The human intestinal microbiota have traditionally been compared by analyzing isolates using an anaerobic culture-based method. Consequently, it has been documented that more than 10 12 bacteria cells per g of content (dry matter) (11,12,19,28) and estimated that 400-500 species inhabit in the intestinal tract (22). The composition and activity of indigenous intestinal microbiota are of paramount importance in human immunology, nutrition, and pathological processes and hence the health of the individual (31). However, the cultivable bacteria were 20 to 30% of the total because of the strict anaerobic and complex environment (12,19,28). Thus, our understanding of the microbiota in human intestinal tract is incomplete due to the culture-based method.The culture-independent approach based on molecular-biological techniques has revealed a great diversity of microbiota in environmental samples (1). Phylogenetic analysis based on the PCR cloning strategy has been used to characterize human fecal microbiota (15,28,32). Recently, we reported that the human fecal microbiota could be analyzed by 16S rDNA libraries and a strictly anaerobic culture-based method (12, 13). We detected many novel phylotypes and species that have not yet been characterized and showed phylogenetic correlation between newly isolated strains and 16S rRNA sequences (16S rDNA). In addition, major interindividual differences were evident in the composition of intestinal microbiota as determined by two independent approaches. The culture-based method is inadequate for the understanding of the whole human intestinal microbiota; thus it is necessary to use molecular-biological techniques like the 16S rDNA library.According to the analysis of culture-based methods, the composition of the human fecal microbiota changes with age (3, 21). Mitsuoka and Hayakawa (21) reported a significant increase in the number of lactobacilli and clostridia and a significant decrease in the number of bifidobacteria in the elderly compared with younger individuals. Recently, Hold et al. (15) Abstract: Fecal microbiota in six elderly individuals were characterized by the 16S rDNA libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis. Random clones of 16S rRNA gene sequences were isolated after PCR amplification with universal primer sets from total genomic DNA extracted from feces of three elderly individuals. These clones were partially sequenced (about 500 bp). T-RFLP analysis was performed using 16S rDNA amplified from six subjects. The lengths of the terminal restriction fragment (T-RF) were analyzed after digestion by HhaI and MspI. Among 240 clones obtained, approximately 46% belonged to 27 known species. About 54% of the other clones were 56 novel "phylotypes" (at least 98% homology of clone sequence). These libraries included 83 species or phylotypes. In addition, about 13% (30 phylotypes) of these phylotypes were newly discovered in these libraries. A large number of species that are not yet known exist in the feces of elderly individuals...

The Clostridium-like organisms TO-931 T and HD-17, isolated from human faeces, have high levels of bile acid 7α-dehydroxylating activity. Sequencing of their 16S rDNA demonstrated that they belong to cluster XI of the genus Clostridium and that they represent a new and distinct line of descent. Clostridium bifermentans and Clostridium sordellii in cluster XI also possess bile acid 7α-dehydroxylating activity. DNA-DNA hybridization experiments with the isolates, TO-931 T and HD-17, and C. bifermentans and C. sordellii revealed that the isolates are a single species distinct from C. bifermentans and C. sordellii. On the basis of phylogenetic analysis, using 16S rDNA sequences, and DNA-DNA hybridization analysis, it is concluded that strains TO-931 T and HD-17 are members of a new species of the genus Clostridium, for which the name Clostridium hiranonis is proposed. The type strain is strain TO-931 T (l JCM 10541 T l DSM 13275 T ).

Although the micro-organisms forming the cutaneous microbiota are considered to play important roles in the modification and prevention of skin diseases, a comprehensive analysis of their composition has not yet been carried out because of difficulties in determining yet-to-be-cultured micro-organisms in the samples. Swab-scrubbed forehead skin samples of five healthy volunteers were analysed by profiling 16S rRNA genes, as well as by conventional culture methods, to provide a profile of the cutaneous microbiota that included yet-to-be-cultured bacteria from normal human skin. Cluster analyses of the 16S rRNA gene sequences indicated a marked increase in diversity compared with that derived from the culture methods. Nineteen previously recognized species and 13 novel phylotypes were obtained from the analysis of 416 clones. In addition to well-known bacteria such as Staphylococcus epidermidis and Propionibacterium acnes, phylotype A, the 16S rRNA gene of which is 97 % similar to that of Methylophilus methylotrophus, was detected in three of the five samples, in one of which it was the predominant clone. Culture-independent genetic profiling of 16S rRNA genes for detecting human cutaneous microbiota has allowed us to detect potentially novel components of the cutaneous microbiota in humans.

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