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Six strains of Gram-stain-negative, obligately anaerobic, non-spore-forming, non-motile rods were isolated from human faeces. Based on phylogenetic characteristics, the six isolates were included in the family Ruminococcaceae , and divided into three groups. The six isolates showed 16S rRNA gene sequence similarity values lower than 96.2 % to the closely related species, Oscillibacter ruminantium GH1T, Oscillibacter valericigenes Sjm18-20T and Dysosmobacter welbiomis J115T. Coherently with the 16S rRNA gene sequence results, the in silico DNA–DNA hybridization and average nucleotide identity values clearly indicated that strains MM35T, MM50T and MM59T belong to different species from the closely related three species. Based on phenotypic features and phylogenetic positions, three novel species, Vescimonas coprocola gen. nov., sp. nov., Vescimonas fastidiosa gen. nov., sp. nov. and Pusillimonas faecalis gen. nov., sp. nov. are proposed. The type strain of V. coprocola is strain MM50T (=JCM 34012T=DSM 111893T). The type strain of V. fastidiosa is strain MM35T (=JCM 34016T=DSM 111899T). The type strain of P. faecalis is strain MM59T (=JCM 34011T=DSM 111669T). The DNA G+C contents estimated according to the whole genomes of strains MM35T, MM50T and MM59T were 56.4, 58.2 and 55.2 mol%, respectively.

Two strictly anaerobic strains (MB11T and MB56) were isolated from common marmoset (Callithrixjacchus) faeces. Cells of the two strains were Gram-stain-negative, pleomorphic short (strain MB11T) or long (strain MB56) rods. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates were related to the genus Phascolarctobacterium . They had 16S rRNA gene sequences similarities lower than 93 % to previously described species, Phascolarctobacterium faecium ACM 3679T and Phascolarctobacterium succinatutens YIT 12067T, and 98.7 % between themselves. DNA–DNA hybridization values showed that strains MB11T and MB56 were the same species. The genomic DNA G+C content of strains MB11T and MB56 were 47.3–47.4 mol% and 47.7–48.0 mol%. The isolates had different enzymatic activities compared with P. succinatutens JCM 16074T and different major cellular fatty acids compared with P. faecium ACM 3679T. Substrate availability revealed that they utilized not only succinate, but also pyruvate. With pyruvate supplementation, they produced both propionate and acetate, while only propionate production occured with succinate. As suggested by the phylogenic and physiological properties of strains MB11T and MB56, we propose the name Phascolarctobacterium wakonense sp. nov. with the type strain MB11T (=JCM 32899T=DSM 107697T).

Chronic diarrhea in laboratory-bred marmosets poses a serious health problem during experiments. Despite a growing demand for laboratory-bred experimental marmosets, the mechanisms underlying the development of diarrhea and measures for its treatment and prevention remain unclear. To explore the factors affecting development of chronic diarrhea in laboratory-bred marmosets, the gut microbiota composition (GMC) of 58 laboratory-bred marmosets, including 19 animals with chronic diarrhea, was analyzed using terminal restriction fragment length polymorphism. We found that the GMCs in these animals cluster into two groups that differ significantly in rate of chronic diarrhea (56.5% in one group, Cluster 1, and 17.1% in Cluster 2). Additionally, a higher α-diversity and a lower proportion of Bifidobacterium spp. according to quantitative PCR was found the animals in the Cluster 1 than in those in Cluster 2. Taken together, our findings indicate that there is a relationship between GMC and development of chronic diarrhea in laboratory-bred marmosets. This is the first study to highlight the potential of assessing GMC in relation to development of chronic diarrhea in laboratory-bred marmosets.

The development of gut microbiota during infancy is an important event that affects the health status of the host; however, the mechanism governing it is not fully understood. l-Amino acid oxidase 1 (LAO1) is a flavoprotein that catalyzes the oxidative deamination of particular l-amino acids and converts them into keto acids, ammonia, and H

A large number of commensal bacteria inhabit the intestinal tract, and interbacterial communication among gut microbiota is thought to occur. In order to analyze symbiotic relationships between probiotic strains and the gut microbiota, a ring with a membrane filter fitted to the bottom was used for in vitro investigations. Test strains comprising probiotic nitto strains (Lactobacillus acidophilus NT and Bifidobacterium longum NT) and type strains (L. acidophilus JCM1132T and B. longum JCM1217T) were obtained from diluted fecal samples using the membrane filter to simulate interbacterial communication. Bifidobacterium spp., Streptococcus pasteurianus, Collinsella aerofaciens, and Clostridium spp. were the most abundant gut bacteria detected before coculture with the test strains. Results of the coculture experiments indicated that the test strains significantly promote the growth of Ruminococcus gnavus, Ruminococcus torques, and Veillonella spp. and inhibit the growth of Sutterella wadsworthensis. Differences in the relative abundances of gut bacterial strains were furthermore observed after coculture of the fecal samples with each test strain. Bifidobacterium spp., which was detected as the dominant strain in the fecal samples, was found to be unaffected by coculture with the test strains. In the present study, interbacterial communication using bacterial metabolites between the test strains and the gut microbiota was demonstrated by the coculture technique. The detailed mechanisms and effects of the complex interbacterial communications that occur among the gut microbiota are, however, still unclear. Further investigation of these relationships by coculture of several fecal samples with probiotic strains is urgently required.

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.

The human intestinal microbiota (HIM) settles from birth and continues to change phenotype by some factors (e.g. host’s diet) throughout life. However, the effect of extreme life environment on human HIM composition is not well known. To understand HIM fluctuation under extreme life environment in humans, fecal samples were collected from six Japanese men on a long Antarctic expedition. They explored Antarctica for 3 months and collected their fecal samples at once-monthly intervals. Using terminal restriction fragment length polymorphism (T-RFLP) and real time polymerase chain reaction (PCR) analysis, the composition of HIM in six subjects was investigated. Three subjects presented restoration of HIM after the expedition compared versus before and during the expedition. Two thirds samples collected during the expedition belonged to the same cluster in dendrogram. However, all through the expedition, T-RFLP patterns showed interindividual variability. Especially, Bifidobacterium spp. showed a tendency to decrease during and restore after the expedition. A reduction of Bifidobacterium spp. was observed in five subjects the first 1 month of the expedition. Bacteroides thetaiotaomicron, which is thought to proliferate during emotional stress, significantly decreased in one subject, indicating that other factors in addition to emotional stress may affect the composition of HIM in this study. These findings could be helpful to understand the effect of extreme life environment on HIM., 資料番号: PA1510088000

Two bacterial strains, designated 180-3T and 214-4T, isolated from human faeces were characterized by using a polyphasic taxonomic approach that included analysis of their phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these strains represented members of the genus Butyricimonas . These strains shared 97.9 % 16S rRNA gene sequence similarity with each other and were related to Butyricimonas virosa JCM 15149T (97 % sequence similarity) and Butyricimonas synergistica JCM 15148T (94–95 %). Although strain 180-3T was related to (but distinct from) B. virosa JCM 15149T and B. synergistica JCM 15148T, with hsp60 gene sequence similarities of 89.4 and 84.6 %, respectively, strain 214-4T exhibited high hsp60 gene sequence similarity (100 %) with B. virosa JCM 15149T and was different from B. synergistica JCM 15148T (83.5 %). DNA–DNA hybridization experiments demonstrated a genomic distinction of strains 180-3T and 214-4T from B. virosa JCM 15149T and B. synergistica JCM 15148T. The strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-stain-negative rods. Growth of the strains was inhibited on medium containing 20 % bile. The two strains produced butyric and isobutyric acids as the end products from glucose, as has been observed in the other two species of the genus Butyricimonas . The major cellular fatty acid of strains 180-3T and 214-4T was iso-C15 : 0. The major menaquinone of the isolates was MK-10 (>50 %). Strains 180-3T and 214-4T have DNA G+C contents of 45 mol%. On the basis of these data, strains 180-3T and 214-4T represent two novel species of the genus Butyricimonas , for which the names Butyricimonas faecihominis sp. nov. and Butyricimonas paravirosa sp. nov., respectively, are proposed. The type strains of B. faecihominis and B. paravirosa are 180-3T ( = JCM 18676T = CCUG 65562T) and 214-4T ( = JCM 18677T = CCUG 65563T), respectively.

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×10(8) colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10(4.8) and 10(5.8) cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.

Strains of Gram-stain-negative, anaerobic, rod-shaped bacteria were isolated from chinchilla (Chinchilla lanigera) faeces, and strain ST166T was investigated taxonomically. Phylogenetic analyses of 16S rRNA gene sequences revealed that strain ST166T belonged to the genus Parabacteroides . Strain ST166T formed a distinct line of descent, and the highest sequence similarity to ST166T was found with Parabacteroides merdae JCM 9497T (95.6 %) and Parabacteroides johnsonii JCM 13406T (95.0 %). Analysis of hsp60 gene sequences also supported these relationships. Based on the phenotypic and phylogenetic characteristics, the novel species Parabacteroides chinchillae sp. nov. is proposed. The type strain of P. chinchillae sp. nov. is ST166T ( = JCM 17104T = CCUG 62154T).

Background In Japan, a variety of traditional dietary habits and daily routines have developed in many regions. The effects of these behaviors, and the regional differences in the composition of the gut microbiota, are yet to be sufficiently studied. To characterize the Japanese gut microbiota and identify the factors shaping its composition, we conducted 16S metagenomics analysis of fecal samples collected from healthy Japanese adults residing in various regions of Japan. Each participant also completed a 94-question lifestyle questionnaire. Results We collected fecal samples from 516 healthy Japanese adults (325 females, 191 males; age, 21–88). Heatmap and biplot analyses based on the bacterial family composition of the fecal microbiota showed that subjects’ region of residence or gender were not strongly correlated with the general composition of the fecal microbiota. Although clustering analysis for the whole cohort did not reveal any distinct clusters, two enterotype-like clusters were observed in the male, but not the female, subjects. In the whole subject population, the scores for bowel movement frequency were significantly correlated with the abundances of Christensenellaceae, Mogibacteriaceae, and Rikenellaceae in the fecal microbiota (P 30), which is consistent with previously published results. However, a previously reported correlation between BMI and bowel movement frequency was not observed. In addition, the abundances of these three families were positively correlated with each other and comprised a correlative network with 14 other bacterial families. Conclusions The present study showed that the composition of the fecal microbiota of healthy Japanese adults at the national level was not strongly correlated with subjects’ area of residence or gender. In addition, enterotype partitioning was ambiguous in this cohort of healthy Japanese adults. Finally, the results implied that the abundances of Christensenellaceae, Mogibacteriaceae, and Rikenellaceae, along with several other bacterial components that together comprised a correlative network, contributed to a phenotype characterized by a high frequency of bowel movements and a lean body type. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0898-x) contains supplementary material, which is available to authorized users.

(1993). An Improved Medium for the Isolation of Lactobacilli from Kefir Grains. Bioscience, Biotechnology, and Biochemistry: Vol. 57, No. 1, pp. 119-120.

Green tea is one of the most popular beverages in the world. Its beneficial health effects and components have been extensively reviewed. However, little is known about the influence of green tea consumption on the human intestinal microbiota (HIM), which plays a crucial role in human health. Ten volunteers who did not usually consume green tea, drank it for 10 days and then stopped drinking it for 7 days. Their fecal samples were collected at three time points: before beginning the 10-day green-tea regime, at the conclusion of that 10 days, and 7 days after stopping the regime. Their fecal samples were analyzed by terminal restriction fragment length polymorphism with specific primer-restriction enzyme systems for HIM and by using a real-time PCR method for the Bifidobacterium species. Although the HIM of each subject was relatively stable, the proportion of Bifidobacterium species played an important role in the classification of their fecal microbiota. Although there were inter-individual differences in the Bifidobacterium species, an overall tendency for the proportion of bifidobacteria to increase because of green tea consumption was noted. However, little change was observed in the composition of Bifidobacterium species in each sample. This suggests that the change in proportion was induced, not by an inter-species transition, but by an intra-species increase and/or decrease. In conclusion, green tea consumption might act as a prebiotic and improve the colon environment by increasing the proportion of the Bifidobacterium species.

Enterotoxigenic Bacteroides fragilis (ETBF) strains have been suggested to be associated with acute and persistent diarrheal disease, inflammatory bowel disease and colorectal cancer, although further epidemiological studies are needed for clarification. Here, a pilot study was performed to examine the effect of the oral administration of yogurt supplemented with a probiotic strain on the cell numbers of fecal ETBF in a healthy population. Among 420 healthy adults, 38 subjects were found to be ETBF carriers, giving a prevalence of approximately 9%. Among them, 32 subjects were enrolled in an open, randomized, parallel-group study to ingest yogurt supplemented with a probiotic strain, Bifidobacterium longum BB536 (BB536Y group), for 8 weeks, with milk provided to the control group (milk group). The cell numbers of ETBF and the dominant species of the B. fragilis group were measured by a quantitative PCR method. Compared with the baseline values, there was a significant decrease in the cell number of ETBF at week 8 in the BB536Y group but not in the milk group. Linear mixed models analysis for longitudinal data revealed a significant difference in the changes of ETBF cell number between the two groups during the intervention phase. These results imply the potential of probiotic yogurt for eliminating ETBF in the microbiota, but its clinical significance needs to be evaluated in the future. This is the first report of a possible effect of probiotic intake on ETBF in the microbiota.

In addition to the crew, microbes also find their way aboard the International Space Station (ISS). Therefore, microbial monitoring is necessary for the health and safety of the crew and for general maintenance of the facilities of this station. Samples were collected from three sites in the Japanese experimental module KIBO on the ISS (air diffuser, handrail, and surfaces) for analysis of fungal biota approximately 1 year after this module had docked with the ISS. Samples taken from KIBO before launch and from our laboratory were used as controls. In the case of KIBO, both microbe detection sheet (MDS) and swab culture tests of orbital samples were negative. The MDS were also examined by field emission-scanning electron microscopy; no microbial structures were detected. However, fungal DNAs were detected by real-time PCR and analyzed by the clone library method; Alternaria sp. and Malassezia spp. were the dominant species before launch and in space, respectively. The dominant species found in specimens from the air conditioner diffuser, lab bench, door push panel, and facility surfaces on our laboratory (ground controls) were Inonotus sp., Cladosporium sp., Malassezia spp., and Pezicula sp., respectively. The fungi in the KIBO were probably derived from contamination due to humans, while those in our laboratory came from the environment (e.g., the soil). In conclusion, the cleanliness in KIBO was equivalent to that in a clean room environment on the ground.

Gram-negative anaerobic rods were isolated from chinchilla (Chinchilla lanigera) faeces and three strains, ST170T, ST180 and ST28T, were investigated taxonomically. On the basis of phylogenetic analyses and specific phenotypic characteristics, the three strains belonged to the genus Bacteroides. Phylogenetic analysis of their 16S rRNA gene sequences revealed that strains ST170T and ST180 formed a single cluster and a distinct line of descent. Strain ST170T exhibited 99.7 % 16S rRNA gene sequence similarity with strain ST180 and 95.1, 94.6 and 94.4 % 16S rRNA gene sequence similarity with Bacteroides massiliensis JCM 13223T, Bacteroides dorei JCM 13471T and Bacteroides vulgatus JCM 5826T, respectively. Strain ST28T also formed a distinct line of descent and exhibited the highest 16S rRNA gene sequence similarity with Bacteroides uniformis JCM 5828T (98.1 %). Low DNA–DNA relatedness (1 %) between strain ST28T and B. uniformis JCM 5828T clearly indicated that they belonged to different species. Analysis of hsp60 sequences also supported these relationships. The DNA G+C contents of strains ST170T and ST28T were 45.2 and 41.0 mol%, respectively. On the basis of phenotypic characteristics and phylogenetic data, two novel species, Bacteroides chinchillae sp. nov. (type strain ST170T = JCM 16497T = CCUG 59335T) and Bacteroides rodentium sp. nov. (type strain ST28T = JCM 16496T = CCUG 59334T), are proposed.

hsp60gene sequences were determined for members of the genusBacteroidesand sequence similarities were compared with those obtained for the 16S rRNA gene. Among the 29Bacteroidestype strains, the mean sequence similarity of thehsp60gene (84.5 %) was significantly less than that of the 16S rRNA gene (90.7 %), indicating a high discriminatory power of thehsp60gene. Species of the genusBacteroideswere differentiated well byhsp60gene sequence analysis, except forBacteroides pyogenesJCM 6294T,Bacteroides suisJCM 6292TandBacteroides tectusJCM 10003T. Thehsp60gene sequence analysis and the levels of DNA–DNA relatedness observed demonstrated that these three type strains are a single species. Consequently,B. suisandB. tectusare heterotypic synonyms ofB. pyogenes. This study suggests that thehsp60gene is an alternative phylogenetic marker for the classification of species of the genusBacteroides.

An equol-producing bacterium, strain DZET, which was isolated from human faeces, was characterized by morphological, biochemical and molecular methods. The isolate was Gram-positive, obligately anaerobic, non-spore-forming, asaccharolytic and rod-shaped. 16S rRNA gene sequence analysis showed 92.8, 91.0, 91.1 and 90.6 % similarities withSlackia faecicanis,Slackia exigua,Slackia heliotrinireducensandSlackia isoflavoniconvertens, respectively. Based on these data, we propose a novel species of the genusSlackia,Slackia equolifacienssp. nov. The major cellular fatty acids are C14 : 0, C18 : 1ω9cand C18 : 1ω9cDMA (dimethyl acetal). The DNA G+C content of the strain is 60.8 mol%. The type strain ofS.equolifacienssp. nov. is DZET(=JCM 16059T=CCUG 58231T).

The comprehensive dynamics of intestinal microbiota including uncultured bacteria in response to probiotic consumption have not been well studied. The aims of this study were twofold: firstly to analyze the impact on intestinal microbiota of yogurt fermented by Bifidobacterium animalis subsp. lactis LKM512, Lactobacillus delbrueckii subsp. bulgaricus LKM1759, and Streptococcus thermophilus LKM1742 (LKM512 yogurt) and placebo fermented by these lactic acid bacterial strains without LKM512; and secondly to investigate the changes in intestinal microbiota that influence the concentration of PA, one of the beneficial metabolites produced by bacteria in the intestine. The LKM512 yogurt/placebo trial was performed in six hospitalized elderly patients (three men and three women with an average age of 80.3 years) and lasted seven weeks with the following schedule: pre-consumption for one week, LKM512 yogurt consumption for two weeks, washout period for two weeks, and placebo consumption for two weeks. The amount of ingested LKM512 yogurt or placebo was 100 g/day/individual. Fecal samples were analyzed using T-RFLP and real-time PCR. The T-RFLP patterns in five of the six volunteers were changed in a similar fashion by LKM512 yogurt consumption, although these patterns were individually changed following consumption of placebo. It was confirmed that B. animalis subsp. lactis was increased dramatically and Lactobacillus spp. tended to be decreased by LKM512 yogurt consumption. Some indigenous uncultured bacteria were increased and some decreased by LKM512 yogurt/placebo consumption. The similar changes in the intestinal microbiota of the elderly caused by consumption of the LKM512 yogurt were found to be influenced by the LKM512 strain itself, and not by the lactic acid bacteria in the yogurt. Moreover, this study suggests that the increase in intestinal PA concentrations caused by LKM512 yogurt consumption is probably dependent on the LKM512 strain colonizing the intestine.

Two bacterial strains, designated MT01(T) and MT12(T), isolated from rat faeces were characterized by using a polyphasic taxonomic approach that included analysis of their phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogeny based on 16S rRNA gene sequences. The 16S rRNA gene sequence analysis showed that these strains were members of the family 'Porphyromonadaceae'. The strains shared 94 % 16S rRNA gene sequence similarity with each other and were related to Odoribacter splanchnicus NCTC 10825(T) (86-87 % sequence similarity). The strains consisted of obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. Growth of the strains was inhibited on medium containing 20 % bile. The two strains produced significant levels of butyric and isobutyric acids as end products from glucose. Although the major cellular fatty acid of these two strains and O. splanchnicus JCM 15291(T) was iso-C(15 : 0), strains MT01(T) and MT12(T) showed a higher level of iso-C(15 : 0) (66 and 74 %, respectively) than did O. splanchnicus JCM 15291(T) (48 %). In addition, the ratios of iso-C(15 : 0) to anteiso-C(15 : 0) in whole-cell methanolysates of the two isolates were very much higher than that of O. splanchnicus JCM 15291(T). The major menaquinone of the isolates was MK-10. This menaquinone composition was different from those of other genera of the family 'Porphyromonadaceae', such as Barnesiella (predominant menaquinones: MK-11 and MK-12), Odoribacter (MK-9), Paludibacter (MK-8), Parabacteroides (MK-9 and MK-10), Porphyromonas (MK-9 and MK-10) and Tannerella (MK-10 and MK-11). Menaquinone composition is therefore an important chemotaxonomic characteristic of these micro-organisms. Strains MT01(T) and MT12(T) have DNA G+C contents of 46 mol%. On the basis of these data, strains MT01(T) and MT12(T) represent two novel species of a novel genus, for which the names Butyricimonas synergistica gen. nov., sp. nov. and Butyricimonas virosa sp. nov., respectively, are proposed. The type strains of B. synergistica and B. virosa are MT01(T) (=JCM 15148(T) =CCUG 56610(T)) and MT12(T) (=JCM 15149(T)=CCUG 56611(T)), respectively.

Inflammatory bowel diseases [IBD], ulcerative colitis [UC] and Crohn's disease [CD], are chronic intestinal disorders of unknown etiology. UC and CD are heterogeneous diseases characterized by various genetic abnormalities that lead to overly aggressive T-cell responses to a subset of commensal enteric bacteria in genetically susceptible individuals. As one of critical factors involved in pathogenesis of IBD, relative imbalance of aggressive and protective bacterial species, termed dysbiosis, has been reported by various literatures. Since early days of microbiology, representatives of microbial species [over 400 species] have been isolated from human gastrointestinal tract, and analyses of dysbiosis in IBD were mainly dependent on culture techniques. However, recent molecular ecological studies based on ribosomal RNA [rRNA] sequences have revealed that cultivation has been able only to access a small fraction of the microbial diversity within the gastrointestinal tract. These techniques enable characterization and quantification of the microbiota. Clone libraries enable identification of the composition of the microbiota. Microbial community structure can be analyzed via fingerprinting techniques, and dot blot hybridization or fluorescent in situ hybridization can analyze abundance of particular taxa. Recent report shows a systematic framework of the microbial diversity in the human gut of more than 1000 different species-level phylogenetic types [phylotypes]. This review focuses on recent advances in the molecular ecological approaches for studying the gut microbiota in IBD.

We investigated associations of species of the Bacteroides fragilis group with Japanese cedar pollinosis (JCPsis). Cell numbers of Bacteroides fragilis and Bacteroides intestinalis were significantly higher in JCPsis subjects than in non-JCPsis subjects before the pollen season. They correlated positively with both symptom scores and JCPsis-specific immunoglobulin E levels.

Three new yeast species, Candida kashinagacola (JCM 15019T = CBS 10903T), C. pseudovanderkliftii (JCM 15025T = CBS 10904T), and C. vanderkliftii (JCM 15029T = CBS 10905T) are described on the basis of comparison of nucleotide sequences of large subunit ribosomal DNA D1/D2 region (LSU rDNA D1/D2). The nearest assigned species of the three new species was Candida llanquihuensis. Candida kashinagacola and C. pseudovanderkliftii differed from C. llanquihuensis by 3.8% nucleotide substitution of the region, while C. vanderkliftii did by 4.4%. Three new species differed in a number of physiological and growth characteristics from any previously assigned species and from one another. A phylogenetic tree based on the sequences of LSU rDNA D1/D2 showed that these new species together with Candida sp. ST-246, Candida sp. JW01-7-11-1-4-y2, Candida sp. BG02-7-20-001A-2-1 and C. llanquihuensis form a clade near Ambrosiozyma species. The new species did not assimilate methanol as a sole source of carbon, which supported the monophyly of these non methanol-assimilating species which are closely related to the methylotrophic yeasts. Candida kashinagacola was frequently isolated from the beetle galleries of Platypus quercivorus in three different host trees (Quercus serrata, Q. laurifolia and Castanopsis cuspidata) located in the sourthern part of Kyoto, Japan, thus indicating that this species may be a primary ambrosia fungus of P. quercivorus. On the other hand, C. pseudovanderkliftii and C. vanderkliftii were isolated only from beetle galleries in Q. laurifolia. Candida vanderkliftii was isolated from beetle gallery of Platypus lewisi as well as those of P. quercivorus. Candida pseudovanderkliftii and C. vanderkliftii are assumed to be auxiliary ambrosia fungi of P. quercivorus.

Two new yeast species of the genus Ambrosiozyma are described on the basis of comparison of nucleotide sequences of large subunit of ribosomal DNA D1/D2 region. Ambrosiozyma kamigamensis and Ambrosiozyma neoplatypodis differ from Ambrosiozyma ambrosiae by 17 nucleotides (3.0%) and 16 nucleotides (2.8%), respectively, out of 565. The two species differ from each other by 13 nucleotides. Ambrosiozyma kamigamensis was isolated from galleries of the ambrosia beetle, Platypus quercivorus, in specimens of Quercus laurifolia and Castanopsis cuspidata located in the southern part of Kyoto, Japan. Ambrosiozyma neoplatypodis was isolated from similar material, but only in Q. laurifolia. Ambrosiozyma kamigamensis can be distinguished from the other Ambrosiozyma species by the inability to assimilate erythritol, whereas A. neoplatypodis can be distinguished by the ability to assimilate both L: -arabinose and nitrate. The type strains of A. kamigamensis and A. neoplatypodis are JCM 14990(T) (=CBS 10899(T)) and JCM 14992(T) (=CBS 10900(T)), respectively. This is the first report of new Ambrosiozyma species since the genus was proposed.

The effect of the presence of ruminal protozoa on the composition of fecal microbiota in cattle was investigated. Six castrated Holstein cattle (mean bodyweight 137 kg) were divided into two groups: three faunated and three unfaunated. The fecal bacterial composition of the faunated and unfaunated cattle was compared using a culture method and by terminal restriction fragment length polymorphism (T-RFLP) analysis. Approximately 0.4 to 2.3% of the bacterial cells detected by microscopy formed colonies on medium 10. The major terminal restriction fragments were detected in the T-RFLP profiles generated by Hha I and Msp I digestion in both the faunated and unfaunated cattle. In particular, the Bacteroides group, the Clostridium coccoides group and the Clostridium leptum subgroup might be the known bacterial groups that protozoa influence by Msp I digestion. From the dendrogram analysis by T-RFLP patterns, the faunated and unfaunated cattle were divided into two clusters, I and II, respectively. These results suggest that absence of protozoa in the rumen changes the composition of fecal bacteria.

Three novel micro-organisms, designated strains OMB115(T), OMB118 and OMB120, were isolated from dental plaque from golden hamsters fed with a high-carbohydrate diet. The three strains were Gram-positive, facultatively anaerobic rods that lacked catalase activity. Analysis of their partial 16S rRNA gene sequences indicated that these isolates belonged to the genus Bifidobacterium. They grew under aerobic conditions and each had a DNA G+C content of 53 mol%. On the basis of phylogenetic analyses involving phenotypic characterization and partial 16S rRNA gene sequencing, strain OMB115(T) represents a novel species of the genus Bifidobacterium, for which the name Bifidobacterium tsurumiense sp. nov. is proposed. The type strain is OMB115(T) (=JCM 13495(T) =DSM 17777(T)).

Recently, highly sensitive molecular-biological approaches using the 16S rRNA gene sequence have been carried out for the detection of bacteria. The aim of this study was to detect bacteremia in febrile patients with ulcerative colitis (UC) using a new molecular approach.Fifteen febrile patients with relapsing UC were enrolled, and 15 healthy volunteers participated as normal controls. Blood samples were analyzed for bacteremia using nested polymerase chain reaction (PCR) with universal primers (27F, 529F, 1492R). We investigated the bacterial DNA by means of terminal restriction fragment length polymorphism (T-RFLP) with five restriction enzymes (Alu I, Hha I, Hae III, Msp I, and Rsa I). A terminal restriction fragment (TRF) profile database was created with the predicted profiles of 63 common bacteria isolated from blood cultures, using computer simulation based on sequence information. TRF lengths were analyzed using the TRF profile database and a T-RFLP profiler.The bacterial gene was detected in 9 out of 15 UC patients (60%) and 8 of out 15 controls (53%). The numbers of Hae III- and Rsa I-digested T-RFs and the average number of five restriction enzyme-digested T-RFs were significantly higher in UC patients than in controls (p=0.0189, 0.0151, 0.0092, respectively). In UC patients, the most prevalent species included the 7 common species in controls and 10 other species.In febrile UC patients with relapse, bacteremia undetected by culture was found at high frequency by the PCR method. Therefore, antibiotic treatment for UC can be approved on the basis of the finding of bacteremia in this study.

Brown rice fermented by Aspergillus oryzae (FBRA) is a fiber-rich food. Effects of dietary administration of FBRA on rat fecal microbiota composition were examined. Male Wistar rats were fed a basal diet or a 5% FBRA- or 10% FBRA-containing diet, and fecal microbiota was analyzed by culture and terminal-restriction fragment length polymorphism (T-RFLP) analysis. The viable cell number of lactobacilli significantly increased after feeding 10% FBRA diet for 3 weeks compared with that in the basal diet group and that in the same group at the beginning of the experiment (day 0). An increase in the viable cell number of lactobacilli was also observed after feeding 10% FBRA for 12 weeks compared with the effect of a basal diet. T-RFLP analysis showed an increase in the percentage of lactobacilli cells in feces of rats fed 10% FBRA for 14 weeks. Lactobacilli strains isolated from rat feces were divided into six types based on their randomly amplified polymorphic DNA (RAPD) patterns, and they were identified as Lactobacillus reuteri, L. intestinalis and lactobacilli species based on homology of the partial sequence of 16S rDNA. FBRA contains lactic acid bacteria, but their RAPD patterns and identified species were different from those in rat feces. These results indicated that dietary FBRA increases the number of lactobacilli species already resident in the rat intestine.

A strain isolated from pleural fluid of a patient with suppurative pleuritis (strain GTC 3021T) was characterized in terms of its phenotypic and biochemical features, cellular fatty acid profile and phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that the isolate was a member of the genus Prevotella. The isolate was related to Prevotella enoeca ATCC 51261T with about 92 % 16S rRNA gene sequence similarity. The strain was an obligately anaerobic, non-pigmenting, non-spore-forming, non-motile, Gram-negative rod. Although the phenotypic and biochemical characteristics of the strain were similar to those of P. enoeca JCM 12259T, the cellular fatty acid composition of the isolate was significantly different from that of P. enoeca JCM 12259T (C18 : 1 ω9c and anteiso-C15 : 0 fatty acid content). Based on these data, we propose a novel Prevotella species, Prevotella pleuritidis sp. nov., with the type strain GTC 3021T (=JCM 14110T =CCUG 54350T). The G+C content of the type strain is 45.4 mol%.

Background Circumscribed palmar or plantar hypokeratosis (CPH) is a rare skin disorder only recently described. Objective To determine the diagnostic features and to provide insight into the pathogenesis of CPH, with analysis of two new Japanese cases. Methods Dermoscopy, immunohistochemistry, electron microscopy, polymerase chain reaction amplification for human papillomavirus (HPV) DNA and 16S microbial rRNA gene profiling were conducted. Results Dermoscopy showed characteristic features using both dry and jelly immersion observation; step-like desquamation and a homogeneous erythema with regularly distributed whitish spots. Immunohistochemistry revealed strong staining with anti-pankeratin antibody (AE1+AE3) and anti-keratin 16 antibody, and decreased expression of keratin 2e. EM revealed a breakage of the corneocytes within their cytoplasm, but structures for cell attachment were intact. HPV and lesion-specific bacteria were not detected. Limitations The number of cases analyzed was two. Conclusion Hyperproliferative epidermal state along with enhanced corneocyte fragility may account for the unique features in CPH.

Three Gram-negative, anaerobic, rod-shaped bacteria (strains CB40, CB41 and CB42T) were isolated from human faeces. Based on phylogenetic analysis and specific phenotypic characteristics, these strains were included in the genus Bacteroides, and 16S rRNA gene sequence analysis indicated that these strains represented a novel species. The strains were most closely related to the type strains of Bacteroides barnesiae and Bacteroides salanitronis, with sequence similarities of 93.4 and 89.8 %, respectively. The G+C content of strain CB42T is 44.7 mol%. Major fatty acids were anteiso-C15 : 0, C16 : 0, iso-C17 : 0 3-OH and C18 : 1 ω9c. On the basis of the data presented, a novel Bacteroides species, Bacteroides coprophilus sp. nov., is proposed, with CB42T (=JCM 13818T=DSM 18228T) as the type strain.

Mouse intestinal bacteria (MIB) is a new operational taxonomic unit (OTU) belonging to the Bacteroides subgroup in the Cytophaga-Flavobacter-Bacteroides (CFB) phylum recently found in the intestine of mice, rats and humans. However, their characters are still unknown since they have not yet been isolated by culture. To understand their habitat characteristics in intestinal tracts, the quantification assays of MIB were established using MIB group-specific primers. The MIB population in the intestine was evaluated as a percentage of the number of 16S rRNA gene copy of MIB. A real-time PCR assay using group specific primers showed the fluctuation of MIB inhabitancy and revealed that the MIB population in the small intestine of mice was significantly lower than the large intestinal contents. Moreover, MIB was found in human feces though the number was lower than in murine. This assay using group-specific primers revealed new information about host-preference of MIB.

A bacterial strain isolated from human faeces, M-165T, was characterized in terms of its phenotypic and biochemical features, cellular fatty acid profile, menaquinone profile and phylogenetic position (based on 16S rRNA gene sequence analysis). A 16S rRNA gene sequence analysis showed that the isolate was a member of the genusParabacteroides. Strain M-165Twas closely related toParabacteroides merdaestrains, showing 98 % sequence similarity. The strain was obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative, rod-shaped and was able to grow on media containing 20 % bile. Although the phenotypic characteristics of the strain M-165Twere similar to those ofP. merdae, the isolate could be differentiated fromP. merdaeby means of API 20A tests forl-arabinose andl-rhamnose fermentation. DNA–DNA hybridization experiments revealed the genomic distinctiveness of the novel strain with respect toP. merdaeJCM 9497T(⩽60 % DNA–DNA relatedness). The DNA G+C content of the strain is 47.6 mol%. On the basis of these data, strain M-165Trepresents a novel species of the genusParabacteroides, for which the nameParabacteroides johnsoniisp. nov. is proposed. The type strain is M-165T(=JCM 13406T=DSM 18315T).

Biological risk assessment of food containing recombinant DNA has exposed knowledge gaps related to the general fate of DNA in the gastrointestinal tract (GIT). Here, a series of experiments is presented that were designed to determine if genetic transformation of the naturally competent bacterium Acinetobacter baylyi BD413 occurs in the GIT of mice and rats, with feed-introduced bacterial DNA containing a kanamycin resistance gene (nptII). Strain BD413 was found in various gut locations in germ-free mice at 10(3)-10(5) CFU per gram GIT content 24-48 h after administration. However, subsequent DNA exposure of the colonized mice did not result in detectable bacterial transformants, with a detection limit of 1 transformant per 10(3)-10(5) bacteria. Further attempts to increase the likelihood of detection by introducing weak positive selection with kanamycin of putative transformants arising in vivo during a 4-week-long feeding experiment (where the mice received DNA and the recipient cells regularly) did not yield transformants either. Moreover, the in vitro exposure of actively growing A. baylyi cells to gut contents from the stomach, small intestine, cecum or colon contents of rats (with a normal microbiota) fed either purified DNA (50 microg) or bacterial cell lysates did not produce bacterial transformants. The presence of gut content of germfree mice was also highly inhibitory to transformation of A. baylyi, indicating that microbially-produced nucleases are not responsible for the sharp 500- to 1,000,000-fold reduction of transformation frequencies seen. Finally, a range of isolates from the genera Enterococcus, Streptococcus and Bifidobacterium spp. was examined for competence expression in vitro, without yielding any transformants. In conclusion, model choice and methodological constraints severely limit the sample size and, hence, transfer frequencies that can be measured experimentally in the GIT. Our observations suggest the contents of the GIT shield or adsorb DNA, preventing detectable exposure of feed-derived DNA fragments to competent bacteria.

Fecal microbiota and polyamine concentration obtained from eleven intractable adult-type atopic dermatitis (AD) patients and thirteen healthy adults were compared. Fecal microbiota were analyzed using terminal-restriction fragment length polymorphism. The fecal microbiota of volunteers were divided into two clusters, A (n=16) and B (n=8), and the number of AD patients was found to be higher in Cluster B than Cluster A, suggesting that there are relationships between the obstinacy of intractable adult-type AD and intestinal microbiota in Cluster B. Fecal spermidine concentration in Cluster B were lower than that in Cluster A significantly (P0.05). Fecal putrescine concentration in Cluster B also tended to be lower than that in Cluster A. Terminal-restriction fragment (T-RF) of 122 bp generated by digestion with Hha I, which were predicted as unknown bacteria, were detected characteristically in Cluster A. In contrast, T-RFs of 368/9 bp generated by digestion with Hha I, which were predicted as Enterobacteriaceae, were detected characteristically in Cluster B. These bacteria are closely associated with intestinal polyamine concentration. These findings raise the possibility that a low concentration of intestinal polyamines produced by intestinal microbiota is one of the important factors in the onset of intractable adult-type AD.

L-Arabinose is a natural, poorly absorbed pentose that selectively inhibits intestinal sucrase activity. Rats were fed for 4 weeks on a 20% sucrose diet containing 1% L-arabinose. Experiment 1 was carried out to investigate the effects of L-arabinose feeding on the intestinal microflora and weights of the cecum, cecal contents and fat pad. In experiment 1, the cecal contents in 5% glucose containing 3% skim milk were frozen at -80°C until needed for incubation, while the cecal contents in experiment 2 were incubated immediately after dissection.The body weights of the rats fed on the 1% L-arabinose diet were not significantly different from those fed on the control diet. The weight of the fat pad tended to be less in the L-arabinose group than in the control group, while the weight of the cecum and amount of ammonia in the cecal contents were significantly higher with L-arabinose ingestion. The amount of total organic acids tended to be highest in the cecal contents of the L-arabinose group.Bifidobacteria were observed in the L-arabinose group in experiments 1 and 2, but not in the control group. The viable count of bifidobacteria was not influenced by preserving the cecal contents at -80°C. The results suggest that L-arabinose feeding influenced the intestinal microflora and induced the production of bifidobacteria.