Diversity of Intestinal Clostridium coccoides Group in the Japanese Population, as Demonstrated by Reverse Transcription-Quantitative PCR

Abstract

We used sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) to quantify the Clostridium coccoides group, which is a major anaerobic population in the human intestine. For this purpose, the C. coccoides group was classified into 3 subgroups and 19 species for expediency in accordance with the existing database, and specific primers were newly developed to evaluate them. Population levels of the C. coccoides group in human feces determined by RT-qPCR were equivalent to those determined by fluorescence in situ hybridization. RT-qPCR analysis of fecal samples from 96 volunteers (32 young children, 32 adults and 32 elderly) by using the 22 new primer sets together with the C. coccoides group-specific primer setm revealed that (i) total counts obtained as the sum of the 3 subgroups and 19 species were equivalent to the results obtained by using the C. coccoides group-specific primer set; (ii) total C. coccoides-group counts in the elderly were significantly lower than those in young children and adults; (iii) genus Blautia was the most common subgroup in the human intestinal C. coccoides-group populations at all age populations tested; (iv) the prevalences of Fusicatenibacter saccharivorans and genus Dorea were significantly higher in adults than in young children and the elderly; and (v) the prevalences of C. scindens and C. hylemonae, both of which produce secondary bile acid in the human intestine, were significantly higher in the elderly than in young children and adults. Hierarchical clustering and principal component analysis showed clear separation of the bacterial components between adult and elderly populations. Taken together, these data suggest that aging plays an important role in the diversity of C. coccoides-group populations in human intestinal microbiota; changes in this diversity likely influence the health of the host.

Sensitive Quantitative Analysis of the Meconium Bacterial Microbiota in Healthy Term Infants Born Vaginally or by Cesarean Section

“…Another advantage is that it provides information on viable cells, whereas the results of DNA-based analyses might also include background DNA (e.g., free bacterial DNA, or DNA of dead bacteria) that may be present in prenatal niches such as amniotic fluid and placenta and also in meconium. We have previously validated that the counts obtained by this RT-qPCR method are equivalent to the bacterial counts enumerated by culturing and fluorescent in situ hybridization (FISH) methods and that the detection sensitivity of RT-qPCR is approximately 100- to 1000-fold higher than that of other molecular methods such as qPCR and terminal restriction fragment length polymorphism (t-RFLP) (Matsuda et al, 2007, 2009; Kubota et al, 2010; Kurakawa et al, 2015b). Therefore, owing to these advantages, we specifically implemented RT-qPCR for the present study.…”

High-fat Diet Induced Dysbiosis & Amelioration by Astaxanthin

“…Clostridium is a major genus of anaerobic endo-spore forming bacteria 53 . Several species in the genus are known pathogens, such as Clostridium botulinum (the causative pathogen of botulism), Clostridium tetani (tetanus) and former member Clostridium difficile (now Clostridioides difficile).…”

The effect of sugar and artificial sweetener on molecular markers of metabolic syndrome: a mice study

“…From total bacteria DNA samples, specific bacteria sequence would be amplified with specific R: 5-CTTTGAGTTTCATTCTTGCGAA -3 for C. coccoides (Kurakawa et al, 2015), and 3.25 µL nuclease free water. G-Storm GS482 Thermal Cycler (G-Storm, Somerton, Somerset, United Kingdom) instrument was used for this reaction with initial condition 94°C for 5 minutes, denaturation 94°C for 30 seconds, annealing condition 55°C for Ccoc according to Tm primer, extension 72°C for 1 minutes, and post elongation 72°C for 10 minutes.…”