Thyroid hormone may predict treatment failure in Kawasaki disease.

BACKGROUND: In systemic inflammatory conditions, inflammatory cytokines can cause low thyroid hormone levels. There are no reports discussing the relation between thyroid hormone levels and response to treatment for Kawasaki disease. METHODS: We investigated 67 patients who underwent treatment in the acute phase of Kawasaki disease. We divided patients into two groups based on their response to initial intravenous immunoglobulin (IVIG) treatment: the responder group (n = 40), and the non-responder group (n = 27). The serum levels of the thyroid hormones free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) were compared before and after treatment in all patients, and between responder and non-responder groups. RESULTS: The FT3, FT4, and TSH levels were low before the initial treatment and increased significantly after treatment (p < 0.05). The FT3, FT4, and TSH levels before treatment were significantly lower in the non-responder group than in the responder group (p < 0.05). Logistic regression analysis suggested that the addition of pre-treatment FT4 values to Gunma score was useful in predicting treatment failure. CONCLUSIONS: Thyroid hormone and TSH levels were lower in the non-responder group than in the responder group in the initial IVIG treatment for Kawasaki disease. This study suggests that Kawasaki disease in the acute phase is associated with low thyroid hormone levels and TSH. It is possible that these hormone levels predict response to the initial IVIG.

Screening of heat-killed lactic acid bacteria based on inhibitory activity against oral bacteria and effects of oral administration of heat-killed Ligilactobacillus salivarius CP3365 on periodontal health in healthy participants: a double-blinded, randomized, placebo-controlled trial.

Objectives: The aims of this study were to select heat-killed lactic acid bacteria (HKL) with antibiotic activity and investigate the efficacy of this bacteria in maintaining periodontal parameters in healthy participants. Materials and methods: An in vitro evaluation was conducted to assess the inhibitory efficacy of lactic acid bacteria against Porphyromonas gingivalis and Fusobacterium nucleatum subsp. nucleatum. The effects of HKL administration on various parameters (plaque control record, bleeding on probing, and probing pocket depth) were assessed in a randomized, placebo-controlled trial. Participants in the test and placebo groups (n = 32) consumed oral tablets containing placebo or HKL daily for 8 weeks. Oral bacteria in supra-plaque and saliva were identified using 16S rRNA gene community profiling analysis. Results: Heat-killed Ligilactobacillus salivarius CP3365 significantly (p < 0.05) decreased the viability of oral bacteria and was selected for clinical trials. Administration of HKL CP3365 significantly (p < 0.05) inhibited increases in each parameter. No changes in the relative abundance of P. gingivalis or F. nucleatum subsp. nucleatum were detected by HKL CP3365, but the relative abundance of oral bacteria (genera Porphyromonas, Fusobacterium, and Haemophilus) was significantly (p < 0.05) decreased. Conclusion: HKL CP3365 effectively inhibited oral bacteria growth and was useful for maintaining periodontal health. Clinical Trial Registration: [https://www.umin.ac.jp/ctr/index.htm], identifier [UMIN000045656].

Inhibitory Effect of the Glycerophosphate Moiety of Lipoteichoic Acid from Lactic Acid Bacteria on Dexamethasone-Induced Atrogin-1 Expression in C2C12 Myotubes.

Atrogin-1, which is an important regulator of ubiquitin-mediated protein degradation in skeletal muscle, is a major marker of muscle loss and disuse muscle atrophy. To investigate which components of lactic acid bacteria (LAB) suppress dexamethasone (DEX)-induced atrogin-1 expression, mouse skeletal muscle C2C12 myotubes were treated with DEX in the presence or absence of components of LAB. Heat-killed cells and lipoteichoic acid (LTA) derived from five LAB strains significantly suppressed DEX-induced atrogin-1 expression. The glycerophosphate (GroP) fraction prepared from chemically-degraded LTA and sn-glycerol-1-phosphate suppressed DEX-induced atrogin-1 expression, whereas the glycolipid anchor fraction of LTA did not. Heat-killed cells obtained by culturing under low-Mn2+ conditions, which generated fewer poly-GroP polymers in LTA, displayed significantly lower inhibitory activity compared to heat-killed cells grown under normal conditions. These results suggested that LTA of LAB contributed to suppressing atrogin-1 expression and that the GroP moiety of LTA was responsible for its inhibitory activity.

Lactobacillus curvatus CP2998 Prevents Dexamethasone-Induced Muscle Atrophy in C2C12 Myotubes.

To investigate whether heat-killed Lactobacillus curvatus CP2998 (CP2998) inhibits glucocorticoid-induced myotube atrophy which is associated with the ubiquitin-proteasome system, mouse skeletal muscle C2C12 myotubes were treated with dexamethasone (DEX) in the presence or absence of CP2998. DEX exposure significantly decreased myotube diameters and increased mRNA expression levels of MuRF1 and MAFbx, E3 ubiquitin ligases. CP2998 treatment restored myotube diameters and dose dependently decreased mRNA expression levels of these E3 ubiquitin ligases. CP2998 treatment also inhibited DEX-induced glucocorticoid dependent transcription. Our results suggest that CP2998 prevents DEX-induced muscle atrophy by suppressing glucocorticoid receptor activation.

M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells.

BACKGROUND: Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG. CONCLUSIONS: M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1.

BACKGROUND: The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). METHODS: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. RESULTS: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. CONCLUSIONS: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.

Instability of KCNE1-D85N that causes long QT syndrome: stabilization by verapamil.

BACKGROUND: A KCNE1 polymorphism, D85N, causes long QT syndrome (LQTS) with a decrease in the slowly activating delayed-rectifier K(+) channel current (IKs ). We examined impacts of D85N polymorphism on KCNE1 protein stability and functions, and tested the ability of various drugs to modify them. METHODS: KCNE1-D85N or the wild-type protein was coexpressed in COS7 cells with KCNQ1 to form K(+) channels. Expression, degradation, and intracellular localization of KCNE1 proteins, as well as the currents conferred by KCNQ1/KCNE1 complexes, were determined using immunoblots, immunofluorescence, and patch-clamp techniques. RESULTS: The protein level of KCNE1-D85N was lower than that of the wild-type, in spite of the comparable levels of their mRNA. KCNE1-D85N was highly ubiquitinated and rapidly degraded as compared to the wild-type; a proteasome inhibitor, MG132, inhibited its degradation and increased its steady-state level. Both KCNE1-D85N and the wild-type proteins were co-immunoprecipitated with KCNQ1. Immunofluorescent signals of KCNE1-D85N accumulated in the endoplasmic reticulum and Golgi apparatus, with reduced levels on the cell membrane. Patch-clamp experiments demonstrated that the membrane current corresponding to IKs was much smaller in cells expressing KCNE1-D85N than in those expressing the wild-type. Verapamil (0.5-10 µM) increased the protein level of KCNE1-D85N, decreased its ubiquitination, slowed its degradation, and enhanced KCNQ1/KCNE1-D85N channel currents. Pretreatment with amiodarone abolished these effects of verapamil. CONCLUSION: KCNE1-D85N is less stable than the wild-type protein, and is rapidly degraded through the ubiquitin-proteasome system. Verapamil may be of a therapeutic value in LQTS patients via preventing degradation of KCNE1-D85N.

Stabilization of Kv1.5 channel protein by bepridil through its action as a chemical chaperone.

While bepridil has been reported to alter the stability of ion channel proteins, the precise mechanism of action remains unclear. We examined the effect of bepridil on the stability of Kv1.5 channel proteins expressed in COS7 cells. Bepridil at 0.3-30 µM increased the protein level of Kv1.5 channels in a concentration-dependent manner. Chase experiments showed that bepridil delayed the degradation process of Kv1.5 channel proteins in the same manner as a proteasomal inhibitor, MG132, did. Bepridil increased the immunofluorescent signal of Kv1.5 channel proteins in the endoplasmic reticulum (ER) and Golgi apparatus and on the cell surface. The cell fraction experiment also showed bepridil-induced increases in Kv1.5 in the ER, Golgi apparatus, and the cell membrane. Bepridil at a lower concentration of 1 µM had no effect on the proteasome activity in vitro. A blocker of the ultrarapid delayed-rectifier K(+) channel current, 4-aminopyridine (4AP), abolished bepridil-induced increases in Kv1.5. Kv1.5-medicated membrane currents measured as 4AP-sensitive currents were increased by bepridil. Taken together, we conclude that bepridil stabilizes Kv1.5 proteins at the ER through an action as a chemical chaperone, thereby increasing the density of Kv1.5 channels in the cell membrane.

Effects of cilnidipine on serum uric acid level and urinary nitrogen monoxide excretion in patients with hypertension.

The effects of cilnidipine on the serum uric acid level and urinary NO excretion in hypertensive patients were investigated. Blood and urine samples of 16 hypertensive outpatients were collected before and 2 months after cilnidipine therapy (10 mg). The serum uric acid level decreased significantly after cilnidipine treatment, while the uric acid-creatinine clearance ratio was unaffected. The cilnidipine medication produced a significant increase in urinary NO excretion, although amlodipine did not change it significantly. Therefore, cilnidipine has a profound antihypertensive effect and may reduce the serum uric acid level and increase NO production in the kidney.

A new microdeletion syndrome of 5q31.3 characterized by severe developmental delays, distinctive facial features, and delayed myelination.

Chromosomal deletion including 5q31 is rare and only a few patients have been reported to date. We report here the first two patients with a submicroscopic deletion of 5q31.3 identified by microarray-based comparative genomic hybridization. The common clinical features of both patients were marked hypotonia,feeding difficulty in infancy, severe developmental delay, and epileptic/nonepileptic encephalopathy associated with delayed myelination. Both patients also shared characteristic facial features,including narrow forehead, low-set and dysmorphic ears, bilateral ptosis, anteverted nares, long philtrum, tented upper vermilion,edematous cheeks, and high arched palate. The deleted region contains clustered PCDHs, including PCDHA [corrected]. and PCDHG, which are highly expressed in the brain where they function to guide neurons during brain development, neuronal differentiation, and synaptogenesis. The common deletion also contains neuregulin 2(NRG2), a major gene for neurodevelopment. We suggest that 5q31.3 deletion is responsible for severe brain developmental delay and distinctive facial features, and that the common findings in these two patients should be recognized as a new microdeletion syndrome. We need further investigations to determine which genes are really responsible for patients' characteristic features

Synthesis and antiviral evaluation of α-D-2',3'-didehydro-2',3'-dideoxy-3'-C-hydroxymethyl nucleosides.

We have identified a selective S(N)2' reaction triggered by iodide ion that leads to the ring-opening of 2,2'-anhydro-α-nucleosides. By applying the method, we have synthesized α-D-2',3'-didehydro-2',3'-dideoxy-3'-C-hydroxymethyl nucleosides, designed as potential antiviral agents.

Fluctuating liver functions in siblings with MPV17 mutations and possible improvement associated with dietary and pharmaceutical treatments targeting respiratory chain complex II.

BACKGROUND/AIMS: To describe the clinical and biological findings of two Japanese siblings with novel MPV17 gene mutations (c.451insC/c.509C > T) manifesting hepatic mitochondrial DNA depletion syndrome. METHODS: We observed these brothers and sought to determine the efficacy of treatment targeting respiratory chain complex II for the younger brother. RESULTS: A 3-month-old boy had presented with profound liver dysfunction, failure to thrive, and watery diarrhea. Although he was then placed on a carbohydrate-rich diet, his liver function thereafter fluctuated greatly in association with viral infections, and rapidly deteriorated to liver failure. He underwent liver transplantation at 17 months of age but died at 22 months of age. The younger brother, aged 47 months at the time of this writing, presented with liver dysfunction from 8 months of age. His transaminase levels also fluctuated considerably fluctuations in association with viral infections. At 31 months of age, treatment with succinate and ubiquinone was initiated together with a lipid-rich diet using ketone milk. Thereafter, his transaminase levels normalized and never fluctuated, and the liver histology improved. CONCLUSIONS: These cases suggested that the clinical courses of patients with MPV17 mutations are greatly influenced by viral infections and that dietary and pharmaceutical treatments targeting the mitochondrial respiratory chain complex II may be beneficial in the clinical management of MPV17 mutant patients.

Diversity of mucosa-associated microbiota in active and inactive ulcerative colitis.

OBJECTIVE: Recent findings indicate that bacteria play an important role in the pathogenesis of inflammatory bowel disease (IBD). However, the exact role of bacteria in ulcerative colitis (UC) has still to be elucidated. The objective of the study was to investigate the potential differences in the intestinal microbiota between patients with UC and control subjects, using terminal restriction fragment length polymorphism (T-RFLP) analysis of the mucosa-associated microbiota from UC patients and non-IBD controls. MATERIAL AND METHODS: Nine active UC patients and 11 non-IBD controls were included in the study. Seven patients with active UC who entered into the inactive phase after antibiotic combination treatment were also classified as patients with inactive UC. Mucosa-associated microbiota was compared between non-IBD controls and UC patients using T-RFLP analysis. Microbiota in both the active and inactive phase was also analyzed in UC patients receiving antibiotic treatment. RESULTS: T-RFLP patterns of mucosa-associated microbiota differed between active UC patients and non-IBD controls. Microbial compositions of active UC patients were significantly less diverse. The difference resulted from loss of commensals. From the viewpoint of disease activity before and after antibiotic combination treatment, T-RFLP patterns were also different between the active and inactive phases in the identical patients. Inactive UC patients possessed more diverse microbial compositions. No specific terminal restriction fragments were observed in UC patients. CONCLUSIONS: T-RFLP analysis showed that the mucosa-associated microbiota of patients with active UC differed from that of non-IBD controls. Active UC patients possessed significantly fewer diverse microbial compositions.

Change of intestinal microbiota with elemental diet and its impact on therapeutic effects in a murine model of chronic colitis.

Elemental diet (ED) has been used as an enteral nutritional therapy for Crohn's disease. However, the precise mechanisms of ED remain unclear. In interleukin-10 (IL-10)-deficient cell-transferred mice, we investigated the change of intestinal microbiota with ED using molecular terminal-restriction fragment length polymorphism (T-RFLP) analysis and culture method, and evaluated its influence on therapeutic effects of ED. ED significantly suppressed intestinal inflammation. The total amount of bacteria in colitis mice fed the regular diet was higher than in normal mice but decreased in colitis mice fed ED. T-RFLP profiles of the ED group markedly differed from those of the regular diet groups. The diversity of bacterial species in the ED group decreased to 60% of that found in the regular diet groups. Among the cultivated bacteria, the change in lactic acid bacteria composition was remarkable. Lactobacillus reuteri and L. johnsonii decreased and Enterococcus faecalis and E. durans increased in the ED group. The culture supernatant of L. reuteri isolates induced significant tumor necrosis factor-alpha (TNF-alpha) and IL-6 activity in RAW 264 cells, while the culture supernatant of E. faecalis and E. durans barely induced their activity. These data suggested that reduction in amount and diversity of intestinal microbiota and decrease of proinflammatory cytokines via a change in composition of lactic acid bacteria by ED seem to contribute to reduction of bowel inflammation in this model.

Terminal restriction fragment length polymorphism analysis of the diversity of fecal microbiota in patients with ulcerative colitis.

BACKGROUND: Terminal restriction fragment length polymorphism (T-RFLP) analysis is a powerful tool to assess the diversity of complexed microbiota. This permits rapid comparison of microbiota from many samples. In this study, we performed T-RFLP analysis of the fecal microbiota from patients with ulcerative colitis (UC). METHODS: Forty-four patients with UC (23 women and 21 men, median age 25 years) and 46 healthy individuals (25 women and 21 men, median age 34 years) were enrolled in this study. DNA was extracted from their stool samples, and the 16S rRNA genes were amplified by PCR. The PCR products were then digested with HhaI and/or MspI restriction enzymes, and the length of the T-RF was determined. RESULTS: The fecal microbial communities were classified in 8 clusters. Almost all the healthy individuals (39 of 46) were included in cluster I, and most of the UC patients could be divided into the other 7 clusters, indicating that fecal bacterial communities are different between healthy individuals and active UC patients. Some T-RFs, derived from the unclassified bacteria, Ruminococcus, Eubacterium, Fusobacterium, gammaproteobacteria, unclassified Bacteroides, and unclassified Lactobacillus, were detected in the UC patients, but not in the healthy individuals. The T-RFLP patterns were also different between the active patients and inactive (remission) patients. The T-RF derived from the unclassified bacteria, Ruminococcus and Eubacterium, and the T-RFs derived from the unclassified bacteria, Eubacterium, and Fusobacterium were predominantly detected in the active patients not the inactive patients. In contrast, the T-RFs derived from Lactobacillus and unclassified Lactobacillus were more predominant in the inactive (remission) patients. In 4 patients with proctitis, the pattern of fecal microbial diversity was very similar. CONCLUSIONS: T-RFLP analyses showed that the diversity of fecal microbiota in patients with UC was different from that in healthy individuals. Unclassified bacteria, as well as known bacteria, can contribute to alterations in the bacterial diversity of UC patients.

Bacteroides barnesiae sp. nov., Bacteroides salanitronis sp. nov. and Bacteroides gallinarum sp. nov., isolated from chicken caecum.

Eight bacterial strains isolated from the caecum of chicken, BL2(T), BL66, EG3, EG6, M27, BL78(T), C35(T) and C43, were characterized by determining their phenotypic characteristics, cellular fatty acid profiles, menaquinone profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these isolates belonged to the genus Bacteroides. One group of five strains (BL2(T), BL66, EG3, EG6 and M27) was related most closely to Bacteroides coprocola JCM 12979(T), with approximately 93 % 16S rRNA gene sequence similarity, and to Bacteroides plebeius JCM 12973(T), with about 92 % similarity, and shared >or=99.6 % similarity with each other. Strain BL78(T) exhibited 90.5 % similarity to B. plebeius JCM 12973(T) and 89.8 % similarity to B. coprocola JCM 12979(T) and differed from the above group of five strains at >or=10 % sequence divergence. Strains C35(T) and C43 were related most closely to Bacteroides eggerthii JCM 12986(T), with 95.1 % sequence similarity, to Bacteroides stercoris JCM 9496(T), with 94.6 % similarity, and to Bacteroides uniformis JCM 5828(T), with 94.4 % similarity, and shared 100 % similarity with each other. From results of phenotypic examination, cellular fatty acid composition analysis, menaquinone composition analysis and DNA G+C contents, the group of five strains as well as strain BL78(T) were shown to differ from the type strains of B. coprocola and B. plebeius. Strain BL78(T) differed from the others based on its menaquinone composition, which included MK-11 and MK-12. Strains C35(T) and C43 could also be differentiated from the type strains of B. eggerthii, B. stercoris and B. uniformis. The group of five strains, strain BL78(T), B. coprocola JCM 12979(T) and B. plebeius JCM 12973(T) showed low levels of DNA-DNA relatedness (<35 %) with each other. High levels of DNA-DNA relatedness were obtained within the group of five strains (>75 %). Strains C35(T) and C43 exhibited a high level of DNA-DNA relatedness (>88 %) with each other, but low levels with B. eggerthii JCM 12986(T) (<40 %), B. stercoris JCM 9496(T) (<37 %) and B. uniformis JCM 5828(T) (<16 %). On the basis of these data, three novel Bacteroides species are proposed: Bacteroides barnesiae sp. nov. (type strain BL2(T)=JCM 13652(T)=DSM 18169(T)), Bacteroides salanitronis sp. nov. (type strain BL78(T)=JCM 13657(T)=DSM 18170(T)) and Bacteroides gallinarum sp. nov. (type strain C35(T)=JCM 13658(T)=DSM 18171(T)).

Effect of maternal consumption of lactobacillus GG on transfer and establishment of fecal bifidobacterial microbiota in neonates.

BACKGROUND: Establishment of the gut microbiota at birth provides a substantial source of microbial stimuli for the maturation of the immune system. Deviations in this process precede the development of specific diseases providing the rationale for the use of probiotics to counteract them. OBJECTIVE: This study was designed to characterize both the mother-infant bifidobacteria transfer at birth and the development of bifidobacteria microbiota during the first weeks of life in infants whose mothers received Lactobacillus rhamnosus GG or placebo. METHODS: Species-specific PCR was used to assess the fecal bifidobacterial composition of mothers before and after delivery and in infants at 5 days and 3 weeks of age. RESULTS: Bifidobacterium longum was the species most commonly found in the mothers. Bifidobacterium catenulatum was the most prevalent group in infants at 5 days of age and B. longum the predominant species at 3 weeks. At 5 days of age, infants whose mothers received L. rhamnosus GG showed a significantly higher occurrence of B. breve and lower of B. adolescentis than those from the placebo group. In addition, L. rhamnosus GG consumption increased the bifidobacterial diversity in infants and reduced the Bifidobacterium microbiota similarity between mother and infant. CONCLUSIONS: These results indicate that specific changes in the transfer and initial establishment of bifidobacteria in neonates take place as consequence of the consumption of L. rhamnosus GG by the mothers.

Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences.

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.

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

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

Bacteroides plebeius sp. nov. and Bacteroides coprocola sp. nov., isolated from human faeces.

Nine strains of Gram-negative, anaerobic rod were isolated from human faeces. Based on phylogenetic analysis and specific phenotypic characteristics, these strains were included within the Bacteroides cluster and were divided into two clusters. Strains from the two clusters showed 16S rRNA gene sequence similarities of 90.4 and 92.7% to the nearest recognized species, Bacteroides vulgatus. The strains also formed two clusters exhibiting a 16S rRNA gene sequence divergence of approximately 6%. DNA-DNA hybridization studies confirmed that the two novel strain clusters were distinct from each other. Based on the phenotypic and phylogenetic findings, two novel species, Bacteroides plebeius sp. nov. and Bacteroides coprocola sp. nov., are proposed, each representing one of the two strain clusters. The DNA G+C content of the type strains were 43.9 mol% for B. plebeius (M12(T)=JCM 12973(T)=DSM 17135(T)) and 42.4 mol% for B. coprocola (M16(T)=JCM 12979(T)=DSM 17136(T)).