Analysis of Human Intestinal Microbiota Using Molecular-Biological Techniques

Abstract

Culture-based approaches have been used for the analysis of human gut microbiota, however, it is difficult to culture 70 to 80% of the bacteria in the human gut. The gut microbiota of adult, elderly, and strictly vegetarian individuals were analyzed by the 16S rRNA gene library and T-RFLP. Among a total of about 1,800 clones obt(ned, approximately 75% of the clones were phylotypes (unexploited bacteria). A large number of species that have not yet been identified exist in the human gut. As a result of phylogenetic analysis, the Clostridium leptum subgroup, the Clostridium coccoides group, and the Bacteroides group were considered to be the predominant bacteria in adult individuals. The C. leptum subgroup, the C. coccoides group, the Bacteroides group, and "Gammaproteobacteria" were detected with high frequency in elderly individuals. In addition, the proportion of the C. coccoides group was lower than that in younger adults. The C. leptum subgroup, the C. coccoides group, the Clostridium rRNA cluster XVIII, and the Bacteroides group were detected in strictly vegetarian individuals. Especially, the Clostridium rRNA cluster XVIII was detected with high frequency. The composition of human gut microbiota was shown by these analyses, and, there were major differences between individuals in the composition of gut microbiota. Microbiota in jejunum, ileum, cecum and recto-sigmoid colon obtained from elderly individuals at autopsy were analyzed using 16S rRNA gene libraries and T-RFLP. The jejunal and ileal microbiota consisted of simple microbial communities that cont(n streptococci, lactobacilli "Gammaproteobacteria", the Enterococcus group, and the Bacteroides group. The cecal and recto-sigmoidal colonic microbiota consisted of complex microbial communities with numerous species (OTUs) that belonged to the C. coccoides group, the C. leptum subgroup, the Bacteroides group and "Gammaproteobacteria". The microbiota group structure was different in each of the four different parts of the human gut. Functional genes were cloned from environmental samples without cultivation of microbes. Novel 1,4-b-xylanase genes, which may contribute to the breakdown of xylan, which cont(ns dietary fiber, were obt(ned directly from mixed genome DNA of fecal microbiota without cultivation. A SOM (Self-Organizing Map) analysis demonstrated that the xylanase gene belongs to the Bacteroidetes.