Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192^T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.     

Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium species

Fecal microbiota of 31 breast-fed, 26 mix-fed, and 11 bottle-fed infants were analyzed by using terminal restriction fragment length polymorphism (T-RFLP), and culture method. We first determined the total and cultivated bacterial counts in infant fecal microbiota. Only approximately 30% of bacteria present in fecal microbiota were cultivable while the remainder was yet-to-be cultured bacteria. Sixty-eight fecal samples were divided into two clusters (I and II) by T-RFLP analysis, and then subdivided into five subclusters (Ia, Ib, IIa, IIb and IIc). There was no clear relationship between clusters and feeding method. A proportion of bifidobacteria was detected in the fecal material by PCR method using species-specific primers. The predominant Bifidobacterium spp. was Bifidobacterium longum longum type (43 samples (63.2%)), followed by B. longum infantis type (23 samples (33.8%)) and B. breve (16 samples (23.5%)). The distribution of Bifidobacterium spp. was similar in the three feeding groups. In contrast, the high incidence of B. breve in cluster I, especially subcluster Ia and B. longum longum type in cluster II, especially subcluster IIa and IIc were characterized by T-RFLP method. Our results showed that the colonization of Bifidobacterium spp. in infant feces correlated with the T-RFLP clusters.     

Population dynamics of Bifidobacterium species in human feces during raffinose administration monitored by fluorescence in situ hybridization-flow cytometry

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.     

Increased amount of Bifidobacterium thermacidophilum and Megasphaera elsdenii in the colonic microbiota of pigs fed a swine dysentery preventive diet containing chicory roots and sweet lupine

AIMS: To investigate which specific bacterial species that were stimulated or inhibited in the proximal colon of pigs when a fructan-rich diet was compared with a diet that contained resistant carbohydrates. The study focussed especially on Bifidobacterial species by using a noncultureable approach. METHODS AND RESULTS: Terminal restriction fragment length polymorphism (T-RFLP) was used to describe differences in the total colonic microbiota as well as in the populations of Bifidobacterium spp. in pigs fed with a fructan-rich diet and a diet containing resistant carbohydrates. The fructan-rich diet has previously been shown to prevent swine dysentery caused by Brachyspira hyodysenteriae. The T-RFLP profiling, 16S rRNA gene cloning and in situ hybridization showed that the pigs fed with the fructan-rich diet had a higher proportion of Bifidobacterium thermacidophilum subsp. porcinum and Megasphaera elsdenii. CONCLUSIONS: These findings suggested that the bacterial fructan fermentation occurring in the porcine colon might be cross-feeding of lactate produced by B. thermacidophilum and used by M. elsdenii. SIGNIFICANCE AND IMPACT OF THE STUDY: B. thermacidophilum and M. elsdenii may be the course of the inhibition of the pathogenic bacteria Brach. hyodysenteriae in colon of pigs when they are fed fructan-rich diets.     

Modifying effects of fermented brown rice on fecal microbiota in rats

Brown rice fermented by Aspergillus oryzae (FBRA) is a fiber-rich food. Effects of dietary administration of FBRA on rat fecal microbiota composition were examined. Male Wistar rats were fed a basal diet or a 5% FBRA- or 10% FBRA-containing diet, and fecal microbiota was analyzed by culture and terminal-restriction fragment length polymorphism (T-RFLP) analysis. The viable cell number of lactobacilli significantly increased after feeding 10% FBRA diet for 3 weeks compared with that in the basal diet group and that in the same group at the beginning of the experiment (day 0). An increase in the viable cell number of lactobacilli was also observed after feeding 10% FBRA for 12 weeks compared with the effect of a basal diet. T-RFLP analysis showed an increase in the percentage of lactobacilli cells in feces of rats fed 10% FBRA for 14 weeks. Lactobacilli strains isolated from rat feces were divided into six types based on their randomly amplified polymorphic DNA (RAPD) patterns, and they were identified as Lactobacillus reuteri, L. intestinalis and lactobacilli species based on homology of the partial sequence of 16S rDNA. FBRA contains lactic acid bacteria, but their RAPD patterns and identified species were different from those in rat feces. These results indicated that dietary FBRA increases the number of lactobacilli species already resident in the rat intestine.     

Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries

The maturation of murine cecal microbiota was determined by terminal restriction fragment polymorphism (T-RFLP) and 16S rRNA gene clone libraries. Cecal microbiota in specific pathogen free (SPF) mice aged four to 10 weeks were collected. The cluster of samples in 4-week-old mice was different from those of other ages based on T-RFLP profiles. The majority of clones obtained in this study belonged to the Clostridium coccoides (C. coccoides) group, the Bacteroides group or the Lactobacillus group. Phylogenetic analysis showed characteristic clusters composed of new operational taxonomic unit (OTU) of the C. coccoides and Bacteroides groups. The existence of a large number of yet unidentified bacteria inhabiting the murine cecum was demonstrated by 16S rRNA gene clone libraries. T-RFLP analysis data were more complex and more sensitive than the patterns generated by computer simulation of 16S rRNA gene clone library analysis data. T-RFLP revealed development with maturation of cecal microbiota including unidentified bacteria of SPF mice.     

Nutritional impacts of dietary oregano and Enviva essential oils on the performance,gut microbiota and blood biochemicals of growing ducks

Nowadays, there is much legislation in the world devoted to restrict the use of synthetic antibiotics in the poultry industry, which could reduce performance rate and production profits. Various phyto-biotic growth promoters have been proposed to serve as antibiotic alternatives with emphasis on plant extracts and essential oils. This study was conducted to assess the impacts of using the oregano essential oil (OEO) (comprised of 5% thymol and 65% carvacrol) and Enviva essential oil (EEO) (4.5% cinnamaldehyde and 13.5% thymol) as phytobiotic feed additives (PFA) on growth performance, cecal microbiota and serum biochemicals of growing ducks. In total, 800 11-day-old ducklings, housed in 20 floor pens, were allotted randomly into five dietary treatments: (i) A basal diet (BD) (control), (ii.) BD+50 mg EEO/kg, (iii.) BD+100 mg EEO/kg, (iv.) BD+150 mg OEO/kg and (v.) BD+300 mg OEO/kg diet. The growth performance traits were studied between 11 and 42 days of age. At the experiment end, 40 ducks were slaughtered (eight/ treatment) and cecal digesta and blood samples were collected to estimate the cecal bacterial populations and serum blood biochemicals. The results indicated that the tested levels of OEO and EEO did not display any significant effect (P>0.05) on the duck’s final BW, BW gain, growth rate, feed intake, feed conversion ratio or survivability rate. Besides, the different levels of EEO and OEO decreased the cecal populations of Coliforms (P<0.01), total aerobes (P<0.01) and lactose-negative Enterobacteria (P<0.05) in comparison with those of the control group. Finally, the tested EEO and OEO levels did not show any significant effect on the serum variables; in terms of total protein, albumin, globulin, total cholesterol, triglycerides, alanine aminotransferase and aspartate aminotransferase. In conclusion, the antimicrobial effect of the OEO and EEO against the cecal microbiota has been proven, while they did not display significant effects on the growth performance or blood variables of growing ducks.     

Complete genome sequence of Bifidobacterium breve CECT 7263, a strain isolated from human milk

Bifidobacterium breve is an actinobacterium frequently isolated from colonic microbiota of breastfeeding babies. Here, we report the complete and annotated genome sequence of a B. breve strain isolated from human milk, B. breve CECT 7263. The genome sequence will provide new insights into the biology of this potential probiotic organism and will allow the characterization of genes related to beneficial properties.     

In vitro effects of selected synbiotics on the human faecal microbiota composition

Synbiotics are recognized means of modulating gut microbiota composition and activities. However, whether synbiotics are superior to prebiotics and probiotics alone in moderating the gut microbiota towards a purportedly healthy composition has not been determined. Eight selected synbiotics (short-chain fructooligosaccharides or fructooligosaccharides, each combined with one of four probiotics, Lactobacillus fermentum ME-3, Lactobacillus plantarum WCFS1, Lactobacillus paracasei 8700:2 or Bifidobacterium longum 46) were added to 24-h pH-controlled anaerobic faecal batch cultures. The prebiotic and probiotic components were also tested alone to determine their respective role within the synbiotic for modulation of the faecal microbiota. Effects upon major groups of the microbiota were evaluated using FISH. Rifampicin variant probiotic strains were used to assess probiotic levels. Synbiotic and prebiotics increased bifidobacteria and the Eubacterium rectale-Clostridium coccoides group. Lower levels of Escherichia coli were retrieved with these combinations after 5 and 10 h of fermentation. Probiotics alone had little effect upon the groups, however. Multivariate analysis revealed that the effect of synbiotics differed from the prebiotics as higher levels of Lactobacillus-Enterococcus were observed when the probiotic was stimulated by the prebiotic component. Here, the synbiotic approach was more effective than prebiotic or probiotic alone to modulate the gut microbiota.     

Increased resistance of mice to Salmonella enterica serovar Typhimurium infection by synbiotic administration of Bifidobacteria and transgalactosylated oligosaccharides

AIMS: The anti-infectious activity of Bifidobacteria in combination with transgalactosylated oligosaccharides (TOS) against Salmonella enterica serovar Typhimurium LT-2 in an opportunistic antibiotic-induced murine infection model in mice was examined. METHODS AND RESULTS: B. breve (strain Yakult) with natural resistance to streptomycin sulphate (SM, MIC: > 4 mg ml(-1)), when given daily at a dose of 108 cfu/mouse orally under SM treatment was constantly excreted at 10(10) cfu g(-1) faeces so long as SM was administered, even at 2 weeks after discontinuing administration of B. breve. Explosive intestinal growth and subsequent extra-intestinal translocation of orally infected LT-2 under SM treatment were inhibited by B. breve colonization, and this anti-infectious activity was strengthened by synbiotic administration of TOS with B. breve. Comparison of anti-Salmonella activity among several Bifidobacterium strains with natural resistance to SM revealed that strains such as B. bifidum ATCC 15696 and B. catenulatum ATCC 27539T conferred no activity, even when they reached high population levels similar those of effective strains such as strain Yakult and B. pseudocatenulatum DSM 20439. Both the increase in the concentration of organic acids and the lowered pH in the intestine due to bifidobacterial colonization correlated with the anti-infectious activity. Moreover, the crude cecal extract of B. breve-colonized mice exerted growth-inhibitory activity against LT-2 in vitro, whereas that of the ineffective B. bifidum-colonized cecum showed much lower activity. CONCLUSIONS: Intestinal colonization by bifidobacteria given exogenously together with TOS during antibiotic treatment prevents the antibiotic-induced disruption of colonization resistance to oral infection with S. enterica serovar Typhimurium, and the metabolic activity needed to produce organic acids and lower the intestinal pH is important in the anti-infectious activity of synbiotics against enteric infection with Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that certain bifidobacteria together with prebiotics may be used for the prophylaxis against opportunistic intestinal infections with antibiotic-resistant pathogens.     

Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Terminal restriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize and compare human fecal microbiota among individuals. T-RFLP patterns of fecal 16S ribosomal DNA (rDNA) PCR products from three adults revealed host-specific bacterial communities and were in good agreement with those reported in our previous study. In addition, we applied T-RFLP analysis for the analysis of complex bifidobacterial communities in human fecal samples. The developed method based on Bifidobacterium genus-specific PCR and T-RFLP could identify more than one bifidobacterial species. T-RFLP patterns of Bifidobacterium genus-specific PCR products from the fecal samples were host-specific as well as those of fecal 16S rDNA PCR products. These results were confirmed by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the genus Bifidobacterium and Bifidobacterium species- and group-specific PCR. Our study demonstrates that T-RFLP analysis is useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals, and that the applied method is useful for rapid and sensitive analysis of bifidobacterial community.     

Alterations and structural resilience of the gut microbiota under dietary fat perturbations

Disequilibrium of the gut microbiota by dietary fat has been implicated in the incidence of overweight or obesity. However, it remains to be elucidated whether dietary fat perturbations in early life have long-lasting impacts on the gut microbiota and to what extent unbalanced diet-induced alterations in childhood are reversible. Accordingly, three groups of 1-day-old hens were used. They were fed with a low-fat diet (LFD), basal diet (BD) and high-fat diet (HFD), respectively, for 6 weeks and then switched to the same normal diets (NDs) for another 19 weeks. At week 6, hens in the LFD and HFD groups were found to have higher body weight, plasma glucose, total cholesterol, triglycerides and low-density lipoprotein cholesterol levels than their counterparts in the BD group, whereas upon switching to NDs, the metabolic deteriorations observed during the LFD consumption were alleviated. Principal component analysis revealed a shift of the gut microbiota structure in the LFD and HFD groups away from that of the BD group at week 6, while the gut microbiota structure of the LFD group was moved back to that of the BD group after reverting to NDs. Additionally, abnormal alterations of obesity-related phylotypes were observed in the LFD and HFD groups, whereas the abundance of these phylotypes in the LFD group was almost reverted to the BD levels over time. Collectively, dietary fat perturbations in early life have long-term impacts on hosts, and the structural resilience of the gut microbiota in hens fed with HFD was lower than that in their LFD counterparts.     

Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-enriched inulin on colonic nitrogen-protein metabolism in healthy humans

Pre- and/or probiotics can cause changes in the ecological balance of intestinal microbiota and hence influence microbial metabolic activities. In the present study, the influence of oligofructose-enriched inulin (OF-IN), Lactobacillus casei Shirota, and Bifidobacterium breve Yakult on the colonic fate of NH3 and p-cresol was investigated. A randomized, placebo-controlled, crossover study was performed in 20 healthy volunteers to evaluate the influence of short- and long-term administration of OF-IN, L. casei Shirota, B. breve Yakult, and the synbiotic L. casei Shirota + OF-IN. The lactose[15N,15N]ureide biomarker was used to study the colonic fate of NH3. Urine and fecal samples were analyzed for 15N content by combustion-isotope ratio mass spectrometery and for p-cresol content by gas chromatography-mass spectrometry. RT-PCR was applied to determine the levels of total bifidobacteria. Both short- and long-term administration of OF-IN resulted in significantly decreased urinary p-cresol and 15N content. The reduction of urinary 15N excretion after short-term OF-IN intake was accompanied by a significant increase in the 15N content of the fecal bacterial fraction. However, this effect was not observed after long-term OF-IN intake. In addition, RT-PCR results indicated a significant increase in total fecal bifidobacteria after long-term OF-IN intake. Long-term L. casei Shirota and B. breve Yakult intake showed a tendency to decrease urinary 15N excretion, whereas a significant decrease was noted in p-cresol excretion. In conclusion, dietary addition of OF-IN, L. casei Shirota, and B. breve Yakult results in a favorable effect on colonic NH3 and p-cresol metabolism, which, in the case of OF-IN, was accompanied by an increase in total fecal bifidobacteria.     

Quantitative assessment of faecal bifidobacterial populations by real-time PCR using lanthanide probes

AIM: To develop real-time quantitative PCR methods, based on the use of probes labelled with a stable fluorescent lanthanide chelate, for the quantification of different human faecal bifidobacterial populations. METHODS AND RESULTS: The designed quantitative PCR assays were found to be specific for the corresponding Bifidobacterium species or groups (Bifidobacterium longum group, Bifidobacterium catenulatum group, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium angulatum, Bifidobacterium bifidum and Bifidobacterium dentium). The detection limits of the methodologies used ranged between 2 x 10(5) and 9 x 10(3) cells g(-1) of faeces. The applicability of the developed assays was tested by analysing 20 human faecal samples. Bif. longum group was found to be the qualitatively and quantitatively predominant bifidobacterial group. CONCLUSIONS: The real-time PCR procedures developed here are specific, accurate, rapid and easy methods for the quantification of Bifidobacterium groups or species in human faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed procedures will facilitate rapid and objective counting of large numbers of samples increasing our knowledge on the role of gut bifidobacterial microbiota in health and disease. This will contribute to the efficient use of intestinal bacterial assays in research, food and pharmaceutical development as well as in the assessment of dietary management of diseases.     

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.     

The Effect of Selected Synbiotics on Microbial Composition and Short-Chain Fatty Acid Production in a Model System of the Human Colon

Background

Prebiotics, probiotics and synbiotics can be used to modulate both the composition and activity of the gut microbiota and thereby potentially affecting host health beneficially. The aim of this study was to investigate the effects of eight synbiotic combinations on the composition and activity of human fecal microbiota using a four-stage semicontinuous model system of the human colon.

Methods and Findings

Carbohydrates were selected by their ability to enhance growth of the probiotic bacteria Lactobacillus acidophilus NCFM (NCFM) and Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04) under laboratory conditions. The most effective carbohydrates for each probiotic were further investigated, using the colonic model, for the ability to support growth of the probiotic bacteria, influence the composition of the microbiota and stimulate formation of short-chain fatty acids (SCFA).The following combinations were studied: NCFM with isomaltulose, cellobiose, raffinose and an oat β-glucan hydrolysate (OBGH) and Bl-04 with melibiose, xylobiose, raffinose and maltotriose. All carbohydrates showed capable of increasing levels of NCFM and Bl-04 during fermentations in the colonic model by 10³–10⁴ fold and 10–10² fold, respectively. Also the synbiotic combinations decreased the modified ratio of Bacteroidetes/Firmicutes (calculated using qPCR results for Bacteroides-Prevotella-Porphyromonas group, Clostridium perfringens cluster I, Clostridium coccoides - Eubacterium rectale group and Clostridial cluster XIV) as well as significantly increasing SCFA levels, especially acetic and butyric acid, by three to eight fold, as compared to the controls. The decreases in the modified ratio of Bacteroidetes/Firmicutes were found to be correlated to increases in acetic and butyric acid (p = 0.04 and p = 0.03, respectively).

Conclusions

The results of this study show that all synbiotic combinations investigated are able to shift the predominant bacteria and the production of SCFA of fecal microbiota in a model system of the human colon, thereby potentially being able to manipulate the microbiota in a way connected to human health.     

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.     

Evaluation of three different forward primers by terminal restriction fragment length polymorphism analysis for determination of fecal bifidobacterium spp. in healthy subjects

The 27F forward primer is frequently used in 16S rRNA gene libraries and T-RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T-RFLP method with three different forward primers (27F, 35F, and 529F) in conjunction with one reverse primer (1492R). T-RFLP analysis of fecal microbiota using 35F and 529F detected higher proportions of the terminal restriction fragment (T-RF) corresponding to Bifidobacterium spp. than that using 27F. 27F is in imperfect agreement while 35F and 529F are in good concordance with the 16S rRNA gene sequences of Bifidobacterium spp., and the latter primers allowed for the detection of T-RFs of Bifidobacterium spp. in fecal samples from five healthy subjects. The T-RFs presumed to be Bifidobacterium spp. were cloned and sequenced, and found to match the 16S rRNA gene sequences of Bifidobacterium spp. Among the five fecal samples, two samples with low frequencies of T-RFs of Bifidobacterium spp. were detected using these forward primers. This probably reflects a low prevalence of Bifidobacterium spp. in these two samples. Our study emphasizes the importance of selecting a suitable forward primer for detection of Bifidobacterium spp.