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 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...

Guggenheimella bovis gen. nov., sp. nov., isolated from lesions of bovine dermatitis digitalis

“…In the present study, we used a rich, liquid medium, OMIZPat, and a limit-dilution culture technique developed for fastidious oral anaerobes (Wyss et al, 1996) to isolate bacterial strains from the advancing front of the lesions of two independent cases of DD in Switzerland. Two 2-yearold Simmental6Red Holstein heifers from small farms (each with about 20 dairy cows and their calves) developed lameness and typical DD about 2 months after returning from their (separate) summering alps.…”

“…Samples were mechanically dispersed and plated to obtain cultures by limit dilution in 96-well flat-bottom microtitre plates, using medium OMIZ-Pat (containing 1 % heatinactivated human serum) supplemented with 1 mg rifampicin, 100 mg fosfomycin, 5 mg polymyxin and 30 mg nalidixic acid l 21 as described by Wyss et al (1996). Novel isolates and reference strains Eubacterium limosum DSM 20543 T , Eubacterium brachy VPI D6B-23 T (Sundquist), Eubacterium nodatum ATCC 33099 T , Eubacterium sulci ATCC 35585 T and Mogibacterium timidum ATCC 33094 were cultured in medium OMIZ-Pat.…”

“…By using culture-independent methods, such as immunocytochemistry and 16S rRNA gene sequence analysis, additional organisms were detected in lesions and were related to Bacteroides levii, Borrelia burgdorferi, Mycoplasma hyopharyngis and several Treponema species (Collighan & Woodward, 1997; Demirkan et al, 1998; Moter et al, 1998). Treponemes have been detected deep in the affected tissue by in situ hybridization using fluorescent 16S rRNAtargeted oligonucleotide probes; however, the eubacterial organisms seen at the front of the lesion appeared not to be treponemes (Moter et al, 1998) and remain unidentified.In the present study, we used a rich, liquid medium, OMIZPat, and a limit-dilution culture technique developed for fastidious oral anaerobes (Wyss et al, 1996) to isolate bacterial strains from the advancing front of the lesions of two independent cases of DD in Switzerland. Two 2-yearold Simmental6Red Holstein heifers from small farms (each with about 20 dairy cows and their calves) developed lameness and typical DD about 2 months after returning from their (separate) summering alps.…”

“…Lesion samples were placed in reducing transport fluid (Loesche et al, 1972) and sent in a cool box to the laboratory, where they were processed within 14 h after surgery. Surgery combined with locally and intravenously administered oxytetracycline resulted in rapid healing (Luginbühl & Kollbrunner, 2000).Samples were mechanically dispersed and plated to obtain cultures by limit dilution in 96-well flat-bottom microtitre plates, using medium OMIZ-Pat (containing 1 % heatinactivated human serum) supplemented with 1 mg rifampicin, 100 mg fosfomycin, 5 mg polymyxin and 30 mg nalidixic acid l 21 as described by Wyss et al (1996). Novel isolates and reference strains Eubacterium limosum DSM 20543 T , Eubacterium brachy VPI D6B-23 T (Sundquist), Eubacterium nodatum ATCC 33099 T , Eubacterium sulci ATCC 35585 T and Mogibacterium timidum ATCC 33094 were cultured in medium OMIZ-Pat.…”

“…The peptidoglycan of OMZ 913 T cells was analysed by GC/MS after total hydrolysis and derivatization according to Groth et al (1996). API ZYM tests, growth assays and protein, antigen and glycan analyses on blots after SDS-PAGE were performed as described previously (Bickar & Reid, 1992;Wyss, 1998;Wyss et al, 1996). For immunoblots, sera of heifers JE and RI and the alkaline phosphatase-conjugated anti-bovine IgG mAb BG 18 (Sigma) were used at 1 : 50 and 1 : 1000 dilutions, respectively.…”