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Mitsuo Sakamoto

Publications and source records attributed to Mitsuo Sakamoto.

47 records · Page 3Linked to original sources
PubMed2003

Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis.

Terminal restriction fragment-length polymorphism (T-RFLP) analysis was applied to characterize oral bacterial flora in saliva from 18 healthy subjects and 18 patients with periodontitis. The 16S rRNA genes (rDNAs) of oral bacteria and spirochaetes in saliva were amplified by PCR with a 6'carboxy-fluorescein (6-FAM)-labelled universal forward primer (27F) and a universal reverse primer (1492R) or the Spirochaeta-selective reverse primer. The 16S rDNAs were digested with restriction enzymes with 4 bp recognition sites (HhaI or MspI) and analysed by using an automated DNA sequencer. T-RFLP patterns were numerically analysed using a computer program. From analysis of the oral bacterial community, patterns derived from periodontally healthy subjects and patients with periodontitis were grouped into different clusters, though with some uncertainty. Samples from patients with periodontitis tended to cluster into their respective types (aggressive and chronic periodontitis), although this was not very clear. Analysis of spirochaetal community using T-RFLP showed that the patterns derived from patients with periodontitis were grouped more as compared with the analysis of the oral bacterial community. These results suggest that samples from patients with periodontitis contain an unexpected diversity. T-RFLP patterns of 16S rDNAs from saliva samples of two periodontally healthy subjects over a 5-week period showed host-specific relatively stable oral bacterial flora. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of oral bacterial flora and rapid comparison of the community structure between subjects with and without periodontitis.

Adult↗
PubMed2003

Terminal restriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities.

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 species- and group-specific PCR. Our study demonstrates 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, and that the applied method is useful for rapid and sensitive analysis of bifidobacterial community.

Adult↗
PubMed2003

Molecular analysis of fecal microbiota in elderly individuals using 16S rDNA library and T-RFLP.

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. 16S rDNA libraries and T-RFLP analysis revealed that the majority of bacteria were Bacteroides and relatives, Clostridium rRNA cluster IV, IX, Clostridium rRNA subcluster XIVa, and "Gammaproteobacteria". The proportion of Clostridium rRNA subcluster XIVa was lower than in healthy adults. In addition, although Ruminococcus obeum and its closely related phylotypes were detected in high frequency in healthy young subjects, hardly any were detected in our elderly individuals. "Gammaproteobacteria" were detected at high frequency.

Aged↗
PubMed2002

Detection of novel oral phylotypes associated with periodontitis.

A phylogenetic approach based on 16S rRNA (rDNA) has been recently applied to investigate the diversity of cultivable and uncultivable species in the human oral cavity without cultivation. In a previous study [Sakamoto et al. (2000) Microbiol. Immunol. 44, 643-652], we identified a number of novel oral phylotypes, representing as yet uncultured organisms. The purpose of this study was to design specific PCR primers for five phylotypes AP12, AP21, AP24, AP50, and RP58, which are deeply branched particularly in the phylogenetic tree, and determine the prevalence of these phylotypes in 45 patients with periodontitis and 18 healthy subjects. The specificity of each primer was validated by the sequence analysis of PCR products obtained from saliva and subgingival plaque samples. Among phylotypes tested, phylotype AP24, which is closely related to oral clone DA014 reported previously [Paster et al. (2001) J. Bacteriol. 183, 3770-3783], was significantly associated with saliva and subgingival plaque samples from patients with periodontitis (P<0.01), but the difference was not statistically significant in the presence of other phylotypes. These data suggest that phylotype AP24 may play an important role in periodontal disease.

Base Sequence↗
PubMed2002

Reclassification of Bacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensis corrig., gen. nov., comb. nov.

The characteristics of the fusiform species Bacteroides forsythus, isolated from human periodontal pockets, were examined. 165 rDNA sequence analysis confirmed that B. forsythus was not a species within the genus Bacteroides sensu stricto. Although B. forsythus was phylogenetically related to Bacteroides distasonis and Bacteroides merdae in the phylogenetic tree, the ratio of anteiso-15:0 to iso-15:0 in whole-cell methanolysates of B. forsythus was different from those of B. distasonis, B. merdae and other Bacteroides species. B. forsythus did not grow on medium containing 20% bile, but members of the Bacteroides fragilis group did. B. forsythus was the only species tested that was trypsin-positive in API ZYM tests. The dehydrogenase enzyme pattern was of no use for the differentiation of B. forsythus and the B. fragilis group. On the basis of these data, a new genus, Tannerella, is proposed for Bacteroides forsythus, with one species, Tannerella forsythensis corrig., gen. nov., comb. nov. The type strain of Tannerella forsythensis is JCM 10827T (= ATCC 43037T).

Animals↗
PubMed2002

Unification of Bifidobacterium infantis and Bifidobacterium suis as Bifidobacterium longum.

The relationships between Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis were examined by means of carbohydrate fermentation, DNA-DNA hybridization, ribotyping and random amplified polymorphic DNA-PCR (RAPD-PCR). The levels of DNA-DNA hybridization among the strains of B. infantis, B. longum and B. suis used in this study were 67-81% under optimal conditions (42 degrees C) and 63-85% under stringent conditions (52 degrees C). Although the strains showed varied carbohydrate-fermentation patterns, the three species were divided into three types, namely the infantis type, the longum type and the suis type, by ribotyping and RAPD-PCR. On the basis of these results, strains of B. infantis, B. longum and B. suis were recognized as distinct groups within a single species. It is concluded that B. infantis and B. suis should be unified as B. longum, the latter species being divided into three biotypes, the infantis type, the longum type and the suis type, by molecular methods.

Bifidobacterium↗
PubMed2002

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries.

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.

Animals↗
PubMed2002

Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods.

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture-based method. Direct counts ranged from 1.9 X 10" to 4.0 X 10" cells/g (wet weight), and plate counts totaled 6.6 X 10(10) to 1.2 X 10(11) CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.

Adult↗
PubMed2002

Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and cultivation.

Fecal microbial diversity in a strictly vegetarian woman was determined by the 16S rDNA library method, terminal restriction fragment length polymorphism (T-RFLP) analysis and a culture-based method. The 16S rDNA library was generated from extracted fecal DNA, using bacteria-specific primers. Randomly selected clones were partially sequenced. T-RFLP analysis was performed using amplified 16S rDNA. The lengths of T-RF were analyzed after digestion by HhaI and MspI. The cultivated bacterial isolates were used for partial sequencing of 16S rDNA. Among 183 clones obtained, approximately 29% of the clones belonged to 13 known species. About 71% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). A total of 55 species or phylotypes were identified among the 16S rDNA library, while the cultivated isolates included 22 species or phylotypes. In addition, many new phylotypes were detected from the 16S rDNA library. The 16S rDNA library and isolates commonly included the Bacteroides group, Bifidobacterium group, and Clostridium rRNA clusters IV, XIVa, XVI and XVIII. T-RFLP analysis revealed the major composition of the vegetarian gut microbiota were Clostridium rRNA subcluster XIVa and Clostridium rRNA cluster XVIII. The dominant feature of this strictly vegetarian gut microbiota was the detection of many Clostridium rRNA subcluster XIVa and C. ramosum (Clostridium rRNA cluster XVIII).

Adult↗
PubMed2002

Bacteroides ovatus as the predominant commensal intestinal microbe causing a systemic antibody response in inflammatory bowel disease.

To clarify what bacterial species of commensal intestinal microbes are recognized as the antigens that induce a serum antibody response in patients with inflammatory bowel disease (IBD), 72 subjects consisting of 12 Crohn's disease patients, 30 ulcerative colitis patients, and 30 healthy volunteers were examined for their titers of serum antibody to these intestinal bacteria. In IBD patients, as a result, significant elevations of both the immunoglobulin G (IgG) and IgA titers to Bacteroides ovatus were found. Immunoblotting showed that a definite 19.5-kDa band of B. ovatus was bound to the serum antibody raised in IBD patients. It was thus concluded that B. ovatus causes serum antibody responses in IBD patients, and a 19.5-kDa molecule of this bacterium appears to be the responsible antigen, although the role of this event in pathogenesis remains unclear.

Adult↗