus, DGGE can be used to examine bacterial diversity and population shifts in the pig GIT.     

A longitudinal study of infant faecal microbiota during weaning

For infants, the introduction of food other than breast milk is a high risk period due to diarrheal diseases, and may be corroborated with a shift in the faecal microbiota. This longitudinal study was the first undertaken to understand the effect of the supplementation on the infant's faecal microbiota and particularly the bifidobacteria. Eleven infants were enrolled. Their faecal microbiota were analysed using temporal temperature gradient gel electrophoresis (TTGE) with bacterial and bifidobacterial primers. In parallel, bifidobacterial counts were followed using competitive PCR. Three periods were distinguished: exclusive breastfeeding (Bf period), weaning (i.e. formula-milk addition, W period) and postweaning (i.e. breastfeeding cessation, Pw period). The bifidobacterial counts were not modified, reaching 10.5 (Log10 cells g(-1) wet weight). In the TTGE profiles, the main identified bands corresponded to Escherichia coli, Ruminococcus sp. and Bifidobacterium sp., more precisely Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium breve. For both TTGE profiles, the analysis of the distance suggested a maturation of the faecal microbiota but no correlation could be established with the diet. Despite a high interindividual variability, composition of the faecal microbiota appeared more homogenous after weaning and this point may be correlated with the cessation of breastfeeding.     

PCR/DGGE and 16S rRNA gene library analysis of the colonic microbiota of HLA-B27/beta2-microglobulin transgenic rats

AIMS: To determine the phylogenetic composition of the colonic microbiota of transgenic (TG) HLA-B27 rats using 16S ribosomal RNA (rRNA) gene sequences obtained from denaturing gradient gel electrophoresis (DGGE) gels and sequences from a 16S rRNA gene library. METHODS AND RESULTS: Colonic microbiota of TG and nontransgenic (NT) rats harboured by 10-week-old and 6-month-old animals was screened using PCR/DGGE. Six months old TG rats had marked inflammation of the colon compared with 10-week-old TG and NT rats. The DGGE profiles of rats with inflamed colon were similar from rat to rat (Dice's Similarity Coefficient proximal colon 73%, distal colon 83%) whereas profiles from animals without inflammation were dissimilar (52-64%). Identifications of bacterial origins of 16S rRNA gene sequences obtained from DGGE gels (200 bp) and from 16S rRNA clones (450 bp) of the colonic microbiota of diseased rats gave sequences most closely phylogenetically affiliated with uncultured or unknown bacteria. CONCLUSIONS: PCR/DGGE was shown to be an effective method to compare the colonic microbiota composition of TG and NT rats relative to the progression of inflammatory disease. Sequencing of 16S rRNA gene fragments from DGGE gels or 16S rRNA gene clones from a random library showed that uncultured or unknown bacteria were most commonly detected by both methods. It can be concluded that it would be better in future studies to search for the antigens produced by the gut microbiota against which the dysfunctional immune system reacts rather than seek phylogenetic associations. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR/DGGE can be used as a rapid initial screening method to compare the composition of bacterial communities of initially unknown composition that are associated with the development of intestinal disease.     

Extrusion of wheat or sorghum and/or addition of exogenous enzymes to pig diets influences the large intestinal microbiota but does not prevent development of swine dysentery following experimental challenge

A study was made of dietary influences on the large intestinal microbiota of pigs and on the incidence of swine dysentery (SD) after experimental infection with Brachyspira hyodysenteriae, the aetiological agent of SD. Animals were fed diets based either on wheat (expts 1 and 2) or sorghum (expt 2). Grains were ground and fed either raw or after high temperature and pressure extrusion and/or after addition of exogenous enzymes to the whole diet to reduce the starch and soluble non-starch polysaccharides available for fermentation in the large intestine. Limiting fermentation creates conditions that apparently reduce the incidence of SD after infection with B. hyodysenteriae. The diets were fed to weaned pigs for 4-6 weeks, then half the animals on each diet were killed and gut samples collected for microbiology. The treatments had little effect on bacterial numbers. In expt 1, dietary extrusion of wheat reduced lactobacilli in the large intestine. Addition of enzymes to extruded wheat-based diets in expt 2 reduced facultative anaerobes and increased non-sporing anaerobes. Addition of enzymes to a raw sorghum diet in expt 3 decreased numbers of facultative anaerobes, while extrusion of sorghum increased total anaerobes. Bacteroides spp. and Fusobacterium spp., which act in synergy with B. hyodysenteriae in SD, were isolated at a higher percentage in pigs fed the untreated wheat diet than in pigs fed the treated wheat diets. Following experimental infection the incidence of SD amongst pigs fed treated wheat diets was slightly lower than those fed the untreated diet, but with sorghum-based diets the opposite was found. Overall, the different dietary treatments used did not significantly reduce SD.     

Phylotypes related to Ruminococcus bromii are abundant in the large bowel of humans and increase in response to a diet high in resistant starch

To further understand how diets containing high levels of fibre protect against colorectal cancer, we examined the effects of diets high in nonstarch polysaccharides (NSP) or high in NSP plus resistant starch (RS) on the composition of the faecal microbial community in 46 healthy adults in a randomized crossover intervention study. Changes in bacterial populations were examined using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Bacterial profiles demonstrated changes in response to the consumption of both RS and NSP diets [analysis of similarities (ANOSIM): R=0.341-0.507, P<0.01]. A number of different DGGE bands with increased intensity in response to dietary intervention were attributed to as-yet uncultivated bacteria closely related to Ruminococcus bromii. A real-time PCR assay specific to the R. bromii group was applied to faecal samples from the dietary study and this group was found to comprise a significant proportion of the total community when individuals consumed their normal diets (4.4+/-2.6% of total 16S rRNA gene abundance) and numbers increased significantly (+/-67%, P<0.05) with the RS, but not the NSP, dietary intervention. This study indicates that R. bromii-related bacteria are abundant in humans and may be significant in the fermentation of complex carbohydrates in the large bowel.     

Secretor genotype (FUT2 gene) is strongly associated with the composition of Bifidobacteria in the human intestine

Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa.     

Faecal and caecal microbiota profiles of mice do not cluster in the same way

Polymerase chain reaction (PCR)-based denaturation gradient gel electrophoresis (DGGE) is currently being used for characterizing the composition of the gut microbiota (GM) of mice in order to better control the study variation arising from the GM. At present, faeces are commonly sampled from live animals, while caecum is most commonly sampled from terminated animals. However, there is no knowledge whether the composition at the one site is representative for the other. In this study C57BL/6 mice were observed from the age of four weeks until the age of 10 weeks. Faeces were sampled weekly. Caecum was sampled surgically under anaesthesia and with subsequent ampicillin treatment at the age of six weeks and again after euthanasia at the age of 10 weeks. Faecal and caecal microbiota profiles were determined using DGGE and subjected to subsequent cluster analysis. The mice subjected to surgical caecal sampling clustered separately for two weeks after termination of antibiotics after which they again clustered with the non-surgically sampled mice. Faecal and caecal profiles clustered separately at the age of six weeks, but not at the age of 10 weeks. There were no correlations between faecal or caecal profiles at six or 10 weeks of age, respectively. It is concluded that faecal and caecal microbiota profiles are not representative of each other in mice. Therefore, it is recommendable in studies to sample from several sites specifically decided in relation to the specific model of a study.     

Molecular Monitoring of the Fecal Microbiota of Healthy Human Subjects during Administration of Lactulose and Saccharomyces boulardii

Diet is a major factor in maintaining a healthy human gastrointestinal tract, and this has triggered the development of functional foods containing a probiotic and/or prebiotic component intended to improve the host's health via modulation of the intestinal microbiota. In this study, a long-term placebo-controlled crossover feeding study in which each subject received several treatments was performed to monitor the effect of a prebiotic substrate (i.e., lactulose), a probiotic organism (i.e., Saccharomyces boulardii), and their synbiotic combination on the fecal microbiota of three groups of 10 healthy human subjects differing in prebiotic dose and/or intake of placebo versus synbiotic. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was used to detect possible changes in the overall bacterial composition using the universal V₃ primer and to detect possible changes at the subpopulation level using group-specific primers targeting the Bacteroides fragilis subgroup, the genus Bifidobacterium, the Clostridium lituseburense group (cluster XI), and the Clostridium coccoides-Eubacterium rectale group (cluster XIVa). Although these populations remained fairly stable based on DGGE profiling, one pronounced change was observed in the universal fingerprint profiles after lactulose ingestion. Band position analysis and band sequencing revealed that a band appearing or intensifying following lactulose administration could be assigned to the species Bifidobacterium adolescentis. Subsequent analysis with real-time PCR (RT-PCR) indicated a statistically significant increase (P < 0.05) in total bifidobacteria in one of the three subject groups after lactulose administration, whereas a similar but nonsignificant trend was observed in the other two groups. Combined RT-PCR results from two subject groups indicated a borderline significant increase (P = 0.074) of B. adolescentis following lactulose intake. The probiotic yeast S. boulardii did not display any detectable universal changes in the DGGE profiles, nor did it influence the bifidobacterial levels. This study highlighted the capacity of an integrated approach consisting of DGGE analysis and RT-PCR to monitor and quantify pronounced changes in the fecal microbiota of healthy subjects upon functional food administration.     

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.