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Treponema Maltophilum Sp Wyss, C; Choi, B K; Schüpbach, P; Guggenheim, B; Göbel, U B. Treponema maltophilum sp. nov., a small oral spirochete isolated from human periodontal lesions. Int. J. Syst. Bacteriol. 1996, 46(3):745-52. Postprint available at: http://www.zora.unizh.ch University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.unizh.ch Originally published at: Int. J. Syst. Bacteriol. 1996, 46(3):745-52 Winterthurerstr. 190 CH-8057 Zurich http://www.zora.unizh.ch Year: 1996 Treponema maltophilum sp. nov., a small oral spirochete isolated from human periodontal lesions Wyss, C; Choi, B K; Schüpbach, P; Guggenheim, B; Göbel, U B Wyss, C; Choi, B K; Schüpbach, P; Guggenheim, B; Göbel, U B. Treponema maltophilum sp. nov., a small oral spirochete isolated from human periodontal lesions. Int. J. Syst. Bacteriol. 1996, 46(3):745-52. Postprint available at: http://www.zora.unizh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.unizh.ch Originally published at: Int. J. Syst. Bacteriol. 1996, 46(3):745-52 Treponema maltophilum sp. nov., a small oral spirochete isolated from human periodontal lesions Abstract A novel culture medium for cultivation of fastidious oral anaerobes is described. This medium, OMIZ-Pat, consists of a rich chemically defined basal medium supplemented with asialofetuin, as well as yeast extract and Neopeptone fractions. Addition of 1 mg of rifampin per liter and 100 mg of fosfomycin per liter allowed routine isolation of spirochetes by a limit dilution method in 96-well plates containing liquid OMIZ-Pat. In addition to members of the four previously recognized species of oral treponemes (Treponema denticola, Treponema pectinovorum, Treponema socranskii, and Treponema vincentii), 26 previously undescribed spirochete strains belonging to one group were isolated. We propose the name Treponema maltophilum sp. nov. for these small spirochetes, which have two endoflagella; one endoflagellum is attached at each cell pole, and the endoflagella overlap in the middle of the cell. Growth of these organisms was dependent on a carbohydrate like D-arabinose, L-fucose, D-maltose, L-rhamnose, D-ribose, D-sucrose, or D-trehalose and was inhibited by fetal bovine serum. T. Maltophilum is distinguished from other oral Treponema species by its 16S rRNA sequence, its protein and antigen patterns as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, and its characteristic alpha-glucosidase activity. The strains included in the new species on the basis of their 16S rRNA sequences are heterogeneous with respect to their alpha-fucosidase, and beta-glucuronidase activities, their dependence on N-acetylglucosamine, and their antigens as detected with patient antibodies. Strain BR is designated the type strain, and strains HO2A and PNA1 are reference strains of the new species. INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1996, p. 745–752 Vol. 46, No. 3 0020-7713/96/$04.0010 Copyright q 1996, International Union of Microbiological Societies Treponema maltophilum sp. nov., a Small Oral Spirochete Isolated from Human Periodontal Lesions 1 2 1 1 2 C. WYSS, * B. K. CHOI, P. SCHU¨ PBACH, B. GUGGENHEIM, AND U. B. GO¨ BEL Institute for Oral Microbiology and General Immunology, Center for Dentistry, Oral Medicine and Maxillofacial Surgery of the University of Zu¨rich, CH-8028 Zu¨rich, Switzerland,1 and Universita¨tsklinikum Charite´, Institut fu¨r Mikrobiologie und Hygiene, D-10117 Berlin, Germany2 A novel culture medium for cultivation of fastidious oral anaerobes is described. This medium, OMIZ-Pat, consists of a rich chemically defined basal medium supplemented with asialofetuin, as well as yeast extract and Neopeptone fractions. Addition of 1 mg of rifampin per liter and 100 mg of fosfomycin per liter allowed routine isolation of spirochetes by a limit dilution method in 96-well plates containing liquid OMIZ-Pat. In addition to members of the four previously recognized species of oral treponemes (Treponema denticola, Treponema pec- tinovorum, Treponema socranskii, and Treponema vincentii), 26 previously undescribed spirochete strains be- longing to one group were isolated. We propose the name Treponema maltophilum sp. nov. for these small spirochetes, which have two endoflagella; one endoflagellum is attached at each cell pole, and the endoflagella overlap in the middle of the cell. Growth of these organisms was dependent on a carbohydrate like D-arabinose, L-fucose, D-maltose, L-rhamnose, D-ribose, D-sucrose, or D-trehalose and was inhibited by fetal bovine serum. T. maltophilum is distinguished from other oral Treponema species by its 16S rRNA sequence, its protein and antigen patterns as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immuno- blotting, and its characteristic a-glucosidase activity. The strains included in the new species on the basis of their 16S rRNA sequences are heterogeneous with respect to their a-fucosidase and b-glucuronidase activities, their dependence on N-acetylglucosamine, and their antigens as detected with patient antibodies. Strain BR is designated the type strain, and strains HO2A and PNA1 are reference strains of the new species. Investigators of the etiology of periodontal diseases have tified on the basis of 16S rRNA sequence criteria (6), for which documented the enormous complexity of the microbial popu- we propose the name Treponema maltophilum. lations of dental plaque (22). Hundreds of species have been isolated and identified by cultural techniques. However, it is MATERIALS AND METHODS widely accepted that numerous organisms present in plaque remain entirely unnoticed or are recognized microscopically Bacteria. The following type and reference strains were obtained and main- tained as described previously (37): Treponema denticola CD-1, 51B2, ATCC because of their prominent morphology but have never been 33521, ATCC 35404, and ATCC 35405T (T 5 type strain); Treponema pectino- isolated. This could explain the failure so far to identify bona vorum ATCC 33768T; Treponema socranskii subsp. buccale ATCC 35534T; Trepo- fide periodontopathogens (11, 24). nema socranskii subsp. paredis ATCC 35535T; Treponema socranskii subsp. Recently, we established a library of 16S rRNA sequences socranskii ATCC 35536T; Treponema vincentii LA-1 (5 ATCC 35580) and Ritz A; and Treponema sp. strain ATCC 43242. from the bacteria present in a single sample of subgingival Culture media. To enhance the recovery of organisms that may be more plaque from a periodontitis patient. This library defined in a fastidious than the laboratory-adapted reference strains, a number of supple- culture-independent way the spectrum of genotypes that were ments were added to chemically defined liquid medium OMIZ-W1 (37). Initially, most prevalent in that population (6). When the spirochetes we used an enriched medium, OMIZ-WP, which was prepared from OMIZ-W1 by adding glutathione, asialofetuin (AsF), a methanol-soluble fraction of yeast were considered, this spectrum proved to be far wider than the extract (YEM), and a deanionized fraction of Difco Neopeptone (DANP) (39). spectrum of oral treponemes that have been cultured. Al- After the first results for carbohydrate utilization by strain BRT were obtained, though this approach yielded no information concerning the 2gofD-mannose per liter,2gofD-arabinose per liter,2gofL-fucose per liter, physiological characteristics of these organisms, it opened the 2gofD-trehalose per liter,2gofD-sucrose per liter, and2gofL-rhamnose per liter were added, and the resulting medium was designated OMIZ-Pat. All way for developing rapid analytical tools to specifically detect comparative studies were performed with cells grown in OMIZ-Pat. The solid these genotypes in plaque samples. media used for colony cloning were prepared as described previously for semi- Improved in vitro culture techniques have been developed to solid media (37), except that the final concentration of agarose (catalog no. investigate the physiology of established laboratory strains of 05068; Fluka, Buchs, Switzerland) was increased from 0.25 to 1.5%. Limit dilution culture analysis of plaque bacterial populations. All plaque oral anaerobes under defined conditions, in particular the met- samples were obtained from patients with recurrent, treatment-resistant peri- abolic activities important for plaque ecology, and to detect odontitis. Subgingival plaque samples were collected from dental pockets that possible virulence factors (36, 37, 40). It was expected that were .5 mm deep by using paper points and were transferred into reduced refinements in these culture techniques would expand the transport medium (17). The bacteria were released and dispersed by vortexing. Samples were processed within3hofcollection. All procedures were performed spectrum of fastidious anaerobes amenable to in vitro analysis. in ambient air without prereduction of media. Samples were diluted with OMIZ- In this paper we describe a novel culture technique that Pat to determine the total counts and with OMIZ-Pat supplemented with anti- allowed isolation and characterization of previously uncultured biotics to select for spirochetes. A dilution of 1027 for nonselective medium and oral treponemes. Although phenotypically heterogeneous, these a dilution of 1025 to 1026 for the antibiotic-containing medium usually resulted in growth in about 50% of the wells. The wells in one 96-well plate were
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