Futoshi Nakazawa

Description of Mogibacterium pumilum gen. nov., sp. nov. and Mogibacterium vescum gen. nov., sp. nov., and reclassification of Eubacterium timidum (Holdeman et al. 1980) as Mogibacterium timidum gen. nov., comb, nov.

A new genus, Mogibacterium, is proposed for anaerobic, non-spore-forming, Gram-positive, rod-shaped bacteria which have been isolated from the periodontal pockets of adult human patients with periodontal disease and infected root canals. The novel isolates, strains D2-18T, BA11a-f and D5-2T, were inert in most of the conventional biochemical tests and phenotypically resemble asaccharolytic Eubacterium species. The protein profiles of whole cells on SDS-PAGE gels and Western immunoblotting reaction analysis distinguished these organisms from type strains belonging to the previously described Eubacterium species. The G + C content of the DNA is 45-46 mol% for Mogibacterium pumilum and 46 mol% for Mogibacterium vescum. The levels of DNA-DNA relatedness of these new species to other Eubacterium species, including Eubacterium limosum, Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium saphenum, and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The DNA-DNA hybridization value between M. pumilum and M. vescum was 30%. Eubacterium timidum exhibited DNA homologies with Mogibacterium species which were low (17 and 18%) but clearly higher than with all the other Eubacterium species. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the closest phylogenetic neighbour of Mogibacterium species was E. timidum, and that these three species represent a novel lineage distinct from the previously described genera of Gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, it is also proposed that E. timidum is transferred to the genus Mogibacterium gen. nov. as Mogibacterium timidum gen. nov., comb. nov. (type strain ATCC 33093T).

Cryptobacterium curtum gen. nov., sp. nov., a new genus of gram-positive anaerobic rod isolated from human oral cavities

Novel Eubacterium-like isolates, strains 12-3T and KV43-B, which were isolated from the periodontal pocket of an adult patient with periodontal disease and necrotic dental pulp, respectively, were studied taxonomically and phylogenetically. The morphological and differential biochemical characteristics of these organisms are also described in this paper. These organisms were Gram-positive, anaerobic, non-spore-forming, rod-shaped bacteria that were inert in most of the conventional biochemical tests and closely resembled members of asaccharolytic oral Eubacterium species. On the other hand, protein profiles of whole cells in SDS-PAGE and Western immunoblotting reaction analysis distinguished these isolates from strains of the previously described genus Eubacterium. The G+C content of the DNAs from the novel isolates was 50 and 51 mol%, respectively. The levels of DNA-DNA relatedness to other asaccharolytic oral Eubacterium species, including Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium timidum, Eubacterium saphenum, Eubacterium minutum and Eubacterium exiguum, was less than 11%. These organisms also exhibited a very low level of reassociation with the DNA of Eubacterium limosum, the type species of the genus Eubacterium. The results of 16S rDNA sequence comparisons revealed that these organisms represent a novel lineage distinct from all previously described genera of Gram-positive, rod-shaped bacteria. On the basis of our results, it is suggested that strains 12-3T and KV43-B should be classified in a new genus and species, for which the name Cryptobacterium curtum gen. nov., sp. nov. is proposed. The type strain of Cryptobacterium curtum is 12-3T (= ATCC 700683T).

Eubacterium exiguum sp. nov., isolated from human oral lesions

Eubacterium exiguum sp. nov. is the name proposed for organisms formerly described as Eubacterium group S strains and similar bacteria isolated from various types of oral lesions. This new species was established on the basis of the results of DNA-DNA hybridization experiments and DNA base composition determinations (G + C contents, 60 to 64 mol%). The results of an API ZYM analysis, Western blotting (immunoblotting) reactions, and phenotypic tests are also given. The type strain of E. exiguum is strain S-7.

Taxonomic characterization of Mogibacterium diversum sp. nov. and Mogibacterium neglectum sp. nov., isolated from human oral cavities

Novel isolates, strains HM-7, HM-6, HH-31, P9a-hT and UJB13-d, which were isolated from tongue plaque and necrotic dental pulp, were studied taxonomically and phylogenetically. These organisms were anaerobic, non-spore-forming, gram-positive, rod-shaped bacteria that were inert in most of the conventional biochemical tests and phenotypically resemble Mogibacterium species or asaccharolytic Eubacterium species. The G+C contents of the DNAs from the novel isolates ranged from 41 to 42 mol %. DNA-DNA hybridization studies demonstrated that these strains might be assigned to the genus Mogibacterium but not to the previously described species. It was also apparent that strain HM-7 belonged to the same species as strains HM-6 and HH-31, and that strains P9a-hT and UJB13-d belonged to a second species. The levels of DNA-DNA relatedness to asaccharolytic Eubacterium species, including Eubacterium brachy, Eubacterium nodatum, Eubacterium saphenum and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The results of 16S rDNA sequence comparisons revealed that these organisms represent novel lineages distinct from all previously described species of gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics, DNA-DNA hybridization data and phylogenetic analysis with 16S rRNA gene sequence data, new species are proposed, namely Mogibacterium diversum (for strains HM-7, HM-6 and HH-31) and Mogibacterium neglectum (for strains P9a-hT and UJB13-d). HM-7T (= ATCC 700923T = JCM 11205T) is the type strain of the former and P9a-hT (= ATCC 700924T = JCM 11204T) is the type strain for the latter.

Isolation and identification of 3,6-dideoxy-3-(L-glyceroylamino)-D-glucose, a constituent of the antigenic polysaccharide of Eubacterium saburreum, strain V5

3,6-Dideoxy-3-(L-glyceroylamino)-D-glucose has been identified, for the first time, as a sugar component of the antigenic polysaccharide of Eubacterium saburreum strain V5, principally by n.m.r. and mass spectrometry.

Susceptibility of Enterococcus faecalis to a Combination of Antibacterial Drugs (3Mix) in vitro

Abstract It has been reported that enterococci cause significantly persistent root canal infection, especially after Ca(OH) 2 intracanal dressing, and that they often show tolerance to certain antibacterial drugs. We aimed to evaluate the susceptibility of enterococci to a combination of antibacterial drugs, i.e. ciprofloxacin, metronidazole and minocycline (3Mix), which is used for Lesion Sterilization and Tissue Repair (LSTR) therapy. The minimum inhibitory concentrations (MICs) of ciprofloxacin and minocycline on E. faecalis (6 strains) and E. faecium (1 strain) were 5–20 μg/mL, respectively, and no inhibitory effect was observed with metronidazole. However, 3Mix (100 μg each/mL), as a mixture, inhibited the growth of every strain completely. In addition, 3Mix also inhibited all bacterial growth in faeces (16 samples). The present result strongly indicates that 3Mix is sufficiently able to inhibit enterococcal growth, and may be useful for endodontic treatment, even in cases where enterococci are suspected to cause endodontic disorders.

The structure of the antigenic polysaccharide produced by Eubactrium saburreum T15.

The antigenic polysaccharide was obtained from the cell wall of Eubacterium saburreum strain T15 by trypsin digestion followed by gel permeation and ion-exchange chromatography. Its structure was determined using acid hydrolysis, methylation analysis, and 1D and 2D NMR spectroscopy. It contained L-threo-pent-2-ulose (Xul), D-fucose (Fuc), and D-glycero-D-galacto-heptose (Hep) in 2:3:3 ratio. Methylation analysis indicated an octasaccharide repeating-unit containing five branches. The 1H and 13C signals in NMR spectra of the sugar residues were assigned by COSY, HOHAHA, and HMQC 2D experiments, and the sequence of sugar residues in the repeating unit was determined by NOESY and HMBC experiments. The polysaccharide also contains two O-acetyl groups in the repeating unit, located on the Hep residue. The repeating structure can be written as: [see text for equation]. This is a novel structure in bacterial cell-wall polysaccharides from Gram-positive bacteria.

Phylogenetic Diversity among Unknown Oral Bacterial Species

Abstract Recently many new bacterial genera and species, such as genus Slackia , Eggerthella , Cryptobacterium and Mogibacterium , have been proposed on the basis of phylogenetic analysis and DNA-DNA similarity data. These organisms, isolated from periodontal pockets and other oral infectious lesions, are asaccharolytic anaerobic gram positive rods (AAGPR), and have been proven to be difficult to culture and unreactive in conventional biochemical tests. Molecular systematic techniques, including 16S rRNA gene sequencing analysis, have demonstrated that there is more diversity than previously thought in these AAGPR species. In addition, these sequences dates can be helpful for the construction of specific PCR primers for use in the rapid detection and identification of previously undescribed taxa. A complete description of the microbial flora associated with oral infections could lead to potential benefits in the elucidation of disease causation, and the development of novel diagnostic tools.