Design and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota

Abstract

Among human faecal bacteria, many members of the Clostridium leptum subgroup are fibrolytic and butyrate producing microorganisms thereby contributing to processes important to colonic health. Yet this phylogenetic subgroup remains poorly described to date. To improve detection and description of members of the C. leptum subgroup, the Clep 866 group probe was developed. Its association with probes targeting the Clostridium viride cluster (Cvir 1414) and Eubacterium desmolans species (Edes 635) allowed for the first time the detection of all members found in this phylogenetic group in human faecal microbiota. A species-specific probe was also designed to detect members of the Ruminococcus callidus species (Rcal 733). The design of signature regions was based on alignment of 16S rRNA sequences isolated from faeces of five healthy adults. Furthermore, an oligonucleotide competitor strategy was developed in order to improve the specificity of the probes formerly validated or designed in this study. The oligonucleotide probes were tested using a collection of target and non-target strains using FISH combined with flow cytometry. These new probes were added to a panel of 18 phylogenetic probes selected to describe faecal microbiota composition in 21 human faeces of healthy adults. Clostridium leptum subgroup represented 22% of the total faecal bacteria and codominated with members of Clostridium coccoides group. The cluster Faecalibacterium prausnitzii was the dominant component of the C. leptum subgroup and 20% of the latter subgroup remained unidentified at the species level.

Determinants of the human infant intestinal microbiota after the introduction of first complementary foods in infant samples from five European centres

“…It was not possible to consider the impact of each parameter in each country separately as the numbers in each centre were limited. One month after weaning started, the faecal microbiota was still quite different from that of adults (Lay et al, 2005), most evidently for the Swedish infants where bifidobacteria represented .50 % of total bacteria and the C. leptum group was virtually undetected. This was also true for Spanish infants, although their post-weaning microbiota appeared closest to that of adults (lowest bifidobacteria and highest Bacteroides, C. coccoides and C. leptum proportions).…”

Characterization of the Microbial Communities in Rumen Fluid Inoculated Reactors for the Biogas Digestion of Wheat Straw

“…The well-known cellulolytic genus Clostridium [47] was not detected in the rumen samples, but replaced genus Ruminococcus conducting the cellulose decomposing in the anaerobic reactors. Additionally, the Clostridium-related sequences in this work were affiliated to Clostridium cluster IV (Clostridium leptum), many of which are fibrolytic and butyrate producing microorganisms [57]. Some species in genus Bacteroides have the potential of endoglucanase (carboxymethylcellulase), cellulase, xylanase, and arylb-xylosidase activities [58,59], which may be the reason for their universal presence in lignocellulose-rich environments.…”

Lower Bifidobacteria counts in both duodenal mucosa-associated and fecal microbiota in irritable bowel syndrome patients

“…Quantification of Lactobacilli genera and species EUB 338 Total bacteria [44] Bac 303 Bacteroides-Prevotella group [45,46] Bif 164 Bifidobacterium [12] Erec 482 Clostridium coccoides-Eubacterium rectale group [47] Chis 150 Clostridium histolyticum group [47] Clit 135 Clostridium lituseburense group [47] Cld 73 Clostridium difficile This study: CGCCGC TCTTTACCGAAGT Fprau 645…”

“…Faecalibacterium prausnitzii [46,48] Lab 158 Lactobacillus-Enterococcus group [49] specifically belonging to bifidobacteria was performed using a 5'nuclease (TaqMan) assay as described previously [13,14] . Briefly, a 20 μL PCR amplification mixture containing 10 μL TaqMan Fast Universal Master Mix (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands), optimized concentrations of primers and probes and 2.0 μL isolated DNA was prepared.…”