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 pectinovoiwm, Treponema socranskii, and Treponema vincentii), 26 previously undescribed spirochete strains belonging to one group were isolated. We propose the name Treponema mahphilum 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 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 P-glucuronidase activities, their dependence on N-acetylglucosamine, and their antigens as detected with patient antibodies. Strain BR is designated the type strain, and strains H02A and PNAl are reference strains of the new species.Investigators of the etiology of periodontal diseases have documented the enormous complexity of the microbial populations of dental plaque (22). Hundreds of species have been isolated and identified by cultural techniques. However, it is widely accepted that numerous organisms present in plaque remain entirely unnoticed or are recognized microscopically because of their prominent morphology but have never been isolated. This could explain the failure so far to identify bona fide periodontopathogens (11, 24).Recently, we established a library of 16s rRNA sequences from the bacteria present in a single sample of subgingival plaque from a periodontitis patient. This library defined in a culture-independent way the spectrum of genotypes that were most prevalent in that population (6). When the spirochetes were considered, this spectrum proved to be far wider than the spectrum of oral treponemes that have been cultured. Although this approach yielded no information concerning the physiological characteristics of these organisms, it opened the way for developing rapid analytical tools to specifically detect these genotypes in plaque samples.Improved in vitro culture techniques have been developed to investigate the physiology of established laboratory strains of oral anaerobes under defined conditions, in particular the metabo...

No abstract

Bacteroidesforsythus is the first wild-type or mutant bacterium found to require exogenous N-acetylmuramic acid for proliferation and maintenance of cell shape. This implies so far unknown pathways for peptidoglycan synthesis and a strict dependence of B. forsythus on other bacteria in its oral habitat. Addition of N-acetylmuramic acid to conventional bacteriological media allows routine cultivation of this fastidious organism.

A highly motile, medium-size, saccharolytic spirochete was isolated from an advanced human periodontal lesion in medium OMIZ-Pat supplemented with 1% human serum. The growth of this organism is dependent on either glucose, maltose, starch, or glycogen. The cells contain six endoflagella, three per pole, which overlap in the central region of the cell body. On the basis of its cell morphology and enzyme activities, as well as its sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein and antigen profiles, this organism is clearly distinct from all previously cultured spirochetes. The presence of a novel species is supported by the 16s rRNA sequence of this organism, which places it in phylotype 19 of Choi et al. (B. K. Choi, B. J. Paster, F. E. Dewhirst, and U. B. Gobel, Infect. Immun. 62:1889-1895, 1994). The only isolate, strain HA2P, is designated the type strain of a novel species, for which we propose the name Treponema amylovorum.The microbial populations of dental plaque have been intensely investigated because of their implication in the development of periodontal diseases (2). Oral spirochetes often predominate in subgingival plaque at diseased sites, but little is known about the population structure of these bacteria and their biological activities. This is mainly due to difficulties encountered in the in vitro cultivation of these fastidious organisms. Recent improvements in culture media and the application of limit dilution techniques have allowed the routine isolation of oral spirochetes (7). Here, we describe a novel oral Treponema species isolated from an advanced periodontal lesion, for which we propose the name Treponema amylovorum sp. nov.; the only isolate, HA2P, is the type strain of this species. MATERIALS AND METHODS Bacteria.The following type and reference strains were maintained as described previously (5, 7): Treponema denticola CD-l,51B2, ATCC 33521, ATCC 35404, and ATCC 35405T; Treponema maltophilum ATCC 51939T, ATCC 51940, and ATCC 51941; Treponemapectinovomm ATCC 33768T; Treponema socranskii subsp. buccale ATCC 35534T; Treponema socranskii subsp. paredis ATCC 35535T; Treponema socranskii subsp. socranskii ATCC 35536T; and Treponema vincentii LA-1 (= ATCC 35580) and Ritz A.Culture media. Medium OMIZ-Pat (7) supplemented with 1% heat-inactivated human serum (catalog no. H1388; Sigma) 100 mg of fosfomycin per liter, and 1 mg of rifampin per liter was used during the isolation of strain HA2PT by limit dilution in 96-well microtiter plates (7). When cultures were supplemented with serum, medium OMIZ-Pat was prepared without asialofetuin. Human serum was heat treated at 56°C for 30 min, clarified by centrifugation and filtration through 0.2-pm-pore-size filters (type Supor 200; Gelman), and stored at -20°C. Solid-agarose cloning and further propagation were done in the absence of antibiotics as described previously (7).Phenotypic characterization. Determinations of enzyme activities and the growth-promoting effects of medium components, as well as other analyses, such as sodiu...

A chemically defined medium, OMIZ (Oral Microbiology and Immunology, Zurich)-W1 was developed. Medium OMIZ-W1 supports the long-term proliferation of a wide range of oral anaerobes, including representative strains of four Treponema species and Porphyromonas gingivaIss. High concentrations of ascorbic acid and ammonium ions proved to be important for the growth of these organisms. T. denticola CD-1 grew in the absence of polyamines and long-chain fatty acids, T. pectinovorum and T. socranskii required polyamines, whereas T. vincentii depended on both polyamines and lecithin for growth. Specific requirements for purines and/or pyrimidines were detected, and these requirements couid be used to distinguish Haemophilus-Actinobacilus group organisms. Some strains of P. gingivalis grew without vitamin K, while others were not satisfied by menadione but required its precursor 1,4-dihydroxy-2-naphthoic acid. Protoporphyrin IX or hemin equally satisfied the porphyrin requirements ofP. gingivalis and Bacteroidesforsythus, whereas ferrous sulfate was more efficiently used as a source of iron than was hemin. The cellular cohesiveness of P. gingivalis increased with high concentrations of hemin in the growth medium. PrevoteUa intermedia, B. forsythus, and several strains of P. gingivalis were more fastidious and required a protein or serum supplement to grow in medium OMIZ-W1.