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

International audienceTarget 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

Correlation between faecal microbial community structure and cholesterol-to-coprostanol conversion in the human gut

Intensity of the cholesterol-to-coprostanol conversion in the intestine, as assessed by the coprostanol-to-cholesterol ratio in faeces, was found highly variable among 15 human volunteers, ranging from absent to almost complete cholesterol conversion. The number of coprostanoligenic bacteria in the same faecal samples, as estimated by the most probable number method, was found to be less than 10(6) cellsg-1 of fresh stools in the low-to-inefficient converters and at least 10(8) cellsg-1 of fresh stools in the highest converters, indicating that the population level of cultivable faecal coprostanoligenic bacteria correlated with the intensity of cholesterol-to-coprostanol conversion in the human gut. Microbial communities of the samples were profiled by temporal temperature gradient gel electrophoresis (TTGE) of bacterial 16S rRNA gene amplicons. Dendrogram analysis of the TTGE profiles using the Pearson product moment correlation coefficient and a unweighted pair group method with arithmetic averages (UPGMA) algorithm clearly separated banding patterns from low-to-inefficient and high converters in two different clusters suggesting a relationship between TTGE profiles and coprostanoligenic activity. Principal components analysis further demonstrated that a large subset of bands rather than some individual bands contributed to this clustering

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g

Differences in fecal microbiota in different European study populations in relation to age, gender, and country: a cross-sectional study

A cross-sectional study on intestinal microbiota composition was performed on 230 healthy subjects at four European locations in France, Germany, Italy, and Sweden. The study participants were assigned to two age groups: 20 to 50 years (mean age, 35 years; n = 85) and >60 years (mean age, 75 years; n = 145). A set of 14 group- and species-specific 16S rRNA-targeted oligonucleotide probes was applied to the analysis of fecal samples by fluorescence in situ hybridization coupled with flow cytometry. Marked country-age interactions were observed for the German and Italian study groups. These interactions were inverse for the predominant bacterial groups Eubacterium rectale-Clostridium coccoides and Bacteroides-Prevotella. Differences between European populations were observed for the Bifidobacterium group only. Proportions of bifidobacteria were two- to threefold higher in the Italian study population than in any other study group, and this effect was independent of age. Higher proportions of enterobacteria were found in all elderly volunteers independent of the location. Gender effects were observed for the Bacteroides-Prevotella group, with higher levels in males than in females. In summary, age-related differences in the microbiota makeup were detected but differed between the study populations from the four countries, each showing a characteristic colonization pattern