Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis Monitoring of Fecal Bifidobacterium Populations in a Prebiotic and Probiotic Feeding Trial

A culture-independent approach based on genus-specific PCR and denaturing gradient gel electrophoresis (DGGE) was used to monitor qualitative changes in fecal bifidobacterial communities in a human feeding trial. DNA was extracted directly from feces and bifidobacterial 16S rDNA sequences were amplified using genus-specific PCR. The PCR fragments were subsequently separated in a sequence-specific manner by DGGE in order to obtain a profile of bifidobacterial fragments. The DGGE profiles revealed that in general, administration for two weeks of galactooligosaccharide and/or Bifidobacterium lactis Bb-12 (8 g and 3 × 10¹⁰ cfu per day, respectively) did not affect the qualitative composition of the indigenous Bifidobacterium population, while B. lactis Bb-12 transiently colonised the gut.     

Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074^T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.     

Intestinal bacterial population of healthy rats during the administration of chitosan and chitooligosaccharides

The effect of the administration of chitosan (CS) and chitooligosaccharides (COS) on rat fecal microbiota was analyzed in this study. The profile of total bacterial population was monitored during 3 weeks of CS or COS application using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons. Quantitative PCR was used for monitoring possible changes in the levels of total bacteria and the levels of individual bacterial groups: Bifidobacteria, Clostridium leptum, Enterobacteriaceae, Lactobacillus–Streptococcus–Enterobacter, and Bacteroides–Prevotella. The DGGE profiles revealed a high complexity and individuality of each tested subject, and variations in the composition of band pattern were observed. CS or COS per os administration changed the profile and structure of the microbial ecosystem of the gastrointestinal tract of healthy rats. COS have, in most cases, an opposite effect compared with CS; only the Bacteroides–Prevotella bacterial group and Enterobacteriaceae were influenced in the same way. The Bifidobacteria group was not influenced by the administration CS and COS.     

3-(6-Chloronicotinyl)-2-nitromethylene-thiazolidine as a New Class of Insecticide Acting against Lepidoptera Species

The growth responses of a variety of human intestinal bacteria to partially hydrolyzed guar gum (PHGG) were investigated in vitro and in vivo. In an in vitro experiment, PHGG moderately enhanced growth of some bacterial strains including Bacteroides ovatus, Clostridium coccoides, C. butyricum, and Peptostreptococcus productus.

Effects of PHGG intake (7 g/volunteer, 3 times per day, for 14 days) on fecal microflora, bacterial metabolites, and pH were investigated using nine healthy human volunteers. The count of Bifidobacterium spp. and the percentage of these species in the total count increased significantly during the PHGG intake periods. Among the acid-forming bacteria, Lactobacillus spp. also increased. The fecal pH and fecal bacterial metabolites such as β-glucuronidase activity, putrefactive products, and ammonia content were significantly decreased by PHGG intake. Two weeks after the end of PHGG intake, the bacterial counts and their biological manifestations appeared to return to the former state.     

Effects of green tea consumption on human fecal microbiota with special reference to Bifidobacterium species

Green tea is one of the most popular beverages in the world. Its beneficial health effects and components have been extensively reviewed. However, little is known about the influence of green tea consumption on the human intestinal microbiota (HIM), which plays a crucial role in human health. Ten volunteers who did not usually consume green tea, drank it for 10 days and then stopped drinking it for 7 days. Their fecal samples were collected at three time points: before beginning the 10‐day green‐tea regime, at the conclusion of that 10 days, and 7 days after stopping the regime. Their fecal samples were analyzed by terminal restriction fragment length polymorphism with specific primer‐restriction enzyme systems for HIM and by using a real‐time PCR method for the Bifidobacterium species. Although the HIM of each subject was relatively stable, the proportion of Bifidobacterium species played an important role in the classification of their fecal microbiota. Although there were inter‐individual differences in the Bifidobacterium species, an overall tendency for the proportion of bifidobacteria to increase because of green tea consumption was noted. However, little change was observed in the composition of Bifidobacterium species in each sample. This suggests that the change in proportion was induced, not by an inter‐species transition, but by an intra‐species increase and/or decrease. In conclusion, green tea consumption might act as a prebiotic and improve the colon environment by increasing the proportion of the Bifidobacterium species.     

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.     

The Development and Stability of the Genus <Emphasis Type="Italic">Bacteriodes</Emphasis> from Human Gut Microbiota in HFA Mice Model

Human flora-associated (HFA) mice are frequently applied in studying the ecology and metabolism of human gut microbiota. However, the development and stability of the genus Bacteriodes, a prominent bacteria group of human gut microbiota, in HFA mice have not yet fully been examined. In this study, PCR-denaturing gradient gel electrophoresis (DGGE) analysis was employed to monitor the Bacteriodes community in the fecal microbiota of six HFA Kunming mice during a period of 3 weeks. Based on the DGGE banding patterns, the majority of prominent bands in the HFA mice DGGE profile were also typical bands in the human DGGE profile, despite the absence of three bands (corresponding to two different B. thetaiotaomicron strains and one B. intestinalis strain) from the human DGGE profile. The Dice coefficient of similarity for the fecal microbiota of HFA mice in comparison to the human donor sample ranged between 74 ± 6% and 81 ± 7%. The phylogeny of bands in the DGGE profile showed that the dominant Bacteriodes species in the fecal microbiota of HFA mice were B. thetaiotaomicron, representing 66.7% of all bands. Our results indicate that the genus Bacteriodes in the fecal microbiota of HFA mice was selected from the human donor and could remain relatively stable over time.     

Effect of apple intake on fecal microbiota and metabolites in humans

The effects of apple intake on the fecal flora, water content, pH, and metabolic activities in eight healthy volunteers and the utilization of apple pectin in vitro were investigated. Although several isolates of Bifidobacterium, Lactobacillus, Enterococcus, and the Bacteroides fragilis group utilized apple pectin, most isolates of Escherichia coli, Collinsela aerofaciense, Eubacterium limosum, and Clostridium perfringens could not. When fecal samples from healthy adults were incubated in liquid broth with apple pectin present or absent, the numbers of Bifidobacterium and Lactobacillus in the former were higher than those in the later. After the intake of apples (2 apples a day for 2 weeks) by eight healthy adult humans, the number of bifidobacteria in feces increased (p < 0.05 on day 7 and p < 0.01 on day 14 of the intake period), and the numbers of Lactobacillus and Streptococcus including Enterococcus tended to increase. However, lecithinase-positive clostridia, including C. perfringens, decreased (p < 0.05), and Enterobacteriaceae and Pseudomonas tended to decrease. Moreover, the concentrations of fecal acetic acid tended to increase on apple intake. The fecal ammonia concentration showed a tendency to reduce and fecal sulfide decreased (p < 0.05) on apple intake. These findings indicate that apple consumption is related to an improved intestinal environment, and apple pectin is one of the effective apple components improving the fecal environment.     

Microbiota profile in feces of breast- and formula-fed newborns by using fluorescence in situ hybridization (FISH)

The development of the gut is controlled and modulated by different interacting mechanisms such as, genetic endowment, intrinsic biological regulatory functions, environment influences and last but no least, the diet influence. Considered together with other endogenous and exogenous factors the type of feeding may interfere greatly in the regulation of the intestinal microbiota. During the last years molecular methods offer a complementarity to the classic culture-based knowledge. FISH has been applied for molecular evaluation of the microbiota in newborns delivered by vaginal delivery. Eleven probes/probe combinations for specific groups of faecal bacteria were used to determine the bacterial composition in faecal samples of newborns infants under different types of feeding. Breast-fed infants harbor a fecal microbiota by more than two times increased in numbers of Bifidobacterium cells when compared to formula-fed infants. After formula-feeding, Atopobium was found in significant counts and the numbers of Bifidobacterium dropped followed by increasing numbers in Bacteroides population. Moreover, under formula feeding the infants microbiota was more diverse.     

Comparative genomics of Bifidobacterium species isolated from marmosets and humans

The genus Bifidobacterium is purported to have beneficial consequences for human health and is a major component of many gastrointestinal probiotics. Although species of Bifidobacterium are generally at low relative frequency in the adult human gastrointestinal tract, they can constitute high proportions of the gastrointestinal communities of adult marmosets. To identify genes that might be important for the maintenance of Bifidobacterium in adult marmosets, ten strains of Bifidobacterium were isolated from the feces of seven adult marmosets, and their genomes were sequenced. There were six B. reuteri strains, two B. callitrichos strains, one B. myosotis sp. nov. and one B. tissieri sp. nov. among our isolates. Phylogenetic analysis showed that three of the four species we isolated were most closely related to B. bifidum, B. breve and B. longum, which are species found in high abundance in human infants. There were 1357 genes that were shared by at least one strain of B. reuteri, B. callitrichos, B. breve, and B. longum, and 987 genes that were found in all strains of the four species. There were 106 genes found in B. reuteri and B. callitrichos but not in human bifidobacteria, and several of these genes were involved in nutrient uptake. These pathways for nutrient uptake appeared to be specific to Bifidobacterium from New World monkeys. Additionally, the distribution of Bifidobacterium in fecal samples from captive adult marmosets constituted as much as 80% of the gut microbiome, although this was variable between individuals and colonies. We suggest that nutrient transporters may be important for the maintenance of Bifidobacterium during adulthood in marmosets.     

Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

A large number of commensal bacteria inhabit the intestinal tract, and interbacterial communication among gut microbiota is thought to occur. In order to analyze symbiotic relationships between probiotic strains and the gut microbiota, a ring with a membrane filter fitted to the bottom was used for in vitro investigations. Test strains comprising probiotic nitto strains (Lactobacillus acidophilus NT and Bifidobacterium longum NT) and type strains (L. acidophilus JCM1132^T and B. longum JCM1217^T) were obtained from diluted fecal samples using the membrane filter to simulate interbacterial communication. Bifidobacterium spp., Streptococcus pasteurianus, Collinsella aerofaciens, and Clostridium spp. were the most abundant gut bacteria detected before coculture with the test strains. Results of the coculture experiments indicated that the test strains significantly promote the growth of Ruminococcus gnavus, Ruminococcus torques, and Veillonella spp. and inhibit the growth of Sutterella wadsworthensis. Differences in the relative abundances of gut bacterial strains were furthermore observed after coculture of the fecal samples with each test strain. Bifidobacterium spp., which was detected as the dominant strain in the fecal samples, was found to be unaffected by coculture with the test strains. In the present study, interbacterial communication using bacterial metabolites between the test strains and the gut microbiota was demonstrated by the coculture technique. The detailed mechanisms and effects of the complex interbacterial communications that occur among the gut microbiota are, however, still unclear. Further investigation of these relationships by coculture of several fecal samples with probiotic strains is urgently required.     

Changes of Fecal <Emphasis Type="Italic">Bifidobacterium</Emphasis> Species in Adult Patients with Hepatitis B Virus-Induced Chronic Liver Disease

The beneficial effects of Bifidobacteria on health have been widely accepted. Patients with chronic liver disease have varying degrees of intestinal microflora imbalance with a decrease of total Bifidobacterial counts. Since different properties have been attributed to different Bifidobacterium species and there is no information available for the detailed changes in the genus Bifidobacterium in patients with chronic liver disease heretofore, it is meaningful to investigate the structure of this bacterium at the species level in these patients. The aim of this study was to characterize the composition of intestinal Bifidobacterium in patients with hepatitis B virus-induced chronic liver disease. Nested-PCR-based denaturing gradient gel electrophoresis (PCR-DGGE), clone library, and real-time quantitative PCR were performed on the fecal samples of 16 patients with chronic hepatitis B (CHB patients), 16 patients with hepatitis B virus-related cirrhosis (HBV cirrhotics), and 15 healthy subjects (Controls). Though there was no significant difference in the diversity among the three groups (P = 0.196), Bifidobacterium dentium seems to be specifically enhanced in patients as the PCR-DGGE profiles showed, which was further validated by clone library and real-time quantitative PCR. In contrast to the B. dentium, Bifidobacterium catenulatum/Bifidobacterium pseudocatenulatum were detected less frequently in the predominant profile and by quantitative PCR in HBV cirrhotics than in the controls, and the level of this species was also significantly different between these two groups (P = 0.023). Although having no quantitative difference among the three groups, Bifidobacterium longum was less commonly detected in HBV cirrhotics than in CHB patients and Controls by quantitative PCR (P = 0.011). Thus, the composition of intestinal Bifidobacterium was deeply altered in CHB and HBV cirrhotic patients with a shift from beneficial species to opportunistic pathogens. The results provide further insights into the dysbiosis of the intestinal microbiota in patients with hepatitis B virus-induced chronic liver disease and might potentially serve as guidance for the probiotics interventions of these diseases.     

Effect of chitosan intake on fecal excretion of dioxins and polychlorinated biphenyls in healthy men

Six healthy male subjects were treated with 0 g, 1 g, 3 g, and 0 g of chitosan for the first, second, third, and fourth of four weeks, respectively. They were administered chitosan before breakfast on the second, third, and fourth days of the week, and fecal specimens were collected corresponding to the prescribed diet consumed for breakfast on the second day to breakfast on the fourth day. Fecal excretion of dioxins and polychlorinated biphenyls (PCBs) was promoted by intake of 3 g of chitosan (p=0.0589 and p<0.05 respectively), and was positively correlated with that of fat (p<0.01 for both). We found that chitosan intake increased the fecal excretion of dioxins and PCBs, as well as that of fat, suggesting that it might be useful for reducing the adverse effects of lipophilic endocrine-disrupting chemicals.     

Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.     

Bacterial Colonization on Fecal Pellets of Harpacticoid Copepods and on Their Diatom Food

Fecal pellets make up a significant fraction of the global flux of organic matter in oceans, and the associated bacterial communities in particular are a potential food source for marine organisms. However, these communities remain largely unknown. In the present study, the bacterial communities on fecal pellets of the benthic copepod Paramphiascella fulvofasciata feeding on the diatoms Navicula phyllepta and Seminavis robusta were analyzed. The aim of this study was to characterize the bacterial communities associated with the diatoms and the fecal pellets by means of DGGE profiling. Furthermore, isolated bacteria were characterized by means of partial 16S rRNA gene sequencing. The composition of the bacterial microflora on fecal pellets was studied in terms of the effect of the original food source, the age of the fecal pellets and the copepod’s identity. Alphaproteobacteria, Flavobacteria, and Bacilli were found on the fecal pellets; whereas on diatoms, exclusively Gammaproteobacteria were identified. Especially after eating N. phyllepta, there was an important increase in bacterial diversity, although the diatom N. phyllepta harbored a less diverse bacterial community than S. robusta. Our data suggest that the additional bacteria originate from the copepod’s digestive tract and largely depends on the initial food source.     

Molecular Characterization of Fecal Microbiota in Patients with Viral Diarrhea

The study provides molecular analyses of fecal microbiota of diarrhea patients infected with four different types of viruses. Fecal specimens from 52 patients with viral diarrhea (13 each of adenovirus, norovirus, rotavirus, and astrovirus) and six healthy individuals were collected and etiological viral agent was confirmed by enzyme immunoassay and specific PCR. To assess the changes in microbial diversity in patients with viral diarrhea, DNA from stool were extracted and characterized by PCR-denaturing gradient gel electrophoresis (DGGE) with universal primers specific for the V3 region of 16S rRNA gene. The strongest bands of the DGGE profiling were excised and sequenced to identify the dominant groups. Bacteroides vulgatus, Bifidobacterium, and Lactobacillus genera were also enumerated by real time PCR. The results revealed that bacterial diversity and similarity in feces from viral diarrhea groups were significantly lower (mean H′/ H _max^￠ H_{ \max }^{\prime } 0.89–0.94, 29–43, respectively) as compared with those of healthy individuals (mean H′/ H _max^￠ H_{ \max }^{\prime } 1.36, 59, respectively). Sequencing of dominant bands affirmed that diarrhea groups were mainly comprised of phylum Firmicutes, such as genera Enterococcus, Peptostreptococcaceae incertae sedi, Streptococcus, Weissella, and Clostridium, and opportunistically pathogenic genus Shigella, while dominant group in healthy individuals was phylum Bacteroidetes. Copy number of Bacteroides vulgatus, Bifidobacterium, and Lactobacillus genera was also reduced significantly in viral diarrhea groups as compared to healthy group. It is concluded that opportunistic pathogens increases, while other species of commensal microbiota decrease significantly in the viral diarrhea patients and dysbacteriosis is dependent on type of virus infection.     

Immobilization of Infant Fecal Microbiota and Utilization in an <Emphasis Type="Italic">in vitro</Emphasis> Colonic Fermentation Model

Bacteria isolated from infant feces were immobilized in polysaccharide gel beads (2.5% gellan gum, 0.25% xanthan gum) using a two-phase dispersion process. A 52-day continuous culture was carried out in a single-stage chemostat containing precolonized beads and fed with a medium formulated to approximate the composition of infant chyme. Different dilution rates and pH conditions were tested to simulate the proximal (PCS), transverse (TCS), and distal (DCS) colons. Immobilization preserved all nine bacterial groups tested with survival rates between 3 and 56%. After 1 week fermentation, beads were highly colonized with all populations tested (excepted Staphylococcus spp. present in low numbers), which remained stable throughout the 7.5 weeks of fermentation, with variations below 1 log unit. However, free-cell populations in the circulating liquid medium, produced by immobilized cell growth, cell-release activity from gel beads, and free-cell growth, were altered considerably by culture conditions. Compared to the stabilization period, PCS was characterized by a considerable and rapid increase in Bifidobacterium spp. concentrations (7.4 to 9.6 log CFU/mL), whereas Bifidobacterium spp., Lactobacillus spp., and Clostridium spp. concentrations decreased and Staphylococcus spp. and coliforms increased during TCS and DCS. Under pseudo-steady-state conditions, the community structure developed in the chemostat reflected the relative proportions of viable bacterial numbers and metabolites generally encountered in infant feces. This work showed that a complex microbiota such as infant fecal bacteria can be immobilized and used in a continuous in vitro intestinal fermentation model to reproduce the high bacterial concentration and bacterial diversity of the feces inoculum, at least at the genera level, with a high stability during long-term experiment.     

Comparative Evaluation of Fecal Fat Excretion Induced by Orlistat and Chitosan

Objective: To compare the effects of chitosan and orlistat on fecal fat excretion. Research Methods and Procedure: A randomized, open‐label, two‐period sequential design study was used. A total of 12 healthy adult volunteers within 20% of their ideal body weight entered a 7‐day run‐in diet period before being randomized to orlistat (120 mg) or chitosan (890 mg) three times daily for 7 days. Subjects then crossed over treatment regimens for an additional 7‐day period. Subjects followed a standardized diet (2500 kcal/d, 30% as fat) for the entire 21‐day study. Feces were collected on days 4 to 7 of the run‐in period (baseline) and during the two treatment periods. Mean daily fecal fat excretion was measured at baseline and during each treatment regimen. Results: Mean baseline fecal fat excretion for all subjects was 1.36 ± 0.45 g/d. During orlistat treatment, mean fecal fat excretion significantly increased from baseline (+16.13 ± 7.27 g/d; p < 0.001). No significant effect was observed with chitosan (+0.27 ± 1.02 g/d; p = 0.379). Fecal fat excretion was significantly greater with orlistat than with chitosan (p < 0.001; 95% confidence intervals: 11.73; 20.00 g/d). Discussion: This study provides additional evidence of the inhibitory effect of orlistat on dietary fat absorption. Chitosan, however, has no effect on fecal fat excretion.     

Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution

A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.     

Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, ~99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.