Development of a 16S rRNA-based Probe for Clostridium ramosum, C. spiroforme and C. cocleatum and its Application for the Quantification in Human Faeces from Volunteers of Different Age Groups
Wildeboer-Veloo, Alida C.M.
Harmsen, Hermie J.M.
Degener, John E.
Welling, Gjalt W.
- Publisher: Microbial Ecology in Health and Disease
Microbial Ecology in Health and Disease
(issn: 1651-2235, eissn: 1651-2235)
A 16S rRNA targeted oligonucleotide probe was developed for the quantitative detection of Clostridium ramosum, C. spiroforme and C. cocleatum in human faecal samples from volunteers of different age groups using fluorescent in situ hybridization (FISH). Some strains of C. spiroforme are able to produce binary toxins, while C. ramosum is believed to play a role in the conversion of bilirubin to urobilinogen. Little is known about the role of C. cocleatum in the intestinal tract. Previous enumerations of these clostridia were done by culture techniques. The probe was designed using 16S rRNA sequence data of reference strains and was shown to be specific. Application of the probe to faecal samples showed an increase of C. ramosum from </1x10<sup>7</sup> to 1.3x10<sup>9</sup> cells/g of wet weight faeces, in 0.5-1-month and 7-10-month-old babies, corresponding to <0.01% to 3.5% of the total microbiota, respectively. The percentage of C. ramosum decreased to 0.2% of all bacteria present in the faeces of children between 3 and 4 years old. An increase to 0.9% of the total microbiota was observed again in faecal samples from elderly subjects. C. spiroforme and C. cocleatum are present at an average of 0.2% until the age of 4 years. None were observed in babies younger than 6 months. In adults and elderly volunteers (>75 years) the percentage declined to 0.02%. The results indicate that C. spiroforme and C. cocleatum do not play a numerically important role in faecal samples of adult healthy volunteers. C. ramosum is a numerically important member of the microbiota around the time of weaning of infants.Keywords : faeces, microbiota, 16S rRNA, probe, fluorescent in situ hybridization, weaning, bacteria, Clostridium .