Terminal Restriction Fragment Length Polymorphism Analysis for Human Fecal Microbiota and Its Application for Analysis of Complex Bifidobacterial Communities

Abstract

A large number and variety of bacteria exist in the human intestinal tract. The balance of the intestinal microbiota closely relates to the health of the individual. Hence, understanding the human intestinal microbiota is very important. Traditional methods to determine the composition of the intestinal microbiota require various culture techniques, which are time-consuming and laborious. Therefore, many researchers used various culture-independent methods; including 16S rRNA probe hybridization (3, 23), PCR-related methods (29), and temperature gradient gel electrophoresis (TGGE) analysis (32), to characterize the human intestinal microbiota.A phylogenetic approach based on 16S rRNA (rDNA) has been applied to investigate the diversity of cultivable and non-cultivable species in human fecal samples without cultivation (4,24,30). The 16S rDNA clone library method can provide direct sequence information. We demonstrated previously that the predominant intestinal microbial community consisted of 130 species or "phylotypes"(17), which have been used for clusters of clone sequences that differed from known species by about 2% and were at least 99% similar to members of their cluster, based on analysis of 744 16S rRNA clones (4). However, analysis of individual 16S rDNA clones is an expensive and extremely inefficient approach for comparison of a multitude of bacterial communities.Terminal restriction fragment length polymorphism (T-RFLP) is a alternative molecular approach that allows the assessment of a diversity of complex bacterial communities and rapid comparison of the community structure and diversity of different ecosystems (11). We have already used this method to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis (20). In the field of intestinal microbiology, T-RFLP analysis is potentially useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals, but has not yet been reported to our knowledge.In the present study, we used T-RFLP analysis to characterize and compare human fecal microbiota among Abstract: Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize and compare human fecal microbiota among individuals. T-RFLP patterns of fecal 16S ribosomal DNA (rDNA) PCR products from three adults revealed host-specific bacterial communities and were in good agreement with those reported in our previous study. In addition, we applied T-RFLP analysis for the analysis of complex bifidobacterial communities in human fecal samples. The developed method based on Bifidobacterium genus-specific PCR and T-RFLP could identify more than one bifidobacterial species. T-RFLP patterns of Bifidobacterium genus-specific PCR products from the fecal samples were host-specific as well as those of fecal 16S rDNA PCR products. These results were confirmed by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the genus Bifidobacterium and Bifidobacterium...

The intestinal microbiota and its role in human health and disease

“…The procedure is outlined in Figure 1 and used primers are shown in Table 1. The bacterial 16S rDNA was amplified by PCR using 5'-6 -carboxyfluorescein (6 -FAM) -labeled 27F primer and 1492R primer except for Bifidobacterium (7). Then, PCR products were digested with restriction enzymes HhaI and MspI, and 5'-labeled restriction fragments were examined in a genetic analyzer.…”

“…In our previous studies, intestinal microbiota was analyzed according to the method of Sakamoto et al (7). The procedure is outlined in Figure 1 and used primers are shown in Table 1.…”

Effects of Two Probiotic Lactobacillus Strains on Jejunal and Cecal Microbiota of Broiler Chicken under Acute Heat Stress Condition as Revealed by Molecular Analysis of 16S rRNA Genes

“…This method has also been successfully applied for the assessment of diversity of human oral microbiota and rapid comparison of the community structure between subjects with and without periodontitis (33). In addition, it has been demonstrated that T-RFLP analysis is useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals (13,32). T-RFLP analysis revealed differences among bacterial populations present in mucosa and lumen of the chicken ileum and cecum (8,9).…”

Comparison among Fecal Secondary Bile Acid Levels, Fecal Microbiota and Clostridium scindens Cell Numbers in Japanese

“…The difference in size of a restriction fragment between that predicted and that observed has been shown in many species of bacteria (24,25). Realtime PCR results did not correlate with T-RFLP results (Table 1).…”

“…When the target of PCR is 16S ribosomal DNA, the terminal restriction fragments pattern reflects the taxonomic diversity of the bacteria in the sample. T-RFLP is a rapid and efficient approach for successfully obtaining profiles of the various members of a complex microbial community, for example, human feces (10,25), chicken cecal samples (6), pig colon (19), rat feces (15), oral bacterial flora (24), soil (20,21), and marine sediments (28). The present study is the first to our knowledge to analyze 40 fecal samples using T-RFLP and to compare them using a dendrogram.…”