Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

IgA nephropathy is one of the leading causes of chronic kidney disease in Japan. Since the origin and mechanisms by which IgA nephropathy develops currently remain unclear, a confirmed disease diagnosis is currently only possible by highly invasive renal biopsy. With the background of the salivary microbiome as a rich source of biomarkers for systemic diseases, we herein primarily aimed to investigate the salivary microbiome as a tool for the non-invasive diagnosis of IgA nephropathy. In a comparison of salivary microbiome profiles using 16S rRNA amplicon sequencing, significant differences were observed in microbial diversity and richness between IgA nephropathy patients and healthy controls. Furthermore, recent studies reported that patients with IgA nephropathy are more likely to develop inflammatory bowel diseases and that chronic inflammation of the tonsils triggered the recurrence of IgA nephropathy. Therefore, we compared the salivary microbiome of IgA nephropathy patients with chronic tonsillitis and ulcerative colitis patients. By combining the genera selected by the random forest algorithm, we were able to distinguish IgA nephropathy from healthy controls with an area under the curve (AUC) of 0.90, from the ulcerative colitis group with AUC of 0.88, and from the chronic tonsillitis group with AUC of 0.70. Additionally, the genus Neisseria was common among the selected genera that facilitated the separation of the IgA nephropathy group from healthy controls and the chronic tonsillitis group. The present results indicate the potential of the salivary microbiome as a biomarker for the non-invasive diagnosis of IgA nephropathy.

DOI： 10.1264/jsme2.ME21006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

In this article, Ligilactobacillus salivarius FFIG strains, isolated from the intestinal tract of wakame-fed pigs, are characterized according to their potential probiotic properties. Strains were evaluated by studying their interaction with porcine intestinal epithelial (PIE) cells in terms of their ability to regulate toll-like receptor (TLR)-3- or TLR4-mediated innate immune responses, as well as by assessing their adhesion capabilities to porcine epithelial cells and mucins. These functional studies were complemented with comparative genomic evaluations using the complete genome sequences of porcine L. salivarius strains selected from subgroups that demonstrated different “immune” and “adhesion” phenotypes. We found that their immunomodulatory and adhesion capabilities are a strain-dependent characteristic. Our analysis indicated that the differential immunomodulatory and adhesive activities of FFIG strains would be dependent on the combination of several surface structures acting simultaneously, which include peptidoglycan, exopolysaccharides, lipoteichoic acid, and adhesins. Of note, our results indicate that there is no correlation between the immunomodulatory capacity of the strains with their adhesion ability to mucins and epithelial cells. Therefore, in the selection of strains destined to colonize the intestinal mucosa and modulate the immunity of the host, both properties must be adequately evaluated. Interestingly, we showed that L. salivarius FFIG58 functionally modulated the innate immune responses triggered by TLR3 and TLR4 activation in PIE cells and efficiently adhered to these cells. Moreover, the FFIG58 strain was capable of reducing rotavirus replication in PIE cells. Therefore, L. salivarius FFIG58 is a good candidate for further in vivo studying the protective effect of lactobacilli against intestinal infections in the porcine host. We also reported and analyzed, for the first time, the complete genome of several L. salivarius strains that were isolated from the intestine of pigs after the selective pressure of feeding the animals with wakame. Further genomic analysis could be of value to reveal the metabolic characteristics and potential of the FFIG strains in general and of the FFIG58 strain, in particular, relating to wakame by-products assimilation.

DOI： 10.3390/microorganisms8111659

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OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. METHODS: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. RESULTS: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. CONCLUSIONS: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection.

DOI： 10.1016/j.ijid.2019.11.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.neures.2020.01.001

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DOI： 10.1080/09168451.2019.1704618

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Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder. In addition to the core symptoms of ASD, many patients with ASD also show comorbid gut dysbiosis, which may lead to various gastrointestinal (GI) problems. Intriguingly, there is evidence that gut microbiota communicate with the central nervous system to modulate behavioral output through the gut-brain axis. To investigate how the microbiota composition is changed in ASD and to identify which microbes are involved in autistic behaviors, we performed a 16S rRNA gene-based metagenomics analysis in an ASD mouse model. Here, we focused on a model with human 15q11-13 duplication (15q dup), the most frequent chromosomal aberration or copy number variation found in ASD. Species diversity of the microbiome was significantly decreased in 15q dup mice. A combination of antibiotics treatment and behavioral analysis showed that neomycin improved social communication in 15q dup mice. Furthermore, comparison of the microbiota composition of mice treated with different antibiotics enabled us to identify beneficial operational taxonomic units (OTUs) for ultrasonic vocalization.

DOI： 10.1016/j.neures.2019.12.010

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DOI： 10.1080/09168451.2019.1644149

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DOI： 10.1002/dev.21827

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Butyricimonas faecihominis 30A1, a butyrate-producing bacterium, was isolated from feces of a Japanese Alzheimer's disease patient. Here, we report the draft genome sequence of this organism. This paper is the first published report of the genomic sequence of a Butyricimonas sp.

DOI： 10.1128/MRA.00462-19

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Language：Japanese   Publisher：(公財)腸内細菌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

This report describes the draft genome sequence of <named-content content-type="genus-species">Weissella viridescens</named-content> UCO-SMC3, isolated from <named-content content-type="genus-species">Helix aspersa</named-content> Müller slime. The reads were generated by a whole-genome sequencing (WGS) strategy on an Illumina MiSeq sequencer and were assembled into contigs with a total estimated size of 1,612,814 bp.

DOI： 10.1128/mra.01654-18

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DOI： 10.1186/s12917-018-1754-z

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The herbal medicine berberine (BBR) has been recently shown to be an AMP-activated protein kinase (AMPK) productive activator with various properties that induce anti-inflammatory responses. We investigated the effects of BBR on the mechanisms of mucosal CD4+T cell activation in vitro and on the inflammatory responses in T cell transfer mouse models of inflammatory bowel disease (IBD). We examined the favorable effects of BBR in vitro, using lamina propria (LP) CD4+ T cells in T cell transfer IBD models in which SCID mice had been injected with CD4+CD45RBhigh T cells. BBR suppressed the frequency of IFN-γ- and Il-17A-producing LP CD4+ T cells. This effect was found to be regulated by AMPK activation possibly induced by oxidative phosphorylation inhibition. We then examined the effects of BBR on the same IBD models in vivo. BBR-fed mice showed AMPK activation in the LPCD4+ T cells and an improvement of colitis. Our study newly showed that the BBR-induced AMPK activation of mucosal CD4+ T cells resulted in an improvement of IBD and underscored the importance of AMPK activity in colonic inflammation.

DOI： 10.1038/s41598-019-48331-w

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Primary sclerosing cholangitis (PSC) is a liver disease known for its frequent concurrence with inflammatory bowel disease. Dysbiosis of the gut microbiota in PSC was reported in several studies, but the microbiological features of the salivary microbiota in PSC have not been established. Here we compared the salivary microbial communities of 24 pediatric-onset PSC patients, 16 age-matched ulcerative colitis (UC) patients, and 24 healthy controls (HCs) by analyzing the bacterial 16S rRNA gene sequence data. The species-richness (α-diversity) showed no significant between-group differences, whereas the overall salivary microbiota structure (β-diversity) showed significant differences among the three groups. Taxonomic assignment revealed that the PSC salivary microbiota were characterized by significant decreases in the abundance of Rothia and Haemophilus compared to the HC group, and significantly decreased Haemophilus and increased Oribacterium compared to the UC group. By combining the genera selected by the random forest algorithm in machine learning, followed by confirmation with 10-fold cross-validation, we were able to distinguish the PSC group from the HC group with the area under the curve (AUC) of 0.7423, and from the UC group with the AUC of 0.8756. Our results indicate the potential of salivary microbiota as biomarkers for a noninvasive diagnosis of PSC.

DOI： 10.1038/s41598-018-23870-w

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Gut microbiota-derived metabolites play important roles in health and disease. D-amino acids and their L-forms are metabolites of gut microbiota with distinct functions. In this study, we show the pathophysiologic role of D-amino acids in association with gut microbiota in humans and mice with acute kidney injury (AKI). In a mouse kidney ischemia/reperfusion model, the gut microbiota protected against tubular injury. AKI-induced gut dysbiosis contributed to the altered metabolism of D-amino acids. Among the D-amino acids, only D-serine was detectable in the kidney. In injured kidneys, the activity of D-amino acid oxidase was decreased. Conversely, the activity of serine racemase was increased. The oral administration of D-serine mitigated the kidney injury in B6 mice and D-serine-depleted mice. D-serine suppressed hypoxia-induced tubular damage and promoted posthypoxic tubular cell proliferation. Finally, the D-serine levels in circulation were significantly correlated with the decrease in kidney function in AKI patients. These results demonstrate the renoprotective effects of gut-derived D-serine in AKI, shed light on the interactions between the gut microbiota and the kidney in both health and AKI, and highlight D-serine as a potential new therapeutic target and biomarker for AKI.

DOI： 10.1172/jci.insight.97957

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DOI： 10.1007/s12275-018-8297-7

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Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

DOI： 10.1111/cmi.12846

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

DOI： 10.1111/cmi.12846

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DOI： 10.25968/MSI.2018.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

DOI： 10.1038/nbt.3960

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Intestinal colonization by bacteria of oral origin has been correlated with several negative health outcomes, including inflammatory bowel disease. However, a causal role of oral bacteria ectopically colonizing the intestine remains unclear. Using gnotobiotic techniques, we show that strains of Klebsiella spp. isolated from the salivary microbiota are strong inducers of T helper 1 (T(H)1) cells when they colonize in the gut. These Klebsiella strains are resistant to multiple antibiotics, tend to colonize when the intestinal microbiota is dysbiotic, and elicit a severe gut inflammation in the context of a genetically susceptible host. Our findings suggest that the oral cavity may serve as a reservoir for potential intestinal pathobionts that can exacerbate intestinal disease.

DOI： 10.1126/science.aan4526

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer New York LLC  

Introduction: Adequate amount of proteins from foods are normally needed to maintain muscle mass of the human body. Although protein intakes of Papua New Guinea (PNG) highlanders are less than biologically adequate, protein deficiency related disorders have rarely been reported. It has been postulated that gut microbiota play a role in such low-protein-adaptation. Objective: To explore underlying biological mechanisms of low-protein adaptation among PNG highlanders by investigating metabolomic profiles of faecal water and urine. Methods: We performed metabolome analysis using faecal water extracted from faecal samples of PNG highlanders, PNG non-highlanders and Japanese subjects. We paid special attention to amino acids and other metabolites produced by gut microbiota, as well as to metabolites involved in nitrogen recycling in the human gut. Results: Our results indicated that amino acid levels were higher in faecal water from PNG highlanders than PNG non-highlanders, but amino acid levels did not differ between PNG highlanders and Japanese subjects. Among PNG highlander samples, amino acid levels tended to be higher in those who consumed less protein. Conclusion: We speculated that a greater proportion of urea was excreted to the intestine among the PNG highlanders than other groups, and that the urea was used for nitrogen salvage. Intestinal bacteria are essential for producing ammonia from urea and also for producing amino acids from ammonia, which is a key process in low-protein adaptation. Profiling the gut microbiota of PNG highlanders is an important avenue for further research into the mechanisms of low-protein adaptation.

DOI： 10.1007/s11306-017-1243-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Post-transplant microbial diversity in the gastrointestinal tract is closely associated with clinical outcomes following allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, little is known about the impact of the fecal microbiota before allo-HSCT. We analyzed fecal samples approximately 2 weeks before conditioning among 107 allo-HSCT recipients between 2013 and 2015. Microbial analysis was performed using 16S rRNA gene sequencing. Operational taxonomic unit-based microbial diversity was estimated by calculating the Shannon index. Patients were classified into three groups based on the diversity index: low (&lt;2), intermediate (2, 3), and high (&gt;3) diversity (18 (16.8%), 48 (44.9%), and 41 (38.3%) patients, respectively). There were no significant differences in the 20-month overall survival, cumulative incidence of relapse, and non-relapse mortality among three groups. The cumulative incidence of grade II to IV acute graft-versus-host disease (aGVHD) was similar among the three groups (low 55.6%; intermediate 35.4%; high 48.8%, p = 0.339, at day 100). Furthermore, we found no differences in the cumulative incidence of grade II to IV acute gastrointestinal GVHD among the three groups (low 38.9%; intermediate 21.3%; high 24.4%, p = 0.778, at day 100). Regarding the composition of microbiota before allo-HSCT, aGVHD patients showed a significantly higher abundance of phylum Firmicutes (p &lt; 0.01) and a lower tendency for Bacteroidetes (p = 0.106) than non-aGVHD patients. Maintenance of Bacteroidetes throughout allo-HSCT may be a strategy to prevent aGVHD.

DOI： 10.1007/s00277-017-3069-8

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Language：English   Publisher：BMJ PUBLISHING GROUP  

DOI： 10.1136/gutjnl-2016-312533

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: The common marmoset has been used as an experimental animal for various purposes. Because its average weight ranges from 250 to 500 g, weight loss quickly becomes critical for sick animals. Therefore, effective and non-stressful treatment for chronic diseases, including diarrhoea, is essential.
Case presentation: We report a case in which faecal microbiota transplantation (FMT) led to immediate recovery from chronic and recurrent diarrhoea caused by Clostridium difficile infection. A male common marmoset experienced chronic diarrhoea after antibiotic treatments. The animal experienced severe weight loss, and a faecal sample was confirmed to be C. difficile-positive but was negative for protozoa. Metronidazole was partially effective at the first administration but not after the recurrence of the clinical signs. Then, oral FMT was administered to the subject by feeding fresh faeces from healthy individuals mixed with the marmoset's usual food. We monitored the faeces by categorization into four groups: normal, loose, diarrhoea, and watery. After the first day of FMT treatment, the marmoset underwent a remarkable recovery from diarrhoea, and after the fourth day of treatment, a test for C. difficile was negative. The clinical signs did not recur. The marmoset recovered from sinusitis and bilateral dacryocystitis, which also did not recur, as a by-product of the improvement in its general health caused by the cessation of diarrhoea after the FMT.
Conclusion: This is the first reported case of successful treatment of a marmoset using oral FMT. As seen in human patients, FMT was effective for the treatment of recurrent C. difficile infection in a captive marmoset.

DOI： 10.1186/s12917-017-1070-z

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Crohn's disease (CD) involves chronic inflammation in the gastrointestinal tract due to dysregulation of the host immune response to the gut microbiome. Even though the host-microbiome interactions are likely contributors to the development of CD, a few studies have detected genetic variants that change bacterial compositions and increase CD risk. We focus on one of the well-replicated susceptible genes, tumor necrosis factor superfamily member 15 (TNFSF15), and apply statistical analyses for personal profiles of genotypes and salivary microbiota collected from CD cases and controls in the Ryukyu Islands, southernmost islands of the Japanese archipelago. Our association test confirmed the susceptibility of TNFSF15 in the Ryukyu Islands. We found that the recessive model was supported to fit the observed genotype frequency of risk alleles slightly better than the additive model, defining the genetic effect on CD if a pair of the chromosomes in an individual consists of all risk alleles. The combined analysis of haplotypes and salivary microbiome from a small set of samples showed a significant association of the genetic effect with the increase of Prevotella, which led to a significant increase of CD risk. However, the genetic effect on CD disappeared if the abundance of Prevotella was low, suggesting the genetic contribution to CD is conditionally independent given a fixed amount of Prevotella. Although our statistical power is limited due to the small sample size, these results support an idea that the genetic susceptibility of TNFSF15 to CD may be confounded, in part, by the increase of Prevotella.

DOI： 10.1007/s00439-017-1764-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Emerging evidence has suggested a potential impact of gut microbiota on the pathophysiology of heart failure (HF). However, it is still unknown whether HF is associated with dysbiosis in gut microbiota. We investigated the composition of gut microbiota in patients with HF to elucidate whether gut microbial dysbiosis is associated with HF. We performed 16S ribosomal RNA gene sequencing of fecal samples obtained from 12 HF patients and 12 age-matched healthy control (HC) subjects, and analyzed the differences in gut microbiota. We further compared the composition of gut microbiota of 12 HF patients younger than 60 years of age with that of 10 HF patients 60 years of age or older. The composition of gut microbial communities of HF patients was distinct from that of HC subjects in both unweighted and weighted UniFrac analyses. Eubacterium rectale and Dorea longicatena were less abundant in the gut microbiota of HF patients than in that of HC subjects. Compared to younger HF patients, older HF patients had diminished proportions of Bacteroidetes and larger quantities of Proteobacteria. The genus Faecalibacterium was depleted, while Lactobacillus was enriched in the gut microbiota of older HF patients. These results suggest that patients with HF harbor significantly altered gut microbiota, which varies further according to age. New concept of heart-gut axis has a great potential for breakthroughs in the development of novel diagnostic and therapeutic approach for HF.

DOI： 10.1371/journal.pone.0174099

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

<title>ABSTRACT</title>

<named-content content-type="genus-species">Bifidobacterium lemurum</named-content> DSM 28807^T was isolated from the gastrointestinal tracts of ring-tailed lemurs (<italic>Lemur catta</italic>). Here, we report the first draft genome sequence of this organism.

DOI： 10.1128/genomea.01656-16

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Probiotic
<italic>Lactobacillus acidophilus</italic>
L-55 was isolated from a healthy human gut. Here, we report the draft genome sequence of this organism.

DOI： 10.1128/genomea.01357-16

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC HEMATOLOGY  

Increasing evidence indicates that the gut microbiota is closely associated with acute graft-versus-host disease (aGVHD) in stem cell transplantation (SCT). Fecal microbiota transplantation (FMT) could represent an alternative treatment option for aGVHD. However, FMT for SCT patients carries a potential risk of infection by infused microbiota because of the severely immunosuppressed status. We therefore conducted a pilot study to evaluate the safety of FMT in SCT. A total of 4 patients with steroid-resistant (n = 3) or steroid-dependent gut aGVHD (n = 1) received FMT. No severe adverse events attributed to FMT were observed. All patients responded to FMT, with 3 complete responses and 1 partial response. Temporal dynamics of microbiota seemed to be linked to the gut condition of patients and peripheral effector regulatory T cells also increased during response to FMT. FMT was safely performed in our patients and might offer a novel therapeutic option for aGVHD. This trial was registered at the University Hospital Medical Information Network (https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000017575) as #UMIN000015115.

DOI： 10.1182/blood-2016-05-717652

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Chronic consumption of excess ethanol increases the risk of colorectal cancer. The pathogenesis of ethanol-related colorectal cancer (ER-CRC) is thought to be partly mediated by gut microbes. Specifically, bacteria in the colon and rectum convert ethanol to acetaldehyde (AcH), which is carcinogenic. However, the effects of chronic ethanol consumption on the human gut microbiome are poorly understood, and the role of gut microbes in the proposed AcH-mediated pathogenesis of ER-CRC remains to be elaborated. Here we analyse and compare the gut microbiota structures of non-alcoholics and alcoholics. The gut microbiotas of alcoholics were diminished in dominant obligate anaerobes (e.g., Bacteroides and Ruminococcus) and enriched in Streptococcus and other minor species. This alteration might be exacerbated by habitual smoking. These observations could at least partly be explained by the susceptibility of obligate anaerobes to reactive oxygen species, which are increased by chronic exposure of the gut mucosa to ethanol. The AcH productivity from ethanol was much lower in the faeces of alcoholic patients than in faeces of non-alcoholic subjects. The faecal phenotype of the alcoholics could be rationalised based on their gut microbiota structures and the ability of gut bacteria to accumulate AcH from ethanol.

DOI： 10.1038/srep27923

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CD4(+) T cells that express the forkhead box P3 (FOXP3) transcription factor function as regulatory T (T-reg) cells and hinder effective immune responses against cancer cells(1-3). Abundant Treg cell infiltration into tumors is associated with poor clinical outcomes in various types of cancers(3-7). However, the role of T-reg cells is controversial in colorectal cancers (CRCs), in which FOXP3(+) T cell infiltration indicated better prognosis in some studies(6-9). Here we show that CRCs, which are commonly infiltrated by suppression-competent FOXP3(hi) T-reg cells, can be classified into two types by the degree of additional infiltration of FOXP3(lo) nonsuppressive T cells(10). The latter, which are distinguished from FOXP3(+) T-reg cells by non-expression of the naive T cell marker CD45RA and instability of FOXP3, secreted inflammatory cytokines. Indeed, CRCs with abundant infiltration of FOXP3(lo) T cells showed significantly better prognosis than those with predominantly FOXP3(hi) T-reg cell infiltration. Development of such inflammatory FOXP3(lo) non-T-reg cells may depend on secretion of interleukin (IL)-12 and transforming growth factor (TGF)-beta by tissues and their presence was correlated with tumor invasion by intestinal bacteria, especially Fusobacterium nucleatum. Thus, functionally distinct subpopulations of tumor-infiltrating FOXP3(+) T cells contribute in opposing ways to determining CRC prognosis. Depletion of FOXP3(hi) T-reg cells from tumor tissues, which would augment antitumor immunity, could thus be used as an effective treatment strategy for CRCs and other cancers, whereas strategies that locally increase the population of FOXP3(lo) non-T-reg cells could be used to suppress or prevent tumor formation.

DOI： 10.1038/nm.4086

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<italic>Leuconostoc mesenteroides</italic>
213M0 was isolated from traditional fermented mare milk airag in Bulgan Aimag, Mongolia. This strain produces a listericidal bacteriocin-like inhibitory substance. Here, we report the draft genome sequence of this organism.

DOI： 10.1128/genomea.00178-16

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<italic>Leuconostoc mesenteroides</italic>
406 was isolated from the traditional fermented mare milk airag in Tuv Aimag, Mongolia. This strain produces an antilisterial bacteriocin. Here, we report the draft genome sequence of this organism.

DOI： 10.1128/genomea.00166-16

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The human gut microbiome has profound influences on the host's health largely through its interference with various intestinal functions. As recent studies have suggested diversity in the human gut microbiome among human populations, it will be interesting to analyse how gut microbiome is correlated with geographical, cultural, and traditional differences. The Japanese people are known to have several characteristic features such as eating a variety of traditional foods and exhibiting a low BMI and long life span. In this study, we analysed gut microbiomes of the Japanese by comparing the metagenomic data obtained from 106 Japanese individuals with those from 11 other nations. We found that the composition of the Japanese gut microbiome showed more abundant in the phylum Actinobacteria, in particular in the genus Bifidobacterium, than other nations. Regarding the microbial functions, those of carbohydrate metabolism were overrepresented with a concurrent decrease in those for replication and repair, and cell motility. The remarkable low prevalence of genes for methanogenesis with a significant depletion of the archaeon Methanobrevibacter smithii and enrichment of acetogenesis genes in the Japanese gut microbiome compared with others suggested a difference in the hydrogen metabolism pathway in the gut between them. It thus seems that the gut microbiome of the Japanese is considerably different from those of other populations, which cannot be simply explained by diet alone. We postulate possible existence of hitherto unknown factors contributing to the population-level diversity in human gut microbiomes.

DOI： 10.1093/dnares/dsw002

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Language：Japanese   Publisher：(公社)日本薬学会  

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<italic>Lactobacillus equigenerosi</italic>
NRIC 0697
^T
was isolated from the gastrointestinal tracts of healthy thoroughbreds. This strain produced unique spherical or oval cells, which is rare in the genus
<italic>Lactobacillus</italic>
. Here, we report the draft genome sequence of this strain.

DOI： 10.1128/genomea.01679-15

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<italic>Streptococcus orisasini</italic>
SH06 was isolated from a healthy thoroughbred gastrointestinal tract. Here, we report the draft genome sequence of this organism. This paper is the first published report of the genomic sequence of
<italic>S. orisasini</italic>
.

DOI： 10.1128/genomea.01566-15

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<italic>Bifidobacterium aesculapii</italic>
DSM 26737
^T
was isolated from feces of baby common marmoset. Here, we report the draft genome sequence of this organism. This paper is the first published report of the genomic sequence of
<italic>B. aesculapii</italic>
.

DOI： 10.1128/genomea.01463-15

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A taxonomic study was performed on 15 bacterial isolates from the caeca of gnotobiotic mice that had been fed with thermophile-fermented compost. The 15 isolates were thermophilic, Gram-stain-positive, facultatively anaerobic, endospore-forming bacteria, and were most closely related to Bacillus thermoamylovorans CNCM I-1378(T). The 16S rRNA gene sequence of strain N-11(T), selected as representative of this new group, showed a similarity of 99.4 % with Bacillus thermoamylovorans CNCM I-1378(T), 94.7 % with Bacillus thermolactis R-6488(T), and 94.4 % with Bacillus kokeshiiformis MO-04(T). The isolates were then classified into two distinct groups based on a (GTG) 5-fingerprint analysis. Two isolates, N-11(T) and N-21, were the representatives of these two groups, respectively. The N-11(T) and N-21 isolates showed 66-71 % DNA-DNA relatedness with one other, but had less than 37 % DNA-DNA relatedness with B. thermoamylovorans LMG 18084(T). The other 13 isolates showed DNA-DNA relatedness values above 74 % with the N-11(T) isolate. All 15 isolates grew at 25-60 degrees C (optimum 50 degrees C), pH 6-8 (optimum pH 7) and were capable of growing on a medium containing 6 % (w/v) NaCl (optimum 0.5 %). The 15 isolates could be distinguished from B. thermoamylovorans LMG 18084(T) because they showed Tween 80 hydrolysis activity and did not produce acid from melibiose. The major fatty acids were anteiso-C-15 : 0, C-16 : 0, iso-C-15 : 0, iso-C-14 : 0 and iso-C-16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and several unidentified phospholipids. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The menaquinone was MK-7. The DNA G + C content was 37.9 mol%. Based on the phenotypic properties, the 15 strains represent a novel species of the genus Bacillus, for which the name Bacillus hisashii sp. nov. is proposed. The type strain is N-11(T) (=NRBC 110226(T) =LMG 28201(T)).

DOI： 10.1099/ijsem.0.000516

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Lactobacillus acetotolerans RIB 9124 (NBRC 13120) was isolated from putrefied (hiochi) Japanese sake. Here we report the complete genome sequence of this organism. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a L. acetotolerans strain. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2015.09.006

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Bifidobacterium angulatum JCM 7096T was isolated from human feces. Here we report the complete genome sequence of this organism. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a B. angulatum strain. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2015.06.412

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

DOI： 10.1016/j.cell.2015.08.058

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Bifidobacterium bifidum JCM 1255(T) was isolated from feces of a breast-fed infant. Here we report the complete genome sequence of this organism. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2015.06.413

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Bifidobacterium breve JCM 1192(T) was isolated from infant feces. Here, we report the complete genome sequence of this organism. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2015.06.414

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Bifidobacterium catenulatum JCM 1194(T) was isolated from human feces. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a B. catenulatum strain. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2015.06.415

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Bifidobacterium pseudocatenulatum JCM 1200(T) was isolated from infant feces. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a B. pseudocatenulatum strain. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2015.06.416

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

DOI： 10.1128/genomea.00481-15

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

DOI： 10.1128/genomea.00485-15

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

OBJECTIVES: The objective of this study was to investigate comparatively the influence of proton-pump inhibitors (PPI) administration on three bacterial communities in the oral cavity, stomach, and colon along the alimentary tract.
METHODS: Forty-five subjects including 18 patients taking PPI were enrolled. Stimulated saliva, gastric fluid (GF), and feces were obtained from each subject for the microbiota analysis through bacterial 16S rRNA gene profiling using the pyrosequencing method.
RESULTS: The species richness (alpha diversity) was similar among these three microbiota, whereas the interindividual diversity (beta diversity) was much higher in the fecal microbiota compared with that in the others. The UniFrac analysis indicated that the salivary and GF microbiota were similar to one another; however, both differed greatly from the fecal microbiota in the overall bacterial community structure. In the comparison between PPI-users and PPI-nonusers, a bacterial cell number increase of similar to 1,000 times was found in the GF of PPI-users using culturing methods, whereas the bacterial number and composition were nearly identical between the two groups using quantitative PCR and a similarity search based on 16S profiling. The beta diversity significantly increased in both the salivary and GF microbiota of PPI-users compared with PPI-nonusers.
CONCLUSIONS: These results suggest that the GF microbiota has recently moved from the saliva. Bacterial overgrowth in the GF by PPI administration may be due to a lack of killing rather than proliferation of the bacteria in the acid-suppressed stomach. The biological significance of the increase in beta diversity by PPI administration remains unclear.

DOI： 10.1038/ctg.2015.20

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Hepatoprotective effects of Rhizopusoryzae/U-1 aqueous extract (RU) were demonstrated in carbon tetrachloride (CCl4)-induced liver-injured rats. In order to investigate the RU effects, the rats were administered RU at a dose of 10 or 100mg/kg of body weight for 10 days before induction of the liver injury by oral administration of CCl4 (125mg/kg body weight). (i) Pretreatment with RU caused a significant decrease in serum lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities that were increased by the administration of CCl4. (ii) RU pretreatment (100mg/kg) increased 5-bromo-2-deoxyuridine incorporation at 48h after CCl4 treatment in hepatocytes. (iii) Histological hematoxylin and eosin staining of the liver showed that RU pretreatment reduced the damage induced by CCl4 administration. (iv) Reverse transcriptase PCR analysis showed RU retreatment caused a transient but significant increase in hepatocyte growth factor (HGF) and a sustained and significant increase in insulin-like growth factor-I (IGF-I) gene expression in hepatocytes injured by CCl4 treatment. From these results, we conclude that oral pre-administration of RU was effective to suppress liver injury induced by the subsequent oral CCl4 administration, and RU-induced increase in IGF-I and HGF gene expression may be, even in part, involved in biological actions of RU in rats.

DOI： 10.1111/asj.12328

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

DOI： 10.1128/genomea.00286-15

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

DOI： 10.1128/genomea.00285-15

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

DOI： 10.1128/genomea.00284-15

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

DOI： 10.1128/genomea.00255-15

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The pathogenesis of multiple sclerosis (MS), an autoimmune disease affecting the brain and spinal cord, remains poorly understood. Patients with MS typically present with recurrent episodes of neurological dysfunctions such as blindness, paresis, and sensory disturbances. Studies on experimental autoimmune encephalomyelitis (EAE) animal models have led to a number of testable hypotheses including a hypothetical role of altered gut microbiota in the development of MS. To investigate whether gut microbiota in patients with MS is altered, we compared the gut microbiota of 20 Japanese patients with relapsing-remitting (RR) MS (MS20) with that of 40 healthy Japanese subjects (HC40) and an additional 18 healthy subjects (HC18). All the HC18 subjects repeatedly provided fecal samples over the course of months (158 samples in total). Analysis of the bacterial 16S ribosomal RNA (rRNA) gene by using a high-throughput culture-independent pyrosequencing method provided evidence of a moderate dysbiosis in the structure of gut microbiota in patients with MS. Furthermore, we found 21 species that showed significant differences in relative abundance between the MS20 and HC40 samples. On comparing MS samples to the 158 longitudinal HC18 samples, the differences were found to be reproducibly significant for most of the species. These taxa comprised primarily of clostridial species belonging to Clostridia clusters XIVa and IV and Bacteroidetes. The phylogenetic tree analysis revealed that none of the clostridial species that were significantly reduced in the gut microbiota of patients with MS overlapped with other spore-forming clostridial species capable of inducing colonic regulatory T cells (Treg), which prevent autoimmunity and allergies; this suggests that many of the clostridial species associated with MS might be distinct from those broadly associated with autoimmune conditions. Correcting the dysbiosis and altered gut microbiota might deserve consideration as a potential strategy for the prevention and treatment of MS.

DOI： 10.1371/journal.pone.0137429

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Oral administration of Lactobacillus reuteri CP3012 or Lactobacillus acidophilus L-92 for 60 days in rats that were previously administered 3,3',4,4',5-pentachlorobiphenyl (PCB126) orally at a dose of 100 mu g/kg of body weight resulted in a significant decrease in hepatic bioaccumulation of PCB126 ( p &lt; 0.05), with levels of 30.7 +/- 3.7 ng/g and 92.6 +/- 25.0 ng/g of liver tissue, respectively, compared with 133.1 +/- 12.7 ng/g of liver tissue in the controls. The electron paramagnetic resonance signal level of the liver PCB126-specific g = 2.49 species in rats administered L. reuteri CP3012 decreased significantly (p &lt; 0.05). Both the bile acid concentration in the feces and total stool output increased significantly following administration of lactobacilli ( p &lt; 0.05); however, adsorption of PCB126 onto the bacterial cells was not observed. These results suggest that these bacteria inhibit reabsorption of PCB126 with bile acid by blocking enterohepatic circulation through absorbing and/or deconjugating the bile acids in the intestinal tract and by promoting excretion of bile acids from the body, thus reducing PCB126 accumulation in the liver.

DOI： 10.3136/fstr.20.821

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/mu g of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes.

DOI： 10.1002/jobm.201200763

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Language：Japanese   Publisher：(一社)日本腎臓学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Analysis of microbiota in various biological and environmental samples under a variety of conditions has recently become more practical due to remarkable advances in next-generation sequencing. Changes leading to specific biological states including some of the more complex diseases can now be characterized with relative ease. It is known that gut microbiota is involved in the pathogenesis of inflammatory bowel disease (IBD), mainly Crohn's disease and ulcerative colitis, exhibiting symptoms in the gastrointestinal tract. Recent studies also showed increased frequency of oral manifestations among IBD patients, indicating aberrations in the oral microbiota. Based on these observations, we analyzed the composition of salivary microbiota of 35 IBD patients by 454 pyrosequencing of the bacterial 16S rRNA gene and compared it with that of 24 healthy controls (HCs). The results showed that Bacteroidetes was significantly increased with a concurrent decrease in Proteobacteria in the salivary microbiota of IBD patients. The dominant genera, Streptococcus, Prevotella, Neisseria, Haemophilus, Veillonella, and Gemella, were found to largely contribute to dysbiosis (dysbacteriosis) observed in the salivary microbiota of IBD patients. Analysis of immunological biomarkers in the saliva of IBD patients showed elevated levels of many inflammatory cytokines and immunoglobulin A, and a lower lysozyme level. A strong correlation was shown between lysozyme and IL-1 beta levels and the relative abundance of Streptococcus, Prevotella, Haemophilus and Veillonella. Our data demonstrate that dysbiosis of salivary microbiota is associated with inflammatory responses in IBD patients, suggesting that it is possibly linked to dysbiosis of their gut microbiota.

DOI： 10.1093/dnares/dst037

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Bifidobacterial plasmids reported so far are derived from a limited number of strains and plasmids of bifidobacterial type strains isolated from humans are unknown. We found that Bifidobacterium kashiwanohenseJCM 15439 (type strain) isolated from a healthy infant contained two cryptic plasmids, designated pBBKW-1 and pBBKW-2. We determined and analyzed the complete sequences of both plasmids. pBBKW-1 (7716 bp) was predicted to replicate by a rolling-circle mechanism and encode six protein-coding genes, two of which are putative replication proteins. pBBKW-1 seems to be a cointegrate plasmid containing two copies of the plasmid pMG1 from Bifidobacterium longum. pBBKW-2 (2920 bp) was predicted to encode six protein-coding genes and be a theta-type replicating plasmid, which has been reported to be more stable than a rolling circle-type replicating plasmid frequently found in bifidobacteria. Our finding will provide new insights into safe recombinant plasmid constructions for humans.

DOI： 10.1111/asj.12095

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Language：English   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/nature13004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Equine Science  

We previously isolated the commensal bacteria lactobacilli and bifidobacteria from the Thoroughbred intestine and prepared the horse probiotics LacFi™, consisting of Lactobacillus ruminis KK14, L. equi KK 15, L. reuteri KK18, L. johnsonii KK21, and Bifidobacterium boum HU. Here, we found that the fve LacFi™ constituent strains remarkably suppressed pro-inflammatory interleukin-17 production in mouse splenocytes stimulated with interleukin-6 and transforming growth factor-β. The protective effects of the probiotic on impaired intestinal barrier function were evaluated in Caco-2 cells treated with tumor necrosis factor-α. Evaluation of transepithelial resistance showed that all the strains exhibited intestinal barrier protective activity, with significant suppression of barrier impairment by L. reuteri KK18. The LacFi™ constituent strains were detected in neonatal LacFi™-administered Thoroughbred feces using polymerase chain reaction denaturing gradient gel electrophoresis and culture methods. These fve strains were found to be the predominant lactobacilli and bifidobacteria in the intestinal microbiota of LacFi™-administered Thoroughbreds. Administration of LacFi™ to neonatal Thoroughbreds decreased diarrhea incidence from 75.9% in the control group (n=29 neonatal Thoroughbreds) to 30.7% in the LacFi™-administered group (n=101 neonatal Thoroughbreds) immediately after birth to 20 weeks after birth. LacFi™ treatment also prevented diarrhea especially at and around 4 weeks and from 10 to 16 weeks. The duration of diarrhea was also shorter in the probiotics-administered group (7.4 ± 0.8 days) than in the control group (14.0 ± 3.2 days). These results indicate that the LacFi™ probiotics regulates intestinal function and contributes to diarrhea prevention. © 2014 Japanese Society of Equine Science.

DOI： 10.1294/jes.25.37

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Although some bacterial strains show potential to prevent colitis, their mechanisms are not fully understood. Here, we investigated the anti-colitic mechanisms of Bifidobacterium longum subsp. infantis JCM 1222(T), focusing on the relationship between interleukin (IL)-17A secreting CD4(+) T cells and intestinal epithelial costimulatory molecules in mice. Oral administration of JCM 1222(T) to mice alleviated dextran sulfate sodium (DSS)-induced acute colitis. The expression of type 1 helper T (Th1)- and IL-17 producing helper T (Th17)-specific cytokines and transcriptional factors was suppressed by JCM 1222(T) treatment. Intestinal epithelial cells (IECs) from colitic mice induced IL-17A production from CD4(+) T cells in a cell-cell contact-dependent manner, and this was suppressed by oral treatment with JCM 1222(T). Using blocking antibodies for costimulatory molecules, we revealed that epithelial costimulatory molecules including CD80 and CD40, which were highly expressed in IECs from colitic mice, were involved in IEC-induced IL-17A response. Treatment of mice and intestinal epithelial cell line Colon-26 cells with JCM 1222(T) decreased the expression of CD80 and CD40. Collectively, these data indicate that JCM 1222(T) negatively regulate epithelial costimulatory molecules, and this effect might be attributed, at least in part, to suppression of IL-17A in DSS-induced colitis.

DOI： 10.1371/journal.pone.0079735

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Lactobacillus casei, L. paracasei, and L. rhamnosus form a closely related taxonomic group (Lactobacillus casei group) within the facultatively heterofermentative lactobacilli. Here, we report the complete genome sequences of L. paracasei JCM 8130 and L. casei ATCC 393, and the draft genome sequence of L. paracasei COM0101, all of which were isolated from daily products. Furthermore, we re-annotated the genome of L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG), which we have previously reported. We confirmed that ATCC 393 is distinct from other strains previously described as L. paracasei. The core genome of 10 completely sequenced strains of the L. casei group comprised 1,682 protein-coding genes. Although extensive genome-wide synteny was found among the L. casei group, the genomes of ATCC 53103, JCM 8130, and ATCC 393 contained genomic islands compared with L. paracasei ATCC 334. Several genomic islands, including carbohydrate utilization gene clusters, were found at the same loci in the chromosomes of the L. casei group. The spaCBA pilus gene cluster, which was first identified in GG, was also found in other strains of the L. casei group, but several L. paracasei strains including COM0101 contained truncated spaC gene. ATCC 53103 encoded a higher number of proteins involved in carbohydrate utilization compared with intestinal lactobacilli, and extracellular adhesion proteins, several of which are absent in other strains of the L. casei group. In addition to previously fully sequenced L. rhamnosus and L. paracasei strains, the complete genome sequences of L. casei will provide valuable insights into the evolution of the L. casei group.

DOI： 10.1371/journal.pone.0075073

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

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Adlercreutzia equolifaciens DSM 19450 ^T was isolated from human feces and is able to metabolize daidzeins (soybean isoflavonoids) to equol. Here, we report the finished and annotated genome sequence of this organism.

DOI： 10.1128/genomea.00742-13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Manipulation of the gut microbiota holds great promise for the treatment of inflammatory and allergic diseases(1,2). Although numerous probiotic microorganisms have been identified(3), there remains a compelling need to discover organisms that elicit more robust therapeutic responses, are compatible with the host, and can affect a specific arm of the host immune system in a well-controlled, physiological manner. Here we use a rational approach to isolate CD4(+)FOXP3(+) regulatory T (T-reg)-cell-inducing bacterial strains from the human indigenous microbiota. Starting with a healthy human faecal sample, a sequence of selection steps was applied to obtain mice colonized with human microbiota enriched in T-reg-cell-inducing species. From these mice, we isolated and selected 17 strains of bacteria on the basis of their high potency in enhancing T-reg cell abundance and inducing important anti-inflammatory molecules-including interleukin-10 (IL-10) and inducible T-cell co-stimulator (ICOS)-in T-reg cells upon inoculation into germ-free mice. Genome sequencing revealed that the 17 strains fall within clusters IV, XIVa and XVIII of Clostridia, which lack prominent toxins and virulence factors. The 17 strains act as a community to provide bacterial antigens and a TGF-beta-rich environment to help expansion and differentiation of T-reg cells. Oral administration of the combination of 17 strains to adult mice attenuated disease in models of colitis and allergic diarrhoea. Use of the isolated strains may allow for tailored therapeutic manipulation of human immune disorders.

DOI： 10.1038/nature12331

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Obesity has become more prevalent in most developed countries over the past few decades, and is increasingly recognized as a major risk factor for several common types of cancer(1). As the worldwide obesity epidemic has shown no signs of abating(2), better understanding of the mechanisms underlying obesity-associated cancer is urgently needed. Although several events were proposed to be involved in obesity-associated cancer1,3, the exact molecular mechanisms that integrate these events have remained largely unclear. Here we show that senescence-associated secretory phenotype (SASP)(4,5) has crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of deoxycholic acid (DCA), a gut bacterial metabolite known to cause DNA damage(6). The enterohepatic circulation of DCA provokes SASP phenotype in hepatic stellate cells (HSCs)(7), which in turn secretes various inflammatory and tumour-promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Notably, blocking DCA production or reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer(8) or depleted of senescent HSCs, indicating that the DCA-SASP axis in HSCs has key roles in obesity-associated HCC development. Moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with non-alcoholic steatohepatitis(3), indicating that a similar pathway may contribute to at least certain aspects of obesity-associated HCC development in humans as well. These findings provide valuable new insights into the development of obesity-associated cancer and open up new possibilities for its control.

DOI： 10.1038/nature12347

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of &gt;= 10-fold or a quantity difference in &gt;150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.

DOI： 10.1093/dnares/dst006

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Language：Japanese   Publisher：(一社)日本腎臓学会  

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Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pone.0060521

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Language：Japanese   Publisher：麻布大学  

DOI： 10.14944/00003743

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Other Link： http://search.jamas.or.jp/link/ui/2014010547

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In response to phosphate limitation, bacteria employ the Pho regulon, a specific regulatory network for phosphate acquisition. The two-component signal transduction system of PhoRB plays a crucial role in the induction of Pho regulon genes, leading to the adaptation to phosphate starvation. Herein, we identified the PhoRB system in Bacteroides fragilis, a commensal gut bacterium, and evaluated its role in gut colonization and survival in peritoneal abscesses. BF1575 and BF1576 encoded PhoR (sensor histidine kinase) and PhoB (response regulator) in the sequenced B. fragilis strain YCH46, respectively. Transcriptome analysis revealed that deletion of phoB affected the expression of 585 genes (more than 4-fold change) in B. fragilis, which included genes for stress response (chaperons and heat shock proteins), virulence (capsular polysaccharide biosynthesis) and phosphate metabolism. Deletion of phoB reduced the ability of the bacterium to persist in peritoneal abscesses induced by an intra-abdominal challenge of B. fragilis. Furthermore, PhoB was necessary for survival of this anaerobe in peritoneal abscesses but not for in vitro growth in rich media or in intestinal colonization. These results indicate that PhoB plays an important role in the survival of B. fragilis under stressful extraintestinal conditions.

DOI： 10.1371/journal.pone.0053829

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Aqueous extracts of Rhizopus oryzae (Aq-ROU) have a broad range of physiological activity. Here we identified a new physiological effect of Aq-ROU in rat hepatocyte cell line RLN-10. Aq-ROU induced the accumulation of nitrite, a stable metabolite nitric oxide (NO), in cell culture medium and induced potent diaminofluorescein-FM diacetate staining in the cells. Real-time reverse transcriptase (RT)-PCR analysis showed marked inducible NO synthase gene expression. Additionally, markedly enhanced expression of p22phox and temporally increased expression of NADPH oxidase1 indicated that superoxide was produced. Nuclear translocation of nuclear factor-kappa (NF-κ) B p65 increased remarkably following Aq-ROU and following lipopolysaccharide treatment, a potent activator of NF-κB. Ammonium pyrrolidine-1- carbodithioate, an inhibitor of NF-κB, inhibited NO production following Aq-ROU treatment. Our data indicate that Aq-ROU induces NO production and potentially the production of superoxide, which may contribute to the broad range of physiological effects observed for Aq-ROU ingested by animals.

DOI： 10.1271/bbb.120951

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We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with LNAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.

DOI： 10.1271/bbb.120923

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

D-Alanylation of teichoic acid (TA) affects various functions of Gram-positive bacteria, including immunomodulatory effects. We investigated in this study the impact of D-alanine (D-Ala) in TA from Streptococcus thermophilus ATCC 19258(T) on the barrier-protecting effect in human intestinal Caco-2 cells. ATCC 19258(T) suppressed the tumor necrosis factor-alpha-induced decrease in transepithelial electrical resistance (TER), an indicator of the barrier function. The D-alanylation of TA in ATCC 19258(T) was growth phase- and culture temperature-dependent. Treatment of ATCC 19258(T) with Mg2+ decreased the dlt mRNA expression and DAla content in TA and also abolished the suppressive effect on the TER decrease. Supplementation with L-alanine (L-Ala) to the broth led to an increase of L-Ala in ATCC 19258(T) and of the intestinal barrier-protecting effect. Taken together, D-Ala in TA played an important role in the barrier-protecting effect of S. thermophilus in the intestinal epithelium, and these beneficial effects could be enhanced by exogenous L-Ala.

DOI： 10.1271/bbb.110646

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Publishing type：Research paper (scientific journal)   Publisher：Hindawi Limited  

<italic>Lactococcus garvieae</italic>is a major pathogen for fish. Two complete (ATCC 49156 and Lg2) and three draft (UNIUD074, 8831, and 21881) genome sequences of<italic>L. garvieae</italic>have recently been released. We here present the results of a comparative genomic analysis of these fish and human isolates of<italic>L. garvieae</italic>. The pangenome comprised 1,542 core and 1,378 dispensable genes. The sequenced<italic>L. garvieae</italic>strains shared most of the possible virulence genes, but the capsule gene cluster was found only in fish-pathogenic strain Lg2. The absence of the capsule gene cluster in other nonpathogenic strains isolated from mastitis and vegetable was also confirmed by PCR. The fish and human isolates of<italic>L. garvieae</italic>contained the specific two and four adhesin genes, respectively, indicating that these adhesion proteins may be involved in the host specificity differences of<italic>L. garvieae</italic>. The discoveries revealed by the pangenomic analysis may provide significant insights into the biology of<italic>L. garvieae</italic>.

DOI： 10.1155/2012/728276

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Other Link： http://downloads.hindawi.com/journals/ijmicro/2012/728276.xml

Language：English   Publishing type：Part of collection (book)   Publisher：John Wiley and Sons  

DOI： 10.1002/9781118010549.ch19

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

The proinflammatory cytokine interleukin (IL)-17 plays important roles in various inflammatory diseases, and IL-17-producing T helper 17 cells (Th17) have received much attention. For therapy of Th17-mediated diseases, some reports have indicated the clinical efficacy of lactic acid bacteria, including Streptococcus thermophilus. In this study, we examined the mechanism for the suppressive effects of S. thermophilus ST28 on the Th17 response in murine splenocytes stimulated with transforming growth factor (TGF)-beta plus IL-6. Stimulation with TGF-beta plus IL-6 increased mRNA expression of IL-17 and its production in the splenocytes, but ST28 markedly suppressed both. Meanwhile, ST28 increased the mRNA expression of interferon (IFN)-gamma as well as its production. Anti-IFN-gamma completely cancelled the suppressive effect of ST28 on IL-17 production. From these data, it was concluded that IFN-gamma induced by ST28 had an important role on the effect. A genomic DNA (10 mu g/ml) from ST28 effectively suppressed IL-17 production, probably via the Toll-like receptor 9. Therefore, modulation of Th1/Th17 balance would be one of the mechanisms under which S. thermophilus ST28 exerts an anti-inflammatory effect.

DOI： 10.3892/ijmm.2011.755

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Strains HM2-1 and HM2-2(T) were isolated from the faeces of a healthy infant and were characterized by determining their phenotypic and biochemical features and phylogenetic positions based on partial 16S rRNA gene sequence analysis. They were Gram-positive, obligately anaerobic, non-spore-forming, non-gas-producing, and catalase-negative non-motile rods. They did not grow at 15 or 45 degrees C in anaerobic bacterial culture medium, and their DNA G+C content was in the range 56-59 molok. In enzyme activity tests, strains HM2-1 and HM2-2(T) were positive for alpha/beta-galactosidases and alpha/beta-glucosidases but negative for beta-glucuronidase and cystine arylamidase. An analysis of the cell-wall composition of strains HM2-1 and HM2-2T revealed the presence of glutamic acid, alanine and lysine. The presence of fructose-6-phosphate phosphoketolase shows that isolates HM2-1 and HM2-2(T) are members of the genus Bifidobacterium. These two isolates belong to the same species of the genus Bifidobacterium. Strain HM2-2(T) was found to be related to Bifidobacterium catenulatum JCM 1194(T) (97.4% 16S rRNA gene sequence identity: 1480/1520 bp), Bifidobacterium pseudocatenulatum JCM 1200(T) (97.2%: 1472/1514 bp), Bifidobacterium dentium ATCC 27534(T) (96.7 %: 1459/1509 bp) and Bifidobacterium angulatum ATCC 27535(T) (965%: 1462/1515 bp). The predominant cellular fatty acids of strains HM2-1 and HM2-2(T) were 16:0 and 18:1 omega 9c, with proportions greater than 18% of the total. Phylogenetic analyses involving phenotypic characterization, DNA-DNA hybridization and partial 16S rRNA gene sequencing proves that the strains represent a novel species of the genus Bifidobacterium, for which the name Bifidobacterium kashiwanohense sp. nov. is proposed. The type strain is HM2-2(T) (=JCM 15439(T) =DSM 21854(T)).

DOI： 10.1099/ijs.0.024521-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Segmented filamentous bacteria (SFB) are noncultivable commensals inhabiting the gut of various vertebrate species and have been shown to induce Th17 cells in mice. We present the complete genome sequences of both rat and mouse SFB isolated from SFB-monocolonized hosts. The rat and mouse SFB genomes each harbor a single circular chromosome of 1.52 and 1.59 Mb encoding 1346 and 1420 protein-coding genes, respectively. The overall nucleotide identity between the two genomes is 86%, and the substitution rate was estimated to be similar to that of the free-living E. coli. SFB genomes encode typical genes for anaerobic fermentation and spore and flagella formation, but lack most of the amino acid biosynthesis enzymes, reminiscent of pathogenic Clostridia, exhibiting large dependency on the host. However, SFB lack most of the clostridial virulence-related genes. Comparative analysis with clostridial genomes suggested possible mechanisms for host responses and specific adaptations in the intestine.

DOI： 10.1016/j.chom.2011.08.007

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Lactococcus garvieae causes fatal haemorrhagic septicaemia in fish such as yellowtail. The comparative analysis of genomes of a virulent strain Lg2 and a non-virulent strain ATCC 49156 of L. garvieae revealed that the two strains shared a high degree of sequence identity, but Lg2 had a 16.5-kb capsule gene cluster that is absent in ATCC 49156. The capsule gene cluster was composed of 15 genes, of which eight genes are highly conserved with those in exopolysaccharide biosynthesis gene cluster often found in Lactococcus lactis strains. Sequence analysis of the capsule gene cluster in the less virulent strain L. garvieae Lg2-S, Lg2-derived strain, showed that two conserved genes were disrupted by a single base pair deletion, respectively. These results strongly suggest that the capsule is crucial for virulence of Lg2. The capsule gene cluster of Lg2 may be a genomic island from several features such as the presence of insertion sequences flanked on both ends, different GC content from the chromosomal average, integration into the locus syntenic to other lactococcal genome sequences, and distribution in human gut microbiomes. The analysis also predicted other potential virulence factors such as haemolysin. The present study provides new insights into understanding of the virulence mechanisms of L. garvieae in fish.

DOI： 10.1371/journal.pone.0023184

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The human gut is colonized with a wide variety of microorganisms, including species, such as those belonging to the bacterial genus Bifidobacterium, that have beneficial effects on human physiology and pathology(1-3). Among the most distinctive benefits of bifidobacteria are modulation of host defence responses and protection against infectious diseases(4-6). Nevertheless, the molecular mechanisms underlying these effects have barely been elucidated. To investigate these mechanisms, we used mice associated with certain bifidobacterial strains and a simplified model of lethal infection with enterohaemorrhagic Escherichia coli O157:H7, together with an integrated &apos;omics&apos; approach. Here we show that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in certain bifidobacteria contribute to protecting mice against death induced by E. coli O157:H7. We found that this effect can be attributed, at least in part, to increased production of acetate and that translocation of the E. coli O157:H7 Shiga toxin from the gut lumen to the blood was inhibited. We propose that acetate produced by protective bifidobacteria improves intestinal defence mediated by epithelial cells and thereby protects the host against lethal infection.

DOI： 10.1038/nature09646

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HINDAWI PUBLISHING CORPORATION  

The effects of Streptococcus thermophilus ST28 on cytokine production by murine splenocytes stimulated with transforming growth factor-beta plus interleukin- (IL-) 6 were evaluated. The addition of ST28 significantly repressed IL-17 production compared to ATCC 19258 (type strain). ST28 also decreased the number of Th17 cells in the stimulated splenocytes. The anti-inflammatory effects of ST28 administration were evaluated in mice with colitis induced by dextran sodium sulphate (DSS). Oral treatment of mice with ST28 ameliorated the intestinal lesions by DSS. Upon DSS treatment, IL-17 production in lamina propria lymphocytes (LPLs) was induced, but ST28 significantly decreased its production. ST28 also decreased the percentage of Th17 cells in LPL from DSS-induced colitis. The present results imply that ST28 suppresses the Th17 response in inflamed intestines and would be useful in the treatment of Th17-mediated diseases, such as inflammatory bowel disease.

DOI： 10.1155/2011/378417

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Language：English   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC VET SCI  

Nitric oxide (NO) has been reported as a key mediator in enhancing hepatocyte proliferation during liver regeneration. Juvenile hepatocytes have a strong ability to proliferate while still in their undifferentiated state but the mechanism of NO production and its contribution to hepatocyte proliferation are not yet fully understood. The present study was designed to investigate NO production in the normal liver and its contribution to hepatocyte proliferation in juvenile rats. Endogenous NO production was evaluated quantitatively using a spin trap followed by electron paramagnetic resonance spectroscopy with the Fe-N, N-diethyldithiocarbamate complex as an NO-trapping reagent in the rat liver. NO production in the liver significantly peaked at 3 weeks after birth, but NO synthase (NOS) 3 expression did not change between 2 to 5 weeks after birth, while NOS 1 and NOS 2 mRNA were not detected. Hepatocyte proliferation, measured by the incorporation of 5-bromo-2'-deoxyuridine into the DNA, was found to decline significantly when endogenous NO production was inhibited by the administration of the NOS inhibitor N-G-nitro-L-arginine methyl ester. These findings indicate that endogenous NO production peaked at 3 weeks after birth and hepatocyte proliferation declined significantly when NO production was inhibited. Thus, this study provides a novel insight into the contribution of NO to hepatic growth and liver maturation in juveniles.

DOI： 10.1292/jvms.09-0551

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

This study describes refined electroporation parameters for efficient transformation of Bacteroides fragilis by plasmids prepared from laboratory strains of Escherichia coli. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four E. coli-Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable for B. fragilis transformation, and it generated 2- to 900-fold more transformants (about 10(4) transformants per mu g pLYL05 DNA) than the other plasmids. For the 72-h cultivation period tested, B. fragilis cells harvested at 48 h yielded the highest numbers of transformants. The transformation efficiency of pLYL05 increased linearly with the electric field strength over a range from 5.0 to 12.5 kV/cm. At least 3 h of postpulse incubation was required to maximize the transformation efficiency. For deletion of B. fragilis genes by homologous recombination, competent cells grown to early exponential phase and 12 h of postpulse incubation were required for efficient integration of the pLYL05-based suicide vector into the target site. The expected integration was obtained in B. fragilis strain NCTC9343 only when a homologously prepared (i.e., in vivo methylated) suicide vector was used. Spontaneous resolution of the diploid successfully deleted the expected genetic region. Our simple and efficient plasmid transfer method enabled disruption of a B. fragilis gene using in vivo-methylated targeted vectors. Our optimized electroporation parameters provide a useful tool for genetic manipulation of Bacteroides species.

DOI： 10.1128/AEM.02420-09

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC EXPERIMENTAL BIOLOGY MEDICINE  

We previously reported that nitric oxide (NO) is first detected in the uterus of a pregnant rat on gestational day 13.5 (GD13.5) and that NO levels peak on GD17.5. In addition, NO production in the uterus is mainly derived from the decidua and not the myometrium. The aim of the present study was to reveal the role of NO that peaked on GD17.5 of gestation in the decidua. To inhibit NO production, pregnant rats were continuously administered by an nitric oxide synthase inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME) for 48 h. In the control group, saline was infused instead of L-NAME. After treatment, the decidua were obtained from GD13.5, GD17.5 and GD21.5 rats. Apoptosis and activated caspase-3-positive cells were observed by transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemistry, respectively. The caspase-3 enzyme activity was also measured in the cell lysate from the decidua. The numbers of TUNEL-positive cells and activated caspase-3-positive cells each increased and the amount of caspase-3 activity also increased significantly in rats on GD17.5 than in rats in the control group, but no changes were observed in rats on GD13.5 and GD21.5. Furthermore, enzyme activity regarding the initiator caspases, caspase-8 and -9, upstream factors for caspase-3 in the caspase cascade, was measured simultaneously on GD17.5 under the same treatment. Caspase-8 and -9 enzyme activities increased significantly in the control group; an increment of caspase-8 activity was especially prominent. The present results indicate that an inhibitor of NO production caused apoptosis through typical apoptotic signals in the decidua on GD17.5, suggesting that an NO peak in the decidua is essential to cell survival and the maintenance of uterine formation.

DOI： 10.1258/ebm.2009.009285

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Escherichia coli SE15 (O150:H5) is a human commensal bacterium recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B2, which includes the majority of extraintestinal pathogenic E. coli. Here, we report the finished and annotated genome sequence of this organism.

DOI： 10.1128/JB.01543-09

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

We previously isolated five strains of putative lactobacilli from the faeces of a thoroughbred horse (a 4-year-old male). Of the five strains, four were identified as members of existing Lactobacillus species; however, sequence analysis of the 16S rRNA gene revealed that the fifth isolate, DI70(T) showed approximately 97% identity (1325/1366 bp) with the type strain of Lactobacillus delbrueckii. Therefore, we considered the possibility that DI70(T) represents a novel species of the genus Lactobacillus. Cells of strain DI70(T) were Gram-stain-positive, catalase-negative, non-spore-forming, non-motile rods. In phylogenetic trees constructed on the basis of 16S rRNA gene sequences, strain DI70(T) formed a subcluster in the L. delbrueckii phylogenetic group and was closely related to L. delbrueckii, Lactobacillus crispatus and Lactobacillus jensenii. However, analysis of DNA-DNA relatedness showed that DI70(T) was genetically distinct from its phylogenetic relatives. The isolate also exhibited distinct biochemical and physiological characteristics when compared with its phylogenetic relatives. It required anaerobic conditions for growth on agar medium. The results indicate that isolate DI70(T) indeed represents a novel species of the genus Lactobacillus, for which we propose the name Lactobacillus equicursoris sp. nov. The type strain is DI70(T) (=JCM 14600(T) =DSM 19284(T)).

DOI： 10.1099/ijs.0.009290-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Lactobacillus rhamnosus is a facultatively heterofermentative lactic acid bacterium and is frequently isolated from human gastrointestinal mucosa of healthy individuals. L. rhamnosus ATCC 53103, isolated from a healthy human intestinal flora, is one of the most widely used and well-documented probiotics. Here, we report the finished and annotated genome sequence of this organism.

DOI： 10.1128/JB.01287-09

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We clarified nitric oxide (NO) production in the rat uterus by electron paramagnetic resonance spectroscopy and with Fe-N-(dithiocarboxy) sarcosine complex (an NO-trapping reagent). We examined changes in NO production in the whole uterus, decidua, and myometrium (gestational days 13.5-2.1.5). The expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative reverse transcription-polymerase chain reaction. The uterine NO levels were low on day 13.5, peaked on day 17.5, and thereafter decreased significantly. The NO production levels in the decidua and myometrium were the same on day 13.5, but the levels in the decidua were 2- to 4-fold higher than those in the myometrium from day 15.5 onwards. The NOS-2 mRNA expression pattern correlated well with changes in the NO levels in the decidua, whereas the NOS-3 mRNA was expressed constantly during gestation. Thus NOS-2-generated NO in the decidua contributed significantly to uterine NO levels.

DOI： 10.1271/bbb.90222

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

To detect the predominant lactobacilli in the intestinal flora of healthy thoroughbreds, we isolated lactobacilli from the feces of nine thoroughbreds (five males and four females; 0-15-year-old). The isolated lactobacilli comprise 17 species (37 strains), and they were classified into five groups: Lactobacillus salivarius (6 species), L. reuteri (6 species), Lactobacillus delbrueckii (3 species), L. buchneri (1 species) and L. vitulinus (1 species). On the basis of 16S rRNA gene sequences, we identified 3 other phylogenetic relatives belonging to the genus Lactobacillus. These results suggest that the intestinal flora of thoroughbreds may comprise many species of the genus Lactobacillus. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the 340-bp fragments of the 16S rRNA genes from the same nine fecal samples showed that L. hayakitensis, L. equigenerosi and L. equi are contained in all the samples, suggesting that these species are predominant lactobacilli in the intestinal flora of thoroughbreds.

DOI： 10.1111/j.1740-0929.2009.00633.x

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.47.232

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Other Link： https://jlc.jst.go.jp/DN/JALC/00330628370?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC VET SCI  

To monitor changes in NO production over time in the fetal placenta of rats, we used electron paramagnetic resonance spectroscopy with the Fe-N-(dithiocarboxy) sarcosine (Fe-DTCS) complex as an NO-trapping reagent. The expression of nitric oxide synthase (NOS) isoforms was examined in parallel using quantitative RT-PCR. NO production was first detected on day 13.5 of gestation. NO levels reached a peak on day 15.5, then decreased significantly during the last few days of gestation. The pattern of expression of NOS II mRNA was in good agreement with changes in NO levels, whereas NOS III mRNA expression did not change markedly during gestation. Thus, it appears that NO levels in the placenta are NOS II-dependent and differ at different gestational stages.

DOI： 10.1292/jvms.71.495

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

We sequenced and analyzed the genome of a commensal Escherichia coli (E. coli) strain SE11 (O152:H28) recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B1. SE11 harbored a 4.8 Mb chromosome encoding 4679 protein-coding genes and six plasmids encoding 323 protein-coding genes. None of the SE11 genes had sequence similarity to known genes encoding phage- and plasmid-borne virulence factors found in pathogenic E. coli strains. The comparative genome analysis with the laboratory strain K-12 MG 1655 identified 62 poorly conserved genes between these two non-pathogenic strains and 1186 genes absent in MG1655. These genes in SE11 were mostly encoded in large insertion regions on the chromosome or in the plasmids, and were notably abundant in genes of fimbriae and autotransporters, which are cell surface appendages that largely contribute to the adherence ability of bacteria to host cells and bacterial conjugation. These data suggest that SE11 may have evolved to acquire and accumulate the functions advantageous for stable colonization of intestinal cells, and that the adhesion-associated functions are important for the commensality of E. coli in human gut habitat.

DOI： 10.1093/dnares/dsn026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Four bacterial strains, designated ST18(T), HM244, HM250 and D149, were isolated from the fresh faeces of four thoroughbred horses in Japan. Cells were Gram-positive, strictly anaerobic, catalase-negative, non-spore-forming, non-motile rods that occurred in chains. They were placed in the same subcluster based on 16S rRNA gene sequence analysis, phenotypic characteristics and levels of DNA-DNA relatedness. Their DNA G + C content ranged from 36 to 38 mol%. Lactobacillus catenaformis, Lactobacillus vitulinus and Catenibacterium mitsuokai belong to cluster XVII of the Clostridium subphylum. Strain ST18(T) was most closely related to L. catenaformis ATCC 25536(T) in the phylogenetic tree, but these strains shared only 89.9 %/o (1336/1486 bp) 16S rRNA gene sequence similarity. L. catenaformis, L. vitulinus and C. mitsuokai are homofermentative bacteria, whereas ST18(T) produced CO2 from glucose. Whereas the cell-wall pepticloglycan type of L. catenaformis and L. vitulinus was L-LyS-L-Ala(3), that of C. mitsuokai and the subgroup represented by ST18(T) was A1 gamma-. (L-Ala-D-Glu-meso-diaminopimelic acid). On the basis of 16S rRNA gene sequence divergence of more than 10 % from L. catenaformis as well as phenotypic characteristics, strains ST18(T), HM244, HM250 and D149 are considered to represent a novel species of a new genus belonging to the Clostridium subphylum cluster XVII, for which the name Sharpea azabuensis gen. nov., sp. nov. is proposed. The type strain of Sharpea azabuensis is ST18(T) (=JCM 14210(T) =DSM 18934 T).

DOI： 10.1099/ijs.0.65543-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The effect of the endocrine-disrupting chemical 3,3&apos;,4,4&apos;,5-pentachlorobiphyenl (PCB 126) on intestinal microbiota after oral administration, and the improvement of intestinal microbiota and feces quantity by the subsequent administration of Lactobacillus acidophilus or Lactobacillus reuteri was investigated. All the rats were given 100 mu g/kg bodyweight of PCB 126. The changes in bacterial counts were confirmed using a culture method. The administration of PCB 126 tended to decrease the bacterial counts of lactobacilli (10(9.6)-10(10.2) to 10(8.8)-10(9.2)) and bifidobacteria (10(5.3)-10(6.1) to 10(3.6)-10(4.2)), and to increase those of Enterobacteriaceae (10(8.2)-10(9.1) to 10(9.4)-10(10.3)) and staphylococci (10(6.6)-10(7.4) to 10(7.2)-10(8.4)) compared to no PCB 126 administration. After administration of PCB 126, L. acidophilus or L. reuteri orally administered to rats caused Enterobacteriaceae and staphylococci counts to decrease, suggesting that the intestinal microbiota was improved by the lactobacilli. The administration of L. acidophilus and L. reuteri improved the balance of intestinal microbiota, and defecation volume returned to its normal level. L. acidophilus and L. reuteri have a remedial effect on intestinal microbiota affected by PCB 126 and can function to lessen accumulated PCB 126 volume.

DOI： 10.1111/j.1740-0929.2008.00542.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri)CM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

DOI： 10.1093/dnares/dsn009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOLOGY SOC  

Two strains of lactic acid bacteria were isolated from faeces of two actively racing thoroughbred horses. The isolates formed a subcluster in the Lactobacillus reuteri phylogenetic group, closely related to Lactobacillus fermentum, L. gastricus, L. ingluviei and L. mucosae, by phylogenetic analysis based on 16S rRNA gene sequences. Levels of DNA-DNA relatedness revealed that the isolates belonged to the same taxon and were genetically separated from their phylogenetic relatives. Biochemical and physiological characteristics also distinguished the isolates from their phylogenetic relatives. The isolates produced spherical or oval cells, and tetrad-like cells were rarely seen. To the best of our knowledge, this is the first report of this morphological characteristic within the genus Lactobacillus. Thus, the isolates represent an atypical novel species of the genus Lactobacillus, for which the name Lactobacillus equigenerosi sp. nov. is proposed. The type strain is NRIC 0697(T) (=JCM 14505(T) =DSM 18793(T)).

DOI： 10.1099/ijs.0.65250-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

Protection against in vivo infection with Salmonella and enhancement of in vitro superoxide production by peripheral blood neutrophils are two reported effects of treatment with aqueous extracts of the fungus Rhizopus oryzae U-1 (Aq-ROU). Here, we report that Aq-ROU also has antiproliferative activity and can induce apoptosis in a human promyelocytic leukemia cell line, HL-60. During Aq-ROU-induced apoptosis, HL-60 cells undergo genomic DNA fragmentation characteristic of apoptosis after just 6 hr of treatment. Using phosphatidylserine (PS) as an indicator of apoptosis, we also found that the proportion of apoptotic cells increased significantly after 9 hr of treatment. Indeed, induction of apoptosis by Aq-ROU reached 43.3% after 24 hr of treatment, which is comparable to the effects of a known apoptosis-inducer, actinomycin D. Moreover, the activities of caspase-3, -8, and -9 increased in parallel with Aq-ROU treatment, with a peak of activity 9 hr after the initial treatment. Taken together, the results suggest that R. oryzae contains one or more water-soluble factor that can reliably and efficiently induce apoptosis in human cells via activation of caspase-3.

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Two strains, KBL13(T) and GBL13, were isolated as one of intestinal lactobacilli from the faecal specimens from different thoroughbreds of the same farm where they were born in Hokkaido, Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming and non-motile rods. KBL13(T) and GBL13 homofermentatively metabolize glucose, and produce lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA-DNA hybridization, DNA G + C content and biochemical characterization indicated that these two strains, KBL13(T) and GBL13, belong to the same species. In the representative strain, KBL13(T) the DNA G + C content was 34.3 mol%. Lactobacillus salivarius JCM 1231(T) (=ATCC 11741(T); AF089108) is the type strain most closely related to the strain KBL13(T) as shown in the phylogenetic tree, and the 16S rRNA gene sequence identity showed 96.0% (1425/1484 bp). Comparative 16S rRNA gene sequence analysis of this strain indicated that the two isolated strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives, L. salivarius, Lactobacillus aviarius, Lactobacillus saerimneri and Lactobacillus acidipiscis. DNA-DNA reassociation experiments with L. salivarius and L. aviarius confirmed that KBL13(T) represents a novel species, for which the name Lactobacillus hayakitensis sp. nov. is proposed. The type strain is KBL13T (=jCM 14209(T) =DSM 18933(T)).

DOI： 10.1099/ijs.0.65135-0

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Cell- surface Toll- like receptors ( TLRs) initiate innate immune responses, such as inducible nitric oxide synthase ( iNOS) induction, to microorganisms' surface pathogens. TLR2 and TLR4 play important roles in gastric mucosa infected with Helicobacter pylori ( H. pylori), which contains lipopolysaccharide ( LPS) as a pathogen. The present study investigates their physiological roles in the innate immune response of gastric epithelial cells to H. pylori- LPS. Changes in the expression of iNOS, TLR2, and TLR4, as well as downstream activation of mitogen-activated protein kinases and nuclear factor-kappa B (NF-kappa B), were analyzed in normal mouse gastric mucosal GSM06 cells following stimulation with H. pylori-LPS and interferon-gamma. Specific inhibitors for mitogen-activated protein kinases, NF-kappa B, and small interfering RNA for TLR2 or TLR4 were employed. The immunohistochemistry of TLR2 was examined in human gastric mucosa. H. pylori- LPS stimulation induced TLR2 in GSM06 cells, but TLR4 was unchanged. TLR2 induction resulted from TLR4 signaling that propagated through extracellular signal- related kinase and NF-kappa B activation, as corroborated by the decline in TLR4 expression on small interfering RNA treatment and pretreatment with inhibitors. The induction of iNOS and the associated nitric oxide production in response to H. pylori- LPS stimulation were inhibited by declines in not only TLR4 but also TLR2. Increased expression of TLR2 was identified in H. pylori- infected human gastric mucosa. TLR4 signaling initiated by H. pylori- LPS and propagated via extracellular signal- regulated kinase and NF-kappa B activation induced TLR2 expression in gastric epithelial cells. Induced TLR2 cooperated with TLR4 to amplify iNOS induction. This positive correlation may constitute a mechanism for stimulating the innate immune response against various bacterial pathogens, including H. pylori- LPS.

DOI： 10.1152/ajpgi.00096.2007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Rhizopus oryzae U-1 water extract (ROU-we) was administered orally to rats at 10 mg/kg body weight for 9 days. Salmonella enteritidis was inoculated at a dose of 10(9)CFU/animal. The following protective effects of ROU-we against infection were examined: cell counts of Salmonella in organs of infected rats (liver, spleen, and mesenteric lymph nodes); phagocytic capacity of collected peripheral monocytes and peritoneal macrophages; cell counts of respective helper T-cell subclasses (Th0, Th1, and Th2); and the leukocyte percentage in peripheral blood. The Salmonella cell count in the liver of the ROU-we group decreased significantly compared to that of the control group and the peripheral monocytes' phagocytic capacity increased 4.5-fold. Moreover, the ROU-we group's Th1 response was higher than the infected control group. However, the healthy control group and the ROU-we group showed similar Th1/Th2 balance and cell count tendencies. These results suggest that ROU-we activated peripheral monocytes and improved Th1/Th2 balance, thereby strengthening immunity against Salmonella infection.

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Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a 'hot spot' for horizontal gene transfer between microbes.

DOI： 10.1093/dnares/dsm018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOL RES FOUNDATION  

Diversity and compositions of the Lactobacillus, Streptococcus, and Bifidobacterium group in the feces of six healthy, actively racing horses (Thoroughbreds) were analyzed by using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR with primer sets specific for each group. PCR-DGGE analysis of the feces showed that Lactobacillus equi, Lactobacillus johnsonii, a phylogenetic relative of Lactobacillus salivarius, a phylogenetic relative of Lactobacillus gastricus, and Weissella confusa were predominant in almost all of the feces tested, and Streptococcus bovis/Streptococcus equinus was predominant in the Streptococcus group. The Bifidobacterium group was not detected by single-PCR but atypical species of the group were found in three of the six Thoroughbreds tested by nested-PCR. Calculation and estimation of lactic acid bacteria and bifidobacteria revealed that lactic acid bacteria were predominant in the feces and bifidobacteria were minor. These results indicate that the community of lactic acid bacteria and bifidobacteria in horse feces are unique because of the presence of specific species for horse feces and a minority of the Bifidobacterium group. Repeated tests of the feces from the same horse over 3 months showed that the diversity and composition of lactic acid bacteria and bifidobacteria in the feces was basically stable throughout the test period.

DOI： 10.2323/jgam.53.191

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In this study, the main changes in bacterial floral diversity in the gastrointestinal tract of a Thoroughbred foal were monitored by using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). The foal died of catarrhal enteritis of the cecum and large colon. Diarrheal feces and gastrointestinal contents were compared with normal feces. The closest relatives of the bacterium in the samples were Lactobacillus johnsonii (100% similarity), uncultured Bacteroides sp. (92.5% similarity), Bacteroides fragilis (96.3% similarity), and Enterococcus faecium/Enterococcus durans (100% similarity); these were detected by PCR-DGGE using a universal primer set. Monitoring revealed that the numbers of Escherichia coli/Shigella sonnei (97.9% similarity) were significantly higher in the diarrheal feces. Thus, PCR-DGGE is a useful tool for monitoring the main changes in bacterial floral diversity occurring in the gastrointestinal tracts of Thoroughbreds.

DOI： 10.1016/j.jevs.2006.11.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE  

The efficiency with which lysis of five strictly anaerobic and six facultatively anaerobic bacterial species, all well-known human colonic commensals, were lysed was tested using a reference method for general metagenomic analysis and an improved method that involves higher levels of lysozyme and proteinase K, as well as the addition of achromopeptidase. Ten species were lysed with an efficiency of &gt;80% by the reference method, while the lytic efficiency for Clostridium ramosum JCM 1298(T) was &lt;50%. The lytic efficiency of the improved method for C. ramosum JCM 1298(T) was 82.5%. Similarly, five samples of human feces were tested with these methods, as well as with the QIAamp DNA stool mini kit. Although the efficiency of lysis of the microbes recovered from the fecal samples fluctuated depending on the sample in the cases of the reference method (13.384.6%) and QIAamp DNA stool mini kit (38.8-69.2%), the improved method gave stable and high-level lysis (&gt;90%) for all the fecal samples. Accordingly, since the DNA samples isolated by the improved method can reflect nearly true genomic information in the microbial flora, our improved method should be applicable to metagenomic analyses, not only for bacteria in the human intestine but also for bacteria in other environments.

DOI： 10.1264/jsme2.22.214

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Rat livers and microsomes were subjected to electron paramagnetic resonance (EPR) measurements at 77K. The EPR spectra of the livers from the control group, carbon tetrachloride-, 3-methylcholanthrene-, and 3,3',4,4',5-pentachlorobiphenyl (PCB126)-treated rats exhibited an EPR spectrum at g = 2.40, 2.24, and 1.93, which is characteristic of P450 in a resting state. The liver of the PCB126-treated rats showed an additional distinct EPR spectrum at g = 2.49, 2.26, and 1.87 (g = 2.49-species). The heme environmental structure of g = 2.49-species was identified by crystal field analysis using three EPR g-values of the microsome treated with various chemicals. These results indicated that g = 2.49-species is a hemeprotein with cysteine thiolate at the 5th coordination site, and a nitrogenous ligand at the 6th site.

DOI： 10.1271/bbb.60367

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Two Paenibacillus macerans strains, JCM 2500(T) and MCRI 12, exhibited two types of 16S rDNA copies in their genomes, accompanied by a length difference of 12bp at positions 203 to 214 (Escherichia coli numbering). The long-type sequences were newly identified for P. macerans 16S rDNA, and the copy numbers were different between the two strains. Both types of 16S rRNA were expressed in each strain, and it was predicted that the polymorphism at this position is located in helix H10, based on a comparison with the E. coli 16S rRNA secondary structure model.

DOI： 10.1271/bbb.69.1995

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Enterococcus faecalls TH10 is a lactic acid bacterial strain isolated from the Malaysian traditional fermented food, "tempeh", and has antibacterial activity against various pathogens. To identify the antibacterial substance, the butanol extract of the culture supernatant of E. faecalls TH10 was fractionated by HPLC equipped with a reversed-phase partition column, and the elutes were subjected to antibacterial assay. As the activity was observed In a fraction eluted by ca 80% methanol, the fraction was analyzed by gas chromatography/mass spectrometry and 3-phenyllactic acid was identified as the major compound. Fractionation with an optical isomer separation column showed that the preparation contained D- and L-forms of 3-phenyllactic acid at a ratio of 2:1. Authentic 3-phenyllactic acid showed antibacterial activity against various bacteria such as Staphylococcus aureus and Escherichia coli. These results suggest the possibility that 3-phenyllactic acid is a biopreservative.

DOI： 10.4265/bio.9.77

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The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:117 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:117 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6,000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:117 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.

DOI： 10.1271/bbb.68.1027

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Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10degreesC. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10degreesC. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4degreesC, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (&lt;10 CFU/g) in model cooked meat products, the bacterial counts increased to 10(8) CFU/g at 10degreesC after 7 to 12 days.

DOI： 10.1128/AEM.69.6.3668-3671.2003

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Nitric oxide (NO) production in the rat placenta was monitored and quantified by electron paramagnetic resonance (EPR) spectroscopy with hemoglobin and an Fe-N-(dithiocarboxy) sarcosine (DTCS) complex as NO-trapping reagents. Expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative RT-PCR analysis. The EPR spectrum of the placenta with hemoglobin trapping showed a three-line hyperfine structure (g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOS inhibitor L-NAME was administered to pregnant rats. Therefore, the specific signal was definitely identified as being derived from endogenous NO spin-trapped by hemoglobin, and the EPR spectrum showed that the NO adduct existed as a pentacoordinate alpha-NO heme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal (g = 2.038) derived from an NO-Fe-DTCS complex. The height of the triplet signal did not vary significantly with gestational stage during the last few days of gestation. At the gestational stages examined, the level of NOS II mRNA expression was significantly higher than that of NOS III mRNA. NOS II expression in term (day 21.5) placenta was significantly increased compared with that in preterm (day 19.5) placenta (P &lt; 0.01, n = 4 or 5). These results suggest that NOS II is the predominant producer of NO in the placenta and that NOS II-generated NO plays significant roles in the maintenance of placental functions immediately before birth.

DOI： 10.1152/ajpcell.00101.2001

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Language：Japanese   Publisher：The Japanese Society of Swine Science  

The physicochemical properties of a red pigment in Parma ham were examined in this study and compared with those of other myoglobin (Mb) derivatives whose red color is due to oxymyoglobin (O₂Mb) and nitrosylmyoglobin (NOMb). A water extract of Parma ham was prepared and the absorption spectra of the final filtrate were recorded at 350-650nm. The effects of pH on the absorption spectra were studied subsequent to the addition of NaOH or HCl to the water extract. A single absorption peak was noted at 423nm in the Soret's band and 2 peaks at visible wavelengths of 549 and 587nm. The absorption spectra of red pigment had no change at pH 6-10. At pH 5, the red pigment started to precipitate and increasingly more so as pH became more acidic. No typical absorption peaks for metmyoglobin (MetMb) could be seen at 505 and 630nm at any pH. The sterile water extract of Parma ham was kept in a sterilized test tube for 7 days at low (5°C) or room temperature (20°C) under conditions of light exposure or darkness. The red pigment of the water extract was stable in the dark at each temperature during 7 days of storage. To the water extract of Parma ham and each of the O₂Mb and NOMb solutions, ferricyanide was added. O₂Mb and NOMb were oxidized by ferrycianide to MetMb, but the spectrum of the water extract from Parma ham had no change. The water extract of Parma ham and the O₂Mb and NOMb solutions were heated at 40-70°C for 30min followed by measurement of the absorption spectrum. The heated extract was filtered under sterile conditions. For the water extract, absorption of the spectral peaks decreased, though the spectrum of this pigment was maintained essentially during heating, in contrast to those of O₂Mb and NOMb; precipitation of the pigment was noted with increasing temperature. The pigment precipitated with foreign proteins denatured by heating, but could be detected in its acetone extract.

DOI： 10.5938/youton.36.124

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Longissimus dorsi muscle from six pigs (24 h post-mortem) was cut into portions of similar size and shape (c. 700 g) and vacuum-packed in polyfilm. The muscle specimens were divided into three samples, one frozen at -20°C, another at -80°C and the third served as the control (not frozen). The meat sample frozen at -80°C was transferred to the -20°C freezer. After one month, both frozen pork samples were thawed at -2°C and drip loss (%) was measured. Hunter colour, metmyoglobin (MetMb) formation (%), water-holding capacity (WHC), TBA value, transmission value (TM) and myofibril fragmentation were also determined. There was no significant difference in drip loss for the two frozen samples. No MetMb formation could be detected and Hunter values were basically the same for all three samples. WHC, TBA value and TM were essentially the same for all three samples. TBA value was quite low for each frozen sample, indicating that lipid oxidation did not occur during freezing. Histological examination of both frozen samples indicated inter- and intracellular ice crystal formation at -20°C, and intracellular ice at -80°C, the extent being less than at -20°C. At -20°C, ice crystals were larger and muscle fibre diameter smaller than for the control or -80°C sample. Myofibril fragmentation in both frozen samples was significantly higher than in the control. Pork sausage was prepared from all three samples by adding 2% NaCl and 100 ppm NaNO2. Cooking loss and colour forming ratios were essentially the same. The sausage sample made from the -20°C frozen meat was harder than that of the other two samples according to rheological measurement. © 1994.

DOI： 10.1016/0309-1740(94)P1828-J

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The red cell fraction of animal blood is presently used only to a limited degree in food industries. For use of this fraction as a food component, examination was made of the nitrosation of hemoglobin (Hb) for coloration in meat processing and reduction of nitrite content in processed meat products. In this study, the purified Hb fraction (98.1%) was prepared from cattle blood, and optimum reaction conditions for nitrosation of Hb were determined. The prepared nitroso-Hb (NOHb) was added to experimental sausage and assessment was made of its capacity to enhance meat product color. The following results were obtained: 1) More than 80% of the total Hb in a reaction mixture (pH4.5) of 25mM NaNO₂-25mM ascorbic acid (AsA) was rapidly nitrosated at 2°C and 20°C. 2) Presence of glucose at 40% in the Hb reaction mixture improved the stability of the formed NOHb. Stability was maintained for as long as 20 days at 2°C. 3) Added nitrite in a NOHb mixture virtually disappeared and no aerobic bacteria could detected after 3 days of 2°C or 20°C storage with/without glucose, 4) When 0.5% or 1% of the NOHb reaction mixture was added to porcine loin meat with non-meat protein ingredient solution, NaCl, NaNO₂ and Na-AsA, nitroso heme pigment formation was greater than that of the control (without addition of NOHb) meat product, and added NOHb showed quantitative effect on the color formation of sausage. 5) Hunter color values of the sausage remained essentially unchanged for 2 weeks of storage at 2°C The color stability of the NOHb added sample appeared essentially the same as that of the control under fluorescent lighting, and red color was retained more. 6) TBA values were quite low and showed only slight variation, indicating lipid oxidation not to have occurred after 2 weeks of storage when NOHb prepared from cattle blood has been added to sausage.

DOI： 10.2508/chikusan.64.855

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DOI： 10.1080/00021369.1991.10857756

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DOI： 10.1182/blood.V128.22.4577.4577

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メタゲノムからヒト常在菌叢の生態と機能を読み解く常在細菌叢の最新の話題：腸内細菌叢による宿主免疫系への影響や腸脳相関

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DOI： 10.2177/jsci.36.315

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We sequenced and analyzed the genome of Lactobacillus rhamnosus ATCC 53103 (also known as L. rhamnosus GG), a human isolate, which is one of the most studied probiotic strains. The organism harbored a 3.0-Mb chromosome encoding 2,836 protein-coding genes, and contained a remarkable number of proteins involved in carbohydrate and amino acid metabolism and transport, and defense mechanisms compared with other sequenced intestinal lactobacilli. Comparative genome analysis revealed that extensive synteny was found between L. rhamnosus ATCC 53103 and its closely related L. casei ATCC 334 (a cheese isolate), but the L. rhamnosus genome contains numerous insertions, which reflect potential adaptation to the gut environment, including six carbohydrate utilization gene clusters that consist of the genes for PTS-type transporter system, glycoside hydrolase, and transcriptional regulator. Genome analysis also predicted many cell surface adherence proteins, and its genome encodes functional SpaCBA pili shed on the importance of adhesion to mucus during colonization of the human. Collectively, these features in the L. rhamnosus genome are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.

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It is interested in Genus Bifidobacterium shows the probiotic effects and as the model case of taxonomy. The nine species of Bifidobacterium and the closest related species of the type strains isolated from human origin were obtained from the Microbe Division, Japan Collection of Microorganisms (JCM), RIKEN BioResource Center. We have started sequencing of the complete genome of nine species. We have done the complete genome sequencing of B. bifidum, B. breve and B. catenulatum. The contigs of Parascardovia denticolens, Scardovia inopinata and Gardnerella vaginalis is less than fifteen. The draft sequences of B. dentium genomes have been obtained, and the shotgun library of the B. scardovii and B. longum genomes were prepared. We are going to widely comparative genomics in the Family Bifidobacteriaceae, when all the genomes will be finished.

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：JAPAN BIFIDUS FOUNDATION  

Recently, it has become possible to sequence the bacterial genome in a short time and the genome data of bacteria has rapidly increased as a result of the improved performance of sequencers and computers. At the time of writing (May 2007), 19 complete genome sequences of non-pathogenic lactic acid bacteria (LAB) representing 14 species from the order Lactobacillales are available. The comparative genomic analysis of LAB might elucidate new functions in species/strains. Furthermore, transcriptome analysis using microarrays has been applied to the study of LAB. The LAB genome data is an important resource for obtaining new knowledge.<br>

DOI： 10.11209/jim.21.209

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We determined the complete genome sequences of Lactobacillus reuteri JCM 1112^T, Lactobacillus fermentum IFO3956, Lactococcus garvieae (a toxic strain for yellowtail), Lactococcus garvieae (a non-toxic strain for yellowtail) and Lactobacillus rhamnosus.The genome analysis of Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium catenulatum is on going. Probiotics are live microorganisms that confer health benefits on the host. The probiotic L. reuteri and non-probiotic L.fermentum are phylogenetically closest to L.reuteri in the Lactobacillus. To elucidate the molecular basis on the probiotic property exhibited by L.reuteri, comparative analysis of both genomes showed that L.reuteri harbored the unique 48kb genomic island containing the genes necessary for the biosynthesis of a reactive 3-hydroxypropionaldehyde (reuterin), which may influence the microflora balance in the gut. We identified three genes (LR1633-LR1635) with a glycerol dehydratase subunit motif (PFAM PF02286-PF02288) in the L.reuteri genome. These genes are designated gupCDE (glycerol utilizing enzymes) in this study. The gupCDE knock-out L.reuteri (L.reuteri Δ gupCDE) was obtained, and the reuterin synthesis will be conducted by glycerol dehydratase in the glycerol metabolism in vivo by the gnotobiotic mice mono-associated with L.reuteri and L.reuteri Δ gupCDE.

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Abstract. We studied the thermo-resistance of E. rhusiopathiae serovar 2 (strain A), E. tonsillarum serovar 7 (strain B), Erysipelothrix spp. serovar 13 (strain C), and serovar 18 (strain D), E. rhusiopathiae serovar N (strain E), and strains which the serovars could not be determined by the usual method but could be determined as E. rhusiopathiae (strains F and G), and determined as Erysipelothrix spp. serovar 13 (strains H and I) by PCR, and, determined the D-values according the grown temperature. Strains D, and F to I, which lack the heat stable antigen or which the serovar could not be determined, showed higher D-values when grown at 37℃ than when the same strains grown at 25℃, while the strains having the heat stable antigen (A to D) did not show any significant differences. Strains of Erysipelothrix spp serovar 13 (C, H and I) showed D-values from 2.3 to 9.6 min, which was higher than those of strains A, B, D, E, F, and G which showed D-values from 0.6 to 1.7 min. Thus, the thermo-resistance of Erysipelothrix spp. might depend on the serovar, and also, it might be considered that heat stable antigens might be involved in the heat resistance of strains of this genus.

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Language：Japanese   Publisher：麻布大学  

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We present the complete genome sequences of two lactic acid bacteria (LAB), probiotic Lactobacillus reuteri (2, 039, 414 bp) and non-probiotic Lactobacillus fermentum (2, 098, 685 bp), which share obligately the heterofermentative property in glucose metabolism and have a phylogenetically closest relation. The two genomes are compared with each other and with other lactobacilli exhibiting facultatively heterofermentative and obligately homofermentative properties. A striking common feature of the two lactobacilli is a lack of a gene encoding 6-phosphofructokinase essential for the Embden-Meyerhof-Pamas (EMP) pathway, so that the pentose phosphate pathway is utilized in them. Furthermore, numerous mobile elements in L. reuteri is found in the genomes. These data imply that a genetic plasticity-environment relationship may account for the diversity between these lactobacilli.

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Other Link： http://id.nii.ac.jp/1112/00004845/

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We analyzed the gastrointestinal flora of active racehorses by culture method and determined by PCR-denaturing gradient gel electrophoresis (DGGE). One active racehorse feces was analyzed by using 17 selective media as the culture method. The bacterial flora, and Lactobacillus, Enterococcus and Bifidobacterium flora in the feces of 6 healthy, actively racing Thoroughbreds were used by PCR-denaturing gradient gel electrophoresis (DGGE) with primer sets universal and specific for each group, respectively. PCR-DGGE analyses of the feces showed that Lactobacillus equi, an organism phylogenetically related to Lactobacillus salivarius, and a strain phylogenetically related to Lactobacillus gastricus were present in almost all of the fecal samples tested, and Streptococcus bovis/Streptococcus equinus was the predominant representative of the Enterococcus group. Single PCR of the samples failed to detect any members of the Bifidobacterium group, but nested PCR identified Bifidobacterium boum, two isolates phylogenetically related to Bifidobacterium urinalis, and Parascardovia denticolens in 3 of the 6 horses tested. B. boum was isolated by the culture method, although the population of Bifidobacterium was very low. On the other hand, L. equi, L. salivarius, Lactobacillus crispatus, Lactobacillus ruminis, Lactobacillus mucosae, Lactobacillus reuteri, Lactobacillus jonsonii, Lactobacillus thermotolerans, Lactobacillus delbrueckii and Lactobacillus vitulinus were isolated as Lactobacillus group from the feces of 3 healthy, actively racing Thoroughbreds. These results indicate that the lactic acid bacterial and bifidobacterial distributions in horse feces are unique because of the presence of species specific to horses and because of the dearth of Bifidobacterium isolates.

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Language：Japanese   Publisher：麻布大学  

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Language：Japanese   Publisher：日本生物工学会  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=301752

Language：Japanese   Publisher：Azabu University  

Mechanical, biochemical and histological evaluations of natural hog and sheep casings were studied to elucidate the effect of connective tissue on the mechanical properties of natural casings. Chinese casings were significant tougher than any other casings (p<0.01). Natural hog and sheep casings were predominantly composed of collagen organized in many layers of sheets of collagen fibers and minor elastin limited in blood vessels. The amounts of collagen, elastin, and proteoglycan, and histological distribution and density of elastin fibers for various casings were essentially the same. These parameters thus would not likely contribute to the strength of natural casings. Chinese casings possessed a significant low heat-solubility of collagen (p<0.01), and a different size and arrangement of collagen fibers. Thus the thermal and structural stabilities of collagen may determine the mechanical properties of casings.

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Other Link： http://id.nii.ac.jp/1112/00004653/

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Adherence of lactic acid bacteria (LAB) to intestinal epithelial cells is one of the important characteristics as probiotics. We tried to express the fluorescence protein genes ECFP (cyan fluorescence) or EYFP (yellow fluorescence) in LAB. The fluorescence was used as a marker of LAB adherence to intestinal epithelial cells. A shuttle vector pKT between E. coli and LAB was constructed the origin of DNA replication from pMB1 for E. coli and pIL253, which is a cloning vector for LAB. pKTCFP and pKTYFP were introduced fluorescence protein genes ECFP or EYFP from commercial pECFP or pEYFP, respectively. L. reuteri was transformed by pKTCFP produced cyan fluorescence protein and fluorescence was detected from this bacterium. Furthermore, pH resistant yellow fluorescence protein gene, pKTEYFP-L68V/Q69K, was constructed, and yellow fluorescence was detected from the transformant of L. reuteri with this plasmid.

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Other Link： http://id.nii.ac.jp/1112/00004636/

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The levels of verotoxin-1 and verotoxin-2 released from verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were estimated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2 and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6,000 mg/L, a minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with treatment by sodium chloride or antibiotics. When the electron paramanetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77K to clarify the mechanism of the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appeared that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated through reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. These findings indicate that nitric oxide derived from sodium nitrite penetrates the cells and inactivates enzymes related to the respiratory chain.

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Other Link： http://id.nii.ac.jp/1112/00004420/

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L. fermentum and L. reuteri were separated from L. fermentum by polyacrylamide gel electrophoresis but not physicochemical phenotypes in 1980. On the other hand, L. fermentum is generally isolated from plant origin fermented foods and silage, whereas L. reuteri is typically isolated from mammalian intestinal tract and feces. Probiotics have recently been the focus of intense lactic acid bacterial researches interest. Although L. fermentum is phenotypically similar to L. reuteri, many research data of probiotics concentrate on L. reuteri not but on L. fermentum. The present studies are aimed at sequencing complete genomes of L. fermentum and L. reuteri, and analyzing those comparative genomes. We have sequenced the complete genomes of these species, and each genome contains approximate 1.8-Mb. The circular chromosomes of L. fermentum and L. reuteri have the average G+C contents of 52% and 39%, respectively. Five 23S rDNAs and six 23S rDNAs exist in the chromosomes of L. fermentum and L. reuteri, respectively. The numbers of insertion sequence (IS) elements and transposons (Tn) of L. fermentum are more than those of L. reuteri. L. reuteri possesses the structural genes determining reuterin (3-hydroxypropionaldehyde), an antimicrobial substance, production, but L. Fermentum does not possess this operon.

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010640330

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The genes, lmrA and lmrP, are identified as multidrug resistant genes in Lactococcus lactis subsp lactis MG1363, and these genes express LmrA and LmrP which are multidrug transporters (Konings et al., 1994-2000^<1-14)>). Multidrug transporters are a representative mechanism of multidrug resistance in bacteria and mammals. The amino acid sequence of LmrA showed 30% homology to the N- and C-terminal halves of the human multidrug resistant P-glycoprotein MDR1. And especially the sequence identities of ATP binding cassette domain of these protein are high (48% and 43% identical, respectively). In this study, attempts were made to confirm the presence of multidrug resistant genes in Lactococcus and Lactobacillus, which are lactic acid bacteria. The complete genomic DNA sequences of both lmrA and lmrP were isolated from 10 strains of Lactococcus lactis subsp. lactis by PCR amplification, whereas the presence of the homologous genomic DNA sequences genes was not confirmed in Lactococcus lactis subsp. cremoris and Lactobacillus. Vector pCAGGS (Niwa et al., 1991^<15)>) was used as the expression vector for lmrA in mammalian cells in this study. The PCR products of lmrA was cloned into pCAGGS in XhoI site, and pCAGGS-lmrA (7,873 bp) was constructed.

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010621575

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010611970

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Language：Japanese   Publisher：日本食肉研究会  

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010591620

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Language：English   Publisher：INST FOOD TECHNOLOGISTS  

With Staphylococcus xylosus FAX-1, metmyoglobin in MRS broth (pH 5.8) was found to undergo conversion to hexacoordinate nitric oxide (NO) complex of Fe(II) myoglobin. When the pH of the MRS culture containing myoglobin changed from 5.8 to 4.0, it affected the conversion from hexacoordinate to pentacoordinate NO complex of Fe(II) myoglobin, This conversion process was reversible. Salami without nitrite or nitrate addition was prepared by inoculating S. xylosus FAX-1, and pentacoordinate NO complex of Fe(II) myoglobin (nitrosylmyoglobin formed in cured meat) was formed in the salami.

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

Ten strains of Lactobacillus fermentum that differed in origin converted metmyoglobin to nitrosylmyoglobin [a pentacoordinate nitric oxide (NO) complex of Fe(II) myoglobin] in MRS broth at pH 4.3. Of the 10 strains, L. fermentum IFO 3956 possessed the strongest capacity to convert metmyoglobin to nitrosylmyoglobin. This strain synthesizes NO enzymatically from the two equivalent guanidino nitrogens of L-arginine. To our knowledge, this demonstrates for the first time the production of NO synthesized from the guanidino nitrogens of L-arginine by lactic acid bacteria. IFO 3956 may possess a bacterial NO synthase.

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Language：Japanese   Publisher：Japanese Society of Animal Science  

Staphylococcus carnosus and Staphylococcus xylosus which were converted from metmyoglobin in MRS broth to a red myoglobin derivative (unknown) formed in Parma ham and nitrosylmyoglobin, respectively were used. Salamis (ca. 240g) without sodium nitrite addition were prepared using these staphylococcal strains as starter cultures. All salamis were ripened at 20°C for 23 days at 80% relative humidity. Salamis inoculated S. carnosus or S. xylosus assumed red color even without nitrite or nitrate addition and with only a small amount of sodium chloride. The physiological features (pH value, residual NO₂^-, water content, water activity and sodium chloride concentration) of these samples were essentially the same as those of cured salami. The bacterial safety of all samples was confirmed on food hygiene, resulting from investigation of bacterial count, acidogenic bacterial count, coliform count, staphylococcal count and salmonella count. Inoculation of S. carnosus or S. xylosus as starter cultures contributed to reduced discoloration of salami with sodium nitrite.

DOI： 10.2508/chikusan.68.787

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Language：English   Publisher：VOLKSWIRTSCHAFTLICHER VERLAG  

The putative citrate permease (Cit-P) plasmid DNA (pN7CP) from Lactococcus lactis subsp. lactis biovar diacetylactis (L. diacetylactis) NIAI N-7 was cloned. Cit-P+ transformants possessed pIL253 with a 3.8-kb fragment (pKAK1) or a 2.0-kb fragment (pKAK7). No XbaI site was present in the 3.8-kb of Xbal-Xbal fragment from pN7CP. It may thus be possible to obtain the 2.0-kb fragment by deleting the 3.8-kb fragment. Assessment was made of citrate-transporting activity in transformants KAK1 and KAK7. Several Cit(-) lactococci were transformed by pKAK7. Although all their transformants except KK-7LM and KK-7SL of Lactococcus lactis subsp. lactis (L. lactis) LM0230 and NIAI SLN were incapable of transporting citrate, transformants KK-7LM and KK-7SL showed the Cit(+) phenotype and citrate-transporting activity. L. diacetylactis and L. lactis were classified under citrate metabolism. However, the present findings strongly indicate some strains of L. lactis to quite likely be the original citrate utilizers but simply to have lost the Cit-P gene.

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Language：English   Publisher：INST FOOD TECHNOLOGISTS  

The red pigment of Parma ham was compared with the myoglobin derivatives present in meat and meat products. Spectral patterns of 75% acetone extracts and electron spin resonance spectra from Parma ham differed from those of the myoglobin derivatives. Staphylococci isolated from Parma ham generated red myoglobin derivative from metmyoglobin. Model fermented sausage prepared by inoculation with the isolates developed a more desirable red color without nitrite or nitrate treatment. The red pigment of Parma ham and the model sausage appeared to be the same myoglobin derivative. The reddening of Parma ham is probably caused by the action of bacteria.

DOI： 10.1111/j.1365-2621.1996.tb10924.x

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DOI： 10.2508/chikusan.65.1026

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Language：English   Presentation type：Poster presentation  

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Venue：高知大学農林海洋科学部  

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Venue：山口大学 吉田キャンパス  

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