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Biomedical subjects

Mitsuo Sakamoto

Publications and source records attributed to Mitsuo Sakamoto.

At least 37 records · Page 2Linked to original sources
PubMed2005

Detection of potentially novel bacterial components of the human skin microbiota using culture-independent molecular profiling.

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.

Bacteria↗
PubMed2005

Detection and quantification of four species of the genus Clostridium in infant feces.

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species-specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle-and mix-fed infants were relatively higher than in breast-fed infants (P< 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to realtime PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1x10(5) to 3x10(7) cells/g wet feces.

Bottle Feeding↗
PubMed2005

Molecular analysis of human oral microbiota.

OBJECTIVES: The application of molecular, mainly 16S ribosomal RNA (rRNA)-based approaches enables researchers to bypass the cultivation step and has proven its usefulness in studying the microbial composition in a variety of ecosystems, including the human oral cavity. In this mini-review, we describe the impact of these culture-independent approaches on our knowledge of the ecology of the human oral cavity and provide directions for future studies that should emphasize the role of specific strains, species and groups of microbes in periodontal disease. MATERIALS AND METHODS: Recent findings are summarized to elucidate the relationship between periodontal disease and human oral microbiota, including as-yet-to-be-cultured organisms. RESULTS: The real-time polymerase chain reaction (PCR) method was developed to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis (formerly Bacteroides forsythus) and Treponema denticola. The checkerboard DNA-DNA hybridization technique allowed enumeration of large numbers of species in very large numbers of samples. 16S rRNA gene clone library analysis revealed the diversity of human oral microbiota and the existence of as-yet-to-be-cultured organisms that are presumed periodontal pathogens. In addition, terminal restriction fragment length polymorphism (T-RFLP) analysis was applied for assessment of diversity of human oral microbiota. CONCLUSION: Culture-independent approaches are useful for studying the microbial ecology in the human oral cavity and should be useful in the future to elucidate the etiology of periodontal disease.

DNA Probes↗
PubMed2005

[A case of leptospirosis caused by Leptospira borgpetersenii serovar sejroe infected in Bali Island, Indonesia].

We report a patient with leptospirosis caused by Leptospira borgpetersenii serovar Sejroe infection on Bali Island, Indonesia. This 33-year-old Japanese man had stayed at a resort hotel on the island from July 8 to July 13 2004. At the hotel, he swam in the pool, walked barefoot, and lied down in the grass. He developed a high fever and headache 7 days after completing his trip, and was admitted to our hospital on July 23. On admission he showed conjunctival suffusion and complained of myalgias. Laboratory findings included granulocytosis and elevated CRP. Plasmodium spp. were not found in blood smears, and no pathogenic bacteria were isolated from blood or fecal cultures. We diagnosed the patient as leptospirosis upon detection of slender coiled organisms with characteristic morphology by darkfield examination of blood sample. Minocycline 100 mg i.v.b.i.d. showed excellent efficacy. A microscopic agglutination test (MAT) during the convalescent stage demonstrated a significant increase in antibodies against L. borgpetersenii serovar Sejroe, confirming the diagnosis of leptospirosis. Despite occurrence of a pandemic of leptospirosis in certain Southeast Asian countries including Indonesia, information concerning pandemic disease is limited. In addition serovars of "imported" cases representing infection in pandemic areas differ widely from those in domestic cases. Adequate laboratory support therefore is crucial for accurate diagnosis of leptospirosis.

Adult↗
PubMed2005

Movement and fixation of intestinal microbiota after administration of human feces to germfree mice.

Human flora-associated (HFA) mice have been considered a tool for studying the ecology and metabolism of intestinal bacteria in humans, although they have some limitations as a model. Shifts in dominant species of microbiota in HFA mice after the administration of human intestinal microbiota was revealed by 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses. Characteristic terminal restriction fragments (T-RFs) were quantified as the proportion of total peak area of all T-RFs. Only the proportion of the T-RF peak at bp 366, identified as the Gammmaproteobacteria group and the family Coriobacteriaceae, was reduced in this study. Increased T-RFs over time at bp 56, 184, and 196 were affiliated with the Clostridium group. However, most of the isolated bacteria with unique population shifts were phylotypes. The vertical transmission of the intestinal microbiota of the mouse offspring was also investigated by dendrogram analysis derived from the similarity of T-RFLP patterns among samples. As a result, the intestinal microbiota of HFA mice and their offspring reflected the composition of individual human intestinal bacteria with some modifications. Moreover, we revealed that human-derived lactobacilli (HDL), which have been considered difficult to colonize in the HFA mouse intestine in previous studies based on culture methods, could be detected in the HFA mouse intestine by using a lactic acid bacterium-specific primer and HDL-specific primers. Our results indicate that the intestinal microbiota of HFA mice represents a limited sample of bacteria from the human source and are selected by unknown interactions between the host and bacteria.

Animals↗
PubMed2005

Direct cloning of genes encoding novel xylanases from the human gut.

The aim of this study was to identify a novel 1,4-beta-xylanase gene from the mixed genome DNA of human fecal bacteria without bacterial cultivation. Total DNA was isolated from a population of bacteria extracted from fecal microbiota. Using PCR, the gene fragments encoding 5 different family 10 xylanases (xyn10A, xyn10B, xyn10C, xyn10D, and xyn10E) were found. Amino acid sequences deduced from these genes were highly homologous with those of xylanases from anaerobic intestinal bacteria such as Bacteroides spp. and Prevotella spp. Self-organizing map (SOM) analysis revealed that xynA10 was classified into Bacteroidetes. To confirm that one of these genes encodes an active enzyme, a full-length xyn10A gene was obtained using nested primers specific to the internal fragments and random primers. The xyn10A gene encoding the xylanase Xyn10A consists of 1146 bp and encodes a protein of 382 amino acids and a molecular weight of 43,552. Xyn10A was a single module novel xylanase. Xyn10A was purified from a recombinant Escherichia coli strain and characterized. This enzyme was optimally active at 40 degrees C and stable up to 50 degrees C at pH 6.5 and over the pH range 4.0-11.0 at 25 degrees C. In addition, 2 ORFs (ORF1 and ORF2) were identified upstream of xyn10A. These results suggested that many unidentified xylanolytic bacteria exist in the human gut and may contribute to the breakdown of xylan which contains dietary fiber.

Algorithms↗
PubMed2005

Culture-independent analysis of fecal microbiota in cattle.

The phylogenetic diversity of the fecal bacterial community in Holstein cattle was determined by 16S ribosomal RNA gene sequence analysis. The sequences were affiliated with the following phyla: Firmicutes (81.3%), Bacteroidetes (14.4%), Actinobacteria (2.5%), and Proteobacteria (1.4%). The Clostridium leptum subgroup was the most phylogenetically diverse group in cattle feces. In addition, a number of previously uncharacterized and unidentified bacteria were recognized in clone libraries.

Animals↗
PubMed2004

Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries.

The maturation of murine cecal microbiota was determined by terminal restriction fragment polymorphism (T-RFLP) and 16S rRNA gene clone libraries. Cecal microbiota in specific pathogen free (SPF) mice aged four to 10 weeks were collected. The cluster of samples in 4-week-old mice was different from those of other ages based on T-RFLP profiles. The majority of clones obtained in this study belonged to the Clostridium coccoides (C. coccoides) group, the Bacteroides group or the Lactobacillus group. Phylogenetic analysis showed characteristic clusters composed of new operational taxonomic unit (OTU) of the C. coccoides and Bacteroides groups. The existence of a large number of yet unidentified bacteria inhabiting the murine cecum was demonstrated by 16S rRNA gene clone libraries. T-RFLP analysis data were more complex and more sensitive than the patterns generated by computer simulation of 16S rRNA gene clone library analysis data. T-RFLP revealed development with maturation of cecal microbiota including unidentified bacteria of SPF mice.

Age Factors↗
PubMed2004

Monitoring the cell number and viability of Lactobacillus helveticus GCL1001 in human feces by PCR methods.

Real-time polymerase chain reaction (PCR) and nested reverse transcription (RT) PCR were applied to demonstrate the viability of lactobacilli in the feces of volunteers fed fermented milk containing lactobacilli. Two sets of specific primers and a TaqMan probe for real-time PCR were constructed using the S-layer gene as a target. After fermented milk ingestion, Lactobacillus helveticus GCL1001 was detected in the feces of 12 volunteers over a few days, with the maximum number being between 10(4.5) and 10(7.8) cells g(-1) of feces. Moreover, mRNA from this strain was detected in the feces of all volunteers by nested RT-PCR. The results show that these methods are applicable to the demonstration of bacterial viability in feces, and that ingested L. helveticus GCL1001 can survive through the gastrointestinal tract.

Colony Count, Microbial↗
PubMed2004

Prevotella shahii sp. nov. and Prevotella salivae sp. nov., isolated from the human oral cavity.

Two bacterial strains, EHS11(T) and EPSA11(T), which were isolated from the human oral cavity, were characterized in terms of phenotypic and biochemical characteristics, cellular fatty acid profiles and phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that each of the isolates belonged to a novel species of the genus Prevotella. Strain EHS11(T) was related to Prevotella loescheii (about 95 % similarity), whereas strain EPSA11(T) was related to Prevotella oris (about 94 % similarity). Both strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. The cellular fatty acid composition of strain EPSA11(T) was very similar to that of P. oris JCM 8540(T). On the other hand, the cellular fatty acid composition of strain EHS11(T) was significantly different from those of other Prevotella species. The predominant fatty acids in strain EHS11(T) are C(18 : 1)omega9c, C(16 : 0) and C(16 : 0) 3-OH, whereas other Prevotella species, except for P. loescheii JCM 8530(T), possess anteiso-C(15 : 0), iso-C(17 : 0) 3-OH and C(18 : 1)omega9c. The predominant fatty acids in P. loescheii JCM 8530(T) are anteiso-C(15 : 0), C(16 : 0) and C(18 : 1)omega9c. DNA-DNA hybridization experiments revealed a genomic distinction of strains EHS11(T) and EPSA11(T) from P. loescheii JCM 8530(T) and P. oris JCM 8540(T). On the basis of these data, two novel Prevotella species are proposed: Prevotella shahii sp. nov. and Prevotella salivae sp. nov. The type strains of P. shahii and P. salivae are EHS11(T) (=JCM 12083(T)=DSM 15611(T)) and EPSA11(T) (=JCM 12084(T)=DSM 15606(T)), respectively.

DNA, Bacterial↗
PubMed2004

Changes in oral microbial profiles after periodontal treatment as determined by molecular analysis of 16S rRNA genes.

Terminal RFLP (T-RFLP) analysis was used to investigate changes in the oral microbiota in saliva and subgingival plaque samples from one patient with aggressive periodontitis (subject A) and two patients with chronic periodontitis (subjects B and C) before and 3 months after periodontal treatment. Substantial changes in the T-RFLP patterns of subgingival plaque samples of subjects B and C were noted after 3 months of improved oral hygiene and full-mouth supra- and subgingival scaling and root planing. However, there was little change in the subgingival microbiota of subject A. Although the proportions of terminal restriction fragments (T-RFs) larger than 1000 bp were notable in the T-RFLP patterns generated after digestion with HhaI of the samples from two subjects before treatment (subject B, 35.5 %; subject C, 29.6 %), the proportions of these T-RFs were significantly reduced or not detected after treatment (subject B, none; subject C, 4.1 %). Real-time PCR showed a significant change in the proportions of target bacteria in subgingival plaque samples of subject B. After 3 months, the Porphyromonas gingivalis population was markedly reduced (3.1 x 10(-3) %), whereas the proportion of Porphyromonas gingivalis before treatment was 7.6 %. The proportions of Tannerella forsythensis, Treponema denticola and Treponema socranskii were also markedly diminished after treatment. Similarly, the proportion of the T-RF presumed to represent Porphyromonas gingivalis was 5.9 % and became undetectable after 3 months. Analysis of 16S rRNA gene clone libraries from subgingival plaque samples of subject B before and after treatment showed a notable change in the subgingival microbiota. These results were in agreement with the T-RFLP analysis data and showed that the T-RFs larger than 1000 bp represent Peptostreptococcus species. Our results indicate that T-RFLP analysis is useful for evaluation of the effects of medical treatment of periodontitis.

Adult↗
PubMed2004

Evaluation of three different forward primers by terminal restriction fragment length polymorphism analysis for determination of fecal bifidobacterium spp. in healthy subjects.

The 27F forward primer is frequently used in 16S rRNA gene libraries and T-RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T-RFLP method with three different forward primers (27F, 35F, and 529F) in conjunction with one reverse primer (1492R). T-RFLP analysis of fecal microbiota using 35F and 529F detected higher proportions of the terminal restriction fragment (T-RF) corresponding to Bifidobacterium spp. than that using 27F. 27F is in imperfect agreement while 35F and 529F are in good concordance with the 16S rRNA gene sequences of Bifidobacterium spp., and the latter primers allowed for the detection of T-RFs of Bifidobacterium spp. in fecal samples from five healthy subjects. The T-RFs presumed to be Bifidobacterium spp. were cloned and sequenced, and found to match the 16S rRNA gene sequences of Bifidobacterium spp. Among the five fecal samples, two samples with low frequencies of T-RFs of Bifidobacterium spp. were detected using these forward primers. This probably reflects a low prevalence of Bifidobacterium spp. in these two samples. Our study emphasizes the importance of selecting a suitable forward primer for detection of Bifidobacterium spp.

Adult↗
PubMed2004

Comparison among fecal secondary bile acid levels, fecal microbiota and Clostridium scindens cell numbers in Japanese.

Bile acid 7alpha-dehydroxylation by intestinal bacteria, which converts cholic acid and chenodeoxycholic acid to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, is an important function in the human intestine. Clostridium scindens is one of the most important bacterial species for bile acid 7alpha-dehydroxylation because C. scindens has high levels of bile acid 7alpha-dehydroxylating activity. We quantified C. scindens and secondary bile acids, DCA and LCA, in fecal samples from 40 healthy Japanese and investigated their correlation. Moreover, we used terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the effect of fecal microbiota on secondary bile acid levels. There was no correlation between C. scindens and secondary bile acid in fecal samples. On the other hand, T-RFLP analysis demonstrated that fecal microbiota associated with high levels of DCA were different from those associated with low levels of DCA, and furthermore that fecal microbiota in the elderly (over 72 years) were significantly different from those in younger adults (under 55 years). These results suggest that intestinal microbiota have a stronger effect on DCA level than does the number of C. scindens cells.

Adult↗
PubMed2004

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.

We examined the effects of probiotic Lactobacillus strains of Lactobacillus agilis JCM 1048 and Lactobacillus salivarius subsp. salicinius JCM 1230 on jejunal and cecal microbiota of broiler chicken under heat stress condition using terminal restriction fragment length polymorphism (T-RFLP) analysis. The jejunal bacterial community was limited to a few bacterial groups, mostly Lactobacillus spp. A relatively abundant and higher prevalence of Lactobacillus spp. were observed in the jejunal and cecal microbiota of the probiotic chickens compared with those of the control chickens under heat stress condition. In general, the probiotic strains did not significantly affect the abundance of L. agilis and L. salivarius in chicken intestine but clearly contributed to increasing their prevalence in the probiotic chickens. The probiotic Lactobacillus strains enriched the diversity of Lactobacillus flora in chicken jejunum and cecum by increasing the abundance and prevalence of Lactobacillus spp. inhabiting the intestine. The richness of Lactobacillus species tended to be similar among the jejunal and cecal microbiota. The bacterial community of cecum was complex and age-dependent. The major components of the cecal microbiota were clostridia and lactobacilli. The Clostridium subcluster XIVa was the most predominant group in chicken cecum. Probiotic Lactobacillus strains restored the microbial balance and maintained the natural stability of indigenous bacterial microbiota following heat stress-induced changes.

Age Factors↗
PubMed2004

[Gnathostomiasis caused by Gnathostoma spinigerum etiologically diagnosed upon extraction of the worm from the skin].

We report a patient with gnathostomiasis in whom a specific diagnosis of Gnathostoma spinigerum infestation was made morphologically upon removal of the worm. A 47-Year-old Japanese male on a business trip to Vietnam ate fried frog with a Vietnamese friend in January 2002, the friend was diagnosed with gnathostomiasis in June 2002. The patient noted swelling of the right leg with migration to the right arm, prompting him to our hospital in February 2003. Hematologic examination showed eosinophilia, and specific anti-gnathostome antibody was detected by a dot enzyme-linked immunosorbent assay (Dot ELISA) in the serum. He was diagnosed as gnathostomiasis, and was given albendazole 400 mg b.i.d. On day 11 of therapy the patient removed a larval worm from the right palmar lesion by pinching with his nails. The worm was identified as G. spinigerum based on morphologic characteristics including number of hooklets on its head-bulb. When gnathostomiasis is suspected, albendazole should be administered before incision of the skin lesion.

Albendazole↗