Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% +/- 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% +/- 7% and 14% +/- 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.     

Enumeration of bacteria from the Clostridium leptum subgroup in human faecal microbiota using Clep1156 16S rRNA probe in combination with helper and competitor oligonucleotides

Target site inaccessibility represents a significant problem for fluorescent in situ hybridisation (FISH) of 16S rRNA oligonucleotide probes. For this reason, the Clep1156 probe targeting 16S rRNA of the Clostridium leptum phylogenetic subgroup used for dot blot experiments could not be used until now for FISH. Considering that bacteria from the C. leptum subgroup are very abundant in the human faecal microbiota and may play a significant role in host health, we have used unlabelled helper and competitor oligonucleotides to improve the 16S rRNA in situ accessibility and specificity of the Clep1156 probe and applied this approach to enumerate C. leptum bacteria in this ecosystem. Nine C. leptum target strains and five non-target strains were selected to develop and validate the helper-competitor strategy. Depending on the target strains, the use of helpers enhanced the fluorescence intensity signal of Clep1156 from 0.4-fold to 8.4-fold with a mean value of 3.6-fold, switching this probe from the brightness class V-VI (masked sites) to III-IV (accessible sites). The simultaneous use of helper and competitor oligonucleotides with Clep1156 probe allowed the expected specificity without disturbing in situ accessibility. Quantified by FISH combined with flow cytometry, C. leptum bacteria in human faecal samples (n=22) represented 19 +/- 7% of bacteria on average [4.9-37.5]. We conclude that helper oligonucleotides are very useful to circumvent the problem of target site in situ accessibility, especially when probe design is limited to only one 16S rRNA area and that helpers and competitors may be efficiently combined.     

Separation of bacteria of the Clostridium leptum subgroup from the human colonic microbiota by fluorescence-activated cell sorting or group-specific PCR using 16S rRNA gene oligonucleotides

Molecular methods based on 16S rRNA gene sequence analyses have shown that bacteria of the Clostridium leptum subgroup are predominant in the colonic microbiota of healthy humans; this subgroup includes bacteria that produce butyrate, a source of energy for intestinal epithelial cells. To improve our understanding of the species within this important group, separation methods using fluorescence-activated cell sorting (FACS) and specific PCR were combined with 16S rRNA gene sequence analyses. FACS was developed for bacteria labelled in situ with two rRNA oligonucleotide probes, namely EUB338-FITC for total bacteria and Clep866-CY5/cp or Fprau645-CY5 for bacteria of the C. leptum subgroup. Bacterial cell sorting allowed a selective recovery of members of the C. leptum subgroup from the human microbiota with efficiencies as high as 95%. Group-specific PCR amplification of the C. leptum subgroup was developed, and temporal thermal gradient gel electrophoresis showed host-specific profiles with low complexity, with a sharing of common bands between individuals and bands stable over 2 months for the same individual. A library of 16S rRNA gene cloned sequences (106 sequences) was prepared with DNA obtained from both separation methods, and 15 distinct phylotypes were identified, among which 10 have no cultivable or currently cultivated representative in reference collections.     

Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes

Bacteroides is a predominant group of the faecal microbiota in healthy adults. To investigate the species composition of Bacteroides by fluorescent in situ hybridisation (FISH) combined with flow cytometry, we developed five species-specific probes targeting the 16S rRNA. Probes were designed to identify cells belonging to Bacteroides distasonis, B. fragilis, B. ovatus, B. vulgatus and B. putredinis. The species-specificity of the probes was assessed against a collection of reference strains from the Cytophaga-Flavobacterium-Bacteroides group. The results of the FISH experiments showed that the probes were specific as they only detected strains of the target species. Determining the fluorescence intensity of each probe relative to that of the EUB 338 probe (domain bacteria) further showed that each species probe easily accessed the targeted site. The probes were applied to quantify the Bacteroides species in faeces collected from 20 healthy adults. All five species were detected in the faecal samples. Cells hybridised with Bfra 998 were the most frequent as they were observed in 90% of individuals (18/20 samples, mean proportion of 3.9 +/- 2.2%). The cells hybridised with Bvulg 1017 were observed in 85% of individuals (17/20 samples) and represented with a mean proportion of 4.2 +/- 6.1%, the most abundant Bacteroides species in human faeces. Cells hybridising with probes for B. ovatus, B. distasonis and B. putredinis were less frequently detected. The large distribution of B. vulgatus and B. fragilis in human faeces is in accordance with previous reports based on culture or molecular studies. This work showed that fluorescent in situ hybridisation is a tool appropriate for a high-resolution analysis of the species composition of complex ecosystems and especially of the Bacteroides group within the faecal microbiota.     

Comparative study of bacterial groups within the human cecal and fecal microbiota

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.     

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.     

Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut.

The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.     

PCR DGGE and RT-PCR DGGE show diversity and short-term temporal stability in the Clostridium coccoides-Eubacterium rectale group in the human intestinal microbiota

As the Clostridium coccoides-Eubacterium rectale (Erec; clostridial phylogenetic cluster XIVa) group is one of the major groups of the human intestinal microbiota, DNA- and RNA-based population analysis techniques (denaturing gradient gel electrophoresis; DGGE) were developed and applied to assess the diversity and temporal stability (6 months-2 years) of this faecal clostridial microbiota in 12 healthy adults. The stability of the Erec group was compared with the stability of the predominant bacterial microbiota, which was also assessed with PCR-DGGE. In addition, the Erec group was quantified with a hybridization-based method. According to our results, the Erec group was diverse in each subject, but interindividual uniqueness was not as clear as that of the predominant bacteria. The Erec group was found to be temporally as stable as the predominant bacteria. Over 200 clones obtained from two samples proved the developed method to be specific. However, the amount of bacteria belonging to the Erec group was not related to the diversity of that same bacterial group. In conclusion, the newly developed DGGE method proved to be a valuable and specific tool for the direct assessment of the stability of the Erec group, demonstrating diversity in addition to short-term stability in most of the subjects studied.     

Colonic microbiota signatures across five northern European countries

The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed. Clostridium coccoides and Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by the Bacteroides (8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed.     

Culture-independent phylogenetic analysis of the faecal flora of the rat

The dominant faecal flora of the rat was determined using randomly cloned 16S rDNA comparative sequence analysis. A total of 109 near full-length 16S rDNA clones were sequenced, representing 69 unique 16S rRNA phylotypes or operational taxonomic units (OTUs). Estimates of species richness indicated that approximately 338 species were present in the faeces, suggesting that only 20% of species were identified. Only two of 39 Gram-negative clones aligned with previously cultured species, the remainder fell into a separate lineage within the Bacteroides-Cytophaga phylum. Several clones within this new group were related to 16S rDNA sequences previously identified from mouse faeces. Lactobacilli were the most abundant Gram-positive species, representing 23% of the total clones but only 7% of OTUs. The remaining Gram-positive clones were distributed among the Clostridium coccoides group (9%), the Clostridium leptum subgroup (18%), and throughout the low GC Gram-positive bacteria (13%). The majority of OTUs (63/69 or 91%) were less than 97% homologous to previously cultured bacteria. Faecal samples were also cultured using a variety of anaerobic media. With the exception of the lactobacilli, the cultured isolates demonstrated low species diversity and poorly reflected the population, as defined through comparative sequence analysis.     

Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces

16S rRNA gene-targeted group-specific primers were designed and validated for specific detection and quantification of the Clostridium leptum subgroup and the Atopobium cluster. To monitor the predominant bacteria in human feces by real-time PCR, we used these specific primers together with four sets of group-specific primers for the Clostridium coccoides group, the Bacteroides fragilis group, Bifidobacterium, and Prevotella developed in a previous study (T. Matsuki, K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka, Appl. Environ. Microbiol. 68:5445-5451, 2002). Examination of DNA extracted from the feces of 46 healthy adults showed that the C. coccoides group was present in the greatest numbers (log10 10.3 +/- 0.3 cells per g [wet weight] [average +/- standard deviation]), followed by the C. leptum subgroup (log10 9.9 +/- 0.7 cells per g [wet weight]), the B. fragilis group (log10 9.9 +/- 0.3 cells per g [wet weight]), Bifidobacterium (log10 9.4 +/- 0.7 cells per g [wet weight]), and the Atopobium cluster (log10 9.3 +/- 0.7 cells per g [wet weight]). These five bacterial groups were detected in all 46 volunteers. Prevotella was found in only 46% of the subjects at a level of log10 9.7 +/- 0.8 cells per g (wet weight). Examination of changes in the population and the composition of the intestinal flora for six healthy adults over an 8-month period revealed that the composition of the flora of each volunteer remained stable throughout the test period.     

Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.     

16S rRNA-based analysis of microbiota from the cecum of broiler chickens.

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.     

What do we mean when we refer to Bacteroidetes populations in the human gastrointestinal microbiota?

Recent large-scale cloning studies have shown that the ratio of Bacteroidetes to Firmicutes may be important in the obesity-associated gut microbiota, but the species these phyla represent in this ecosystem has not been examined. The Bacteroidetes data from the recent Turnbaugh study were examined to determine those members of the phylum detected in human faecal samples. In addition, FISH analysis was performed on faecal samples from 17 healthy, nonobese donors using probe Bac303, routinely used by gut microbiologists to enumerate Bacteroides – Prevotella populations in faecal samples, and another probe (CFB286) whose target range has some overlap with that of Bac303. Sequence analysis of the Turnbaugh data showed that 23/519 clones were chimeras or erroneous sequences; all good sequences were related to species of the order Bacteroidales , but no one species was present in all donors. FISH analysis demonstrated that approximately one-quarter of the healthy, nonobese donors harboured high numbers of Bacteroidales not detected by probe Bac303. It is clear that Bacteroidales populations in human faecal samples have been underestimated in FISH-based studies. New probes and complementary primer sets should be designed to examine numerical and compositional changes in the Bacteroidales during dietary interventions and in studies of the obesity-associated microbiota in humans and animal model systems.     

Dietary fructooligosaccharides alter the cultivable faecal population of rats but do not stimulate the growth of intestinal bifidobacteria

The effect of fructans on the cultivable faecal community of Bio Breeding rats fed diets containing 5% (m/v) food-grade fructooligosaccharide (FOS) was investigated. Culturing of faecal material using chicory inulin as the sole carbohydrate source revealed the presence of a greater diversity of inulin-utilizing bacterial species in FOS-fed rats as compared with the control rats, although both contained species which effectively utilized inulin. The majority of cultivable inulin-utilizing species fell within the Clostridium coccoides group and Clostridium leptum subgroup, some of which were related to previously cultured butyrate-producing bacteria from the intestines of various animals. The impact of FOS on the growth of the indigenous bifidobacteria community and three inulin-utilizing isolates was assessed using real-time polymerase chain reaction. While dietary FOS was found to stimulate the growth of all three inulin-utilizing isolates, no growth stimulation of the indigenous bifidobacteria community occurred over the duration of the feeding trial.     

Reset of a critically disturbed microbial ecosystem: faecal transplant in recurrent Clostridium difficile infection

Recurrent Clostridium difficile infection (CDI) can be effectively treated by infusion of a healthy donor faeces suspension. However, it is unclear what factors determine treatment efficacy. By using a phylogenetic microarray platform, we assessed composition, diversity and dynamics of faecal microbiota before, after and during follow-up of the transplantation from a healthy donor to different patients, to elucidate the mechanism of action of faecal infusion. Global composition and network analysis of the microbiota was performed in faecal samples from nine patients with recurrent CDI. Analyses were performed before and after duodenal donor faeces infusion, and during a follow-up of 10 weeks. The microbiota data were compared with that of the healthy donors. All patients successfully recovered. Their intestinal microbiota changed from a low-diversity diseased state, dominated by Proteobacteria and Bacilli, to a more diverse ecosystem resembling that of healthy donors, dominated by Bacteroidetes and Clostridium groups, including butyrate-producing bacteria. We identified specific multi-species networks and signature microbial groups that were either depleted or restored as a result of the treatment. The changes persisted over time. Comprehensive and deep analyses of the microbiota of patients before and after treatment exposed a therapeutic reset from a diseased state towards a healthy profile. The identification of microbial groups that constitute a niche for C. difficile overgrowth, as well as those driving the reinstallation of a healthy intestinal microbiota, could contribute to the development of biomarkers predicting recurrence and treatment outcome, identifying an optimal microbiota composition that could lead to targeted treatment strategies.     

Comparative Study of Bacterial Groups within the Human Cecal and Fecal Microbiota

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.     

T-RFLP技术在人肠道柔嫩梭菌类群的组成分析中的应用

目的建立能快速筛查和比较大规模人群肠道中柔嫩梭菌类群组成结构的T-RFLP（末端限制性片段长度多态性）。方法采用T-RFLP技术特异性地分析柔嫩梭菌类群的组成,考察该方法的重复性和灵敏度。用该方法对15个志愿者肠道中的柔嫩梭菌类群进行分析,并与DGGE方法分析的结果进行对比。结果T-RFLP方法重复性高,最低能检测到群落中1%的细菌,其结果与DGGE的分析结果具有较好的一致性。结论本研究建立的柔嫩梭菌类群特异性的T-RFLP方法能够对大量肠道样品中柔嫩梭菌类群的结构进行快速、有效的筛查和比较。     

Distribution of genes encoding the trypsin-dependent lantibiotic ruminococcin A among bacteria isolated from human fecal microbiota

Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogenetically related strains that can make up a significant part of the human fecal microbiota.     

Lactobacillus rhamnosus R11 consumed in a food supplement survived human digestive transit without modifying microbiota equilibrium as assessed by real-time polymerase chain reaction

The aim of this study was to evaluate the survival of Lactobacillus rhamnosus R11 and Lactobacillus acidophilus R52 in the human digestive tract and their effects on the microbiota homeostasis. We designed an open human trial including 14 healthy volunteers. A 3-week exclusion period of fermented products was followed by a 12-day consumption period of 4 capsules daily containing 2 x 10(9)L. rhamnosus R11 and 1 x 10(8)L. acidophilus R52, and a 12-day wash-out period. The 2 strains and dominant bacterial groups of the microbiota were quantified by real-time polymerase chain reaction. At the end of the capsule consumption period, high levels of L. rhamnosus R11 were detected in faecal samples from all volunteers, reaching a mean value of 7.1 log(10) colony-forming unit (CFU) equivalents/g of stool. L. acidophilus R52 was detected in the stools of only 1 volunteer, reaching a maximum level of 6.1 log(10) CFU equivalents/g of stool. Dilution plating enumerations performed in parallel provided less consistent and generally lower levels. No significant effect of capsule consumption was observed on microbiota homeostasis for the dominant faecal populations. Mean values of 8.8, 9.2, 9.9 and 10.6 log(10) CFU equivalents/g of stool were obtained for the Clostridium coccoides, Bifidobacterium sp., Bacteroides sp. and Clostridium leptum groups, respectively.