Etsuro Hoshino

Bactericidal efficacy of carbon dioxide laser against bacteria-contaminated titanium implant and subsequent cellular adhesion to irradiated area

The aim of this study was to assess CO2 laser ability to eliminate bacteria from titanium implant surfaces. The changes of the surface structure, the rise in temperature, and the damage of connective tissue cells after laser irradiation were also considered.

Treponema medium sp. nov., Isolated from Human Subgingival Dental Plaque

A new Treponema species, for which we propose the name Treponema medium, was isolated from subgingival plaque from an adult with periodontal disease. The morphological characteristics, differential biochemical characteristics, and protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of this organism are described. The guanine-plus-cytosine content of the DNA of T. medium is 51 mol%. The levels of DNA-DNA relatedness of the new species to other Treponema species, including Treponema denticola, Treponema vincentii, Treponema socranskii, Treponema pallidum, and Treponema phagedenis, are less than 30%. A phylogenetic analysis based on 16S rRNA sequences distinguished the new Treponema strain from strains belonging to previously described Treponema species. The type strain of T. medium is strain G7201.

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).

Bactericidal Efficacy of Metronidazole against Bacteria of Human Carious Dentin in vitro

The bactericidal efficacy of metronidazole against bacteria in carious dentin was clarified by measuring (1) the difference between bacterial recovery from suspensions of carious dentin on metronidazole-containing BHI-Blood agar plates (10 micrograms/ml) and control plates and (2) the difference between bacterial recovery from carious lesions of freshly extracted teeth, covered by alpha-tricalcium phosphate (TCP) cement containing metronidazole (5%) for 1-3 days and that covered by TCP only. More than 10(3) bacteria per milligram sample were recovered from carious dentin. More than 99% of the bacteria were, however, not recovered when samples were inoculated on metronidazole-containing BHI-Blood agar plates or when the lesions were covered by TCP cement containing metronidazole, indicating that metronidazole effectively disinfected the carious dentin.

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.

PCR-restriction fragment length polymorphism analysis of genes coding for 16S rRNA in Veillonella spp.

Restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal DNA (16S rDNA PCR-RFLP) was used to generate restriction profiles of the American Type Culture Collection type strains of the genus Veillonella, i.e., V. atypica, V. caviae, V. criceti, V. dispar, V. parvula, V. ratti, and V. rodentium. Whole-cell protein profiles were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for comparative purposes. The 16S rRNA genes were amplified by PCR, and RFLP analysis of the 16S rDNA was performed with MnlI and Sau3AI. MnlI produced six RFLP patterns for seven type strains, since the patterns for V. atypica and V. caviae were the same. RFLP patterns with Sau3AI could distinguish between V. atypica and V. caviae. The type strains of Veillonella species were easily distinguished by 16S rDNA PCR-RFLP.

Degradation of arginine and other amino acids by butyrate-producing asaccharolytic anaerobic Gram-positive rods in periodontal pockets

The use of 20 amino acids by butyrate-producing asaccharolytic anaerobic Gram-positive rods (AAGPRs) in periodontal pockets, i.e. Eubacterium minutum, Filifactor alocis, E. infirmum, E. sulci and E. saphenum, was studied. E. minutum used only arginine and lysine, and produced substantial amounts of butyrate and ammonia as the main metabolic products from arginine, and acetate, butyrate and ammonia from lysine. Fi. alocis used arginine alone and produced butyrate and ammonia. E. infirmum, E. sulci and E. saphenum used lysine alone and produced acetate, butyrate and ammonia. The growth of these bacterial species was supported and enhanced by arginine and/or lysine enriched to culture media, but not by the other amino acids. Arginine deiminase, ornithine carbamoyltransferase and carbamate kinase activity were detected in the cell-free extract of E. minutum, suggesting that arginine was metabolised to citrulline initially, and subsequently to ornithine and carbamoyl phosphate. Ornithine and carbamoyl phosphate were further converted to butyrate, and carbon dioxide and ammonia, respectively. Enzymatic activity of arginine deiminase and ornithine carbamoyltransferase was not detected in Fi. alocis, indicating that Fi. alocis converted arginine to ornithine directly, not via citrulline, and further to butyrate.

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.

Differentiation of oral Actinomyces species by 16S ribosomal DNA polymerase chain reaction-restriction fragment length polymorphism

16S rDNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to generate restriction profiles of the reference strains, including the American Type Culture Collection type strains, of oral Actinomyces spp., i.e., A. israelii, A. gerencseriae, A. naeslundii genospecies 1 and 2, A. odontolyticus, A. meyeri and A. georgiae, and 23 Actinomyces strains isolated from human dental plaque. The 16S rRNA gene sequences from isolated genomic DNA samples were amplified by PCR. The PCR products were purified and characterized by single digestion with four restriction endonucleases, i.e., MnlI, HaeIII, CfoI, or HpaII. Among them, MnlI was found to discriminate the respective reference strains. The clinical isolates were assigned to one of the species, i.e., A. gerencseriae, A. naeslundii genospecies 1 and 2 and A. odontolyticus, on the basis of their restriction profiles by single digestion with MnlI. Thus, 16S rDNA PCR-RFLP, using MnlI, is a rapid and reliable method for the differentiation of oral Actinomyces spp.