Restriction fragment-length polymorphism analysis of 16S ribosomal RNA genes amplified by polymerase chain reaction for rapid identification of cultivable oral treponemes

Although oral treponemes are among the most frequently found bacteria in periodontal pockets, identification of these organisms can be difficult. In this study, restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA genes (16S rRNA gene PCR-RFLP) was used to generate restriction profiles of reference strains of oral treponemes including Treponema denticola, Treponema socranskii, Treponema vincentii. Treponema pectinovorum and Treponema medium as well as for Treponema phagedenis and Treponema pailidum and five treponeme strains isolated from human peridontal pockets. Before RFLP analysis, the 16S rRNA gene sequences were obtained from the GenBank database, and the analysis of the theoretical banding patterns for HpaII suggested good species discrimination. 16S rRNA gene sequences were amplified from isolated genomic DNA samples by PCR with spirochete-specific primers. The PCR products were then purified and characterized by single digestion with restriction endonuclease HpaII, and this allowed discrimination between the respective reference strains. Five clinical isolates, four T. denticola and one T. socranskii, were assigned on the basic of their restriction profiles by digestion with HpaII. 16S rRNA gene PCR-RFLP using HpaII is a rapid and reliable method for differentiation of cultivable oral treponemes.     

Taxonomic study of spirochetes isolated from human periodontal lesions

Nineteen strains of spirochetes obtained from subgingival plaque of 19 patients with advanced periodontitis were studied morphologically, biochemically, and genetically and compared with type strains of Treponema denticola and Treponema socranskii. The results showed that 16 strains which biochemically resembled T. denticola could be divided into two groups based on G + C content and DNA homology. Two isolates appeared to belong to the T. socranskii species. One intermediate size isolate did not fit any known Treponema species.     

Visualization of an extracellular mucoid layer of Treponema denticola ATCC 35405 and surface sugar lectin analysis of some Treponema species

Slime layers and capsules are common amongst medically relevant bacteria. We herein report that Treponema denticola, which has been associated with periodontitis, synthesizes or acquires an extracellular polysaccharide layer that we have observed through electron microscopy using the polysaccharide-specific dye Alcian blue and phosphotungstate. We have also visualized this extracellular layer by dark-field microscopy of Alcian blue-stained spirochete cells. A representative strain of each of the oral spirochete species T. denticola, Treponema vincentii and Treponema socranskii were differentiated by concanavalin A, phaseolus, lotus A and arachis lectins in a microtiter plate immunoassay for the detection of surface sugars.     

Distribution and characterization of plasmids in oral anaerobic spirochetes

Fifteen oral spirochete strains belonging to the species Treponema denticola, Treponema vencentii and Treponema socranskii as well as 9 fresh clinical isolates were screened for the presence of extrachromosomal plasmid DNA by a modified alkaline lysis procedure. A 2.6–kb plasmid was detected in both T. denticola ATCC 33520 and T. denticola e'. The 2.6–kb plasmid from T. denticola e' was shown to be similar to pTDl, previously reported by Ivic et al. in T. denticola ATCC 33520 on the basis of molecular weight, restriction endonuclease profile and DNA:DNA hybridization. T. denticola ATCC 33520 and T. denticola e'share 65% DNA homology and belong to different serological groups. This dissimilarity has been reconfirmed by specific immunofluorescence using polyclonal and monoclonal antibodies. A plasmid-free T. denticola ATCC 33520 was identified. Comparative studies have shown no antigenic, morphological, or genetic differences between the plasmid-bearing and the plasmid-free strain. In addition, screening of fresh clinical isolates of spirochetes revealed the presence of a 4.2–kb plasmid in 4 of these strains.     

Treponema species associated with abscesses of endodontic origin

Spirochetes have been frequently observed in abscesses of endodontic origin, but they have rarely been identified. This study sought to investigate the prevalence of eight oral treponemes in acute periradicular abscesses using a species-specific nested polymerase chain reaction assay. Purulent exudate was collected by aspiration from 19 cases diagnosed as acute periradicular abscesses and DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first polymerase chain reaction products to detect a specific fragment of the 16S rDNA of each Treponema species. The species-specific nPCR assay used in this study allowed the detection of Treponema denticola in 79%(15 of 19), Treponema socranskii in 26%(5 of 19), Treponema pectinovorum in 21% (4 of 19), Treponema amylovorum in 16% (3 of 19), and Treponema medium in 5% (1 of 19) of the cases. Spirochetal DNA was found in 89% of the cases (17 of 19). The number of Treponema species per case ranged from 1 to 3 (mean, 1.5). Treponema vincentii , Treponema lecithinolyticum and Treponema maltophilum were not detected in any pus sample. The present data lend support to the assertion that Treponema species, particularly T. denticola and T. socranskii , may be involved in the pathogenesis of acute periradicular abscesses.     

Genetic characterization of an oral treponeme isolated from human subgingival plaque

We examined the genetic characteristics of a human oral treponeme isolated from subgingival plaque of a patient with periodontal disease that could not be assigned to any of the previously described oral Treponema species. The guanine-plus-cytosine content of its DNA was 51 mole%. The levels of DNA-DNA relatedness of the organism to other Treponema species, including Treponema denticola, Treponema vincentii, Treponema socranskii, Treponema pallidum and Treponema phagedenis , were less than 30%. This study clearly demonstrated that the isolated treponeme could be completely distinct from the established oral Treponema species genetically. Also, the study indicated remarkable genetic heterogeneity among human oral Treponema species.     

Resistance to human beta-defensins is common among oral treponemes

BACKGROUND/AIMS: Oral treponemes are implicated in the pathogenesis of periodontal disease. We have previously shown that Treponema denticola ATCC type strains and strain GM-1 are resistant to killing by human beta-defensins (hbetaD)-1 and -2. We hypothesize that resistance to beta-defensins is a common feature of oral treponemes, which allows colonization and persistence in the oral cavity. In this study, we tested additional isolates of T. denticola, as well as six other species of treponemes, for resistance to hbetaD-1, -2 and -3. We also examined the four ATCC strains of T. denticola and strain GM-1 for resistance to hbetaD-3. METHODS: Resistance was determined by motility and Alamar Blue assays for metabolic activity. RESULTS: All T. denticola strains tested were resistant to hbetaD-1, -2 and -3, with the exception of strain Ambigua, which was sensitive to hbetaD-2 and -3. All other treponemes except Treponema vincentii were resistant to hbetaD-1. Treponema pectinovorum was sensitive to hbetaD-2, while T. vincentii, T. pectinovorum and Treponema maltophilum were sensitive to hbetaD-3. Escherichia coli was used as a control organism and was killed by all three defensins. CONCLUSION: Resistance to the constitutively expressed hbetaD-1 may assist treponemes in initial colonization of epithelial surfaces, while resistance to the inducible hbetaD-2 and -3 would allow some treponemes to survive in active periodontal lesions.     

Characterization of new periodontal and endodontic isolates of spirochetes

Gas chromatographic analysis of cellular fatty acids (CFA), biochemical reactions, electrophoresis of soluble cellular proteins, as well as immunodiffusion were used to discriminate between 16 fresh spirochete isolates from periodontal and endodontic infections. CFA patterns were compared by the Hewlett Packard MIDI library to all available reference strains, and results of the biochemical tests were compared to VPI's records for treponemal strains. The electrophoretograms of soluble proteins of the fresh isolates were compared to those of previously well described strains of Treponema denticola, T. pectinovorum, T. vincentii, T. socranskii subsp. socranskii, T. socranskii subsp. paredis, T. socranskii subsp. buccale , and T. socranskii 04. Immunodiffusion was carried out by using adsorbed polyclonal rabbit antibodies to representative strains of the species mentioned. These methods separated most clinical strains from approved species strains, suggesting that new species had been isolated.     

Identification of spirochetes (treponemes) in endodontic infections

The purpose of this study was to determine the prevalence of spirochetes in asymptomatic infected root canals and in endodontic abscesses/cellulitis. Aseptic clinical samples were collected using paper points from 54 infected root canals and from aspirates of 84 abscesses/cellulitis. Oligonucleotide primers were produced for PCR identification of Treponema vincentii, T. pectinovorum, T. medium, T. amylovorum, T. denticola, T. maltophilum, and T. socranskii. PCR detected spirochetes in 51 of 84 (60.7%) samples from abscesses/cellulitis and in 20 of 54 (37.0%) samples from asymptomatic infected root canals. T. socranskii was the most frequently detected (44.9%), followed by T. maltophilum (29.7%), T. denticola (28.9%), T. pectinovorum (13.7%), and T. vincentii (5.1%). The number of treponema species detected ranged from 1 to 5 species per sample. The mean numbers of species detected were 2.3 in abscesses/cellulitis and 2.6 in infected root canals. Significant association among species was found between T. maltophilum and T. socranskii, as well as between T. maltophilum and T. denticola by determining the odds ratio (> 2.0).     

Characterization of a 4.2-kb plasmid isolated from periodontopathic spirochetes

Oral anaerobic treponemes are associated with active periodontal disease and may comprise up to 57% of the microbiota in periodontal pockets. Four treponeme strains (designated U2a, U2b, U9b, and U9c) isolated from clinical cases were found to harbor a new 4.2-kb plasmid when plasmid DNA was extracted and purified employing the Qiagen Plasmid Kit. This plasmid differs from the smaller plasmids (2.0-, 2.6-, and 2.7-kb) reported previously by others in Treponema denticola. The newly discovered 4.2-kb plasmid was found to be the same in all four treponeme strains by restriction endonuclease analysis. It is a circular plasmid since restriction with Pst I, Pvu II, Sma I, Xma I, Ava I or Bam HI produced a single band of the same size. Bacterial strain U2b was shown to be Treponema socranskii and U9c to be T. denticola. The plasmid is designated "pTS1". The presence of the same plasmid in different species of the treponemes isolated from the same patient suggests the possibility of a naturally occurring genetic transfer system within the oral spirochetes or their ability to take up and maintain mobilizable plasmids.     

Characterization of oral treponemes isolated from human periodontal pockets

A total of 90 clinical strains of oral treponemes was isolated from subgingival plaque in patients with periodontal disease. They were characterized by biochemical means as well as cell enzyme, protein analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DNA dot blot hybridization. Sixty strains were isolated on Medium 10 (M10), which was fundamentally serum-free. The remainder were isolated on serum-containing media. Isolates were divided into 6 groups according to their biochemical characteristics. Three of the 6 groups were asaccharolytic, and 2 of these 3 groups were Treponema denticola and " Treponema vincenti ". The other 3 groups were saccharolytic and further divided into 9 subgroups. The analyses of cell enzyme, cell protein and dot blot hybridization with the DNA probe of Treponema socranskii indicated that all the saccharolytic groups were T. socranskii or closely related species. This study indicated that the newly characterized saccharolytic oral treponemes could be identified using M10 from the human periodontal pocket.     

Amended biochemical characteristics and phylogenetic position of Treponema medium

Umemoto et al. (1997, Int J Syst Bacteriol 47, pp. 67-72) proposed spirochete strain G7201, isolated from the periodontal pocket of an adult patient, as a new species, Treponema medium. They deposited this strain in the American Type Culture Collection (ATCC) as type strain ATCC 700293T. Recently, ATCC suggested that there is a discrepancy between the previous report and the results obtained by ATCC in biochemical tests on T. medium ATCC 700293T. In this study, we re-examined and verified the biochemical characteristics of T. medium. The fermentation pattern of carbohydrates of T. medium resembled that of Treponema vincentii and Treponema denticola, but T. medium was clearly differentiated from T. vincentii in the production of indole, and from T. denticola in the hydrolysis of esculin. Also, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profile analysis and phylogenetic comparison of 16S rDNA sequences revealed that T. medium is clearly differentiated from any established treponemal species, which supports the validity of the proposal of Treponema medium as a new species.     

Taxonomy and virulence of oral spirochetes

All oral spirochetes are classified in the genus Treponema. This genus is in the family Spirochaetaceae as in Bergey's manual of systematic bacteriology. Other generic members of the family include Spirochaeta, Cristispira and Borrelia. This conventional classification is in accord with phylogenetic analysis of the spirochetes based on 16S rRNA cataloguing. The oral spirochetes fall naturally within the grouping of Treponema. Only four species of Treponema have been cultivated and maintained reliably: Treponema denticola, Treponema pectinovorum, Treponema socranskii and Treponema vincentii. These species have valid names according to the rules of nomenclature except for Treponema vincentii, which only has had effective publication. The virulence factors of the oral spirochetes updated in this mini-review have been discussed within the following broad confines: adherence, cytotoxic effects, iron sequestration and locomotion. T. denticola has been shown to attach to human gingival fibroblasts, basement membrane proteins, as well as other substrates by specific attachment mechanisms. The binding of the spirochete to human gingival fibroblasts resulted in cytotoxicity and cell death due to enzymes and other proteins. Binding of the spirochete to erythrocytes was accompanied by agglutination and lysis. Hemolysis releases hemin, which is sequestered by an outer membrane sheath receptor protein of the spirochete. The ability to locomote through viscous environments enables spirochetes to migrate within gingival crevicular fluid and to penetrate sulcular epithelial linings and gingival connective tissue. The virulence factors of the oral spirochetes proven in vitro underscore the important role they play in the periodontal disease process. This role has been evaluated in vivo by use of a murine model.     

Viscosity-dependent locomotion of oral spirochetes

Spirochetes are thought to remain motile in environments (such as intercellular spaces) that immobilize extracellularly flagellated eubacteria. This attribute suggests that the viscosity of the milieu is of importance to locomotion. We sought to determine the interdependence of oral spirochete locomotion with media viscosity. Video time-lapse microscopy using darkfield optics was used. The motility of the spirochetes in media of different viscosities (various concentrations of Noble agar was measured. Treponema denticola exhibited the fastest speed (18.7 ± 4.4 μ/min) at a viscosity of 30 mPa s. The highest speeds for Treponema vincentii and Treponema socranskii were 41.9 ± 14.9 and 33.4 ± 13.2 μm/min, respectively, at 88 mPa s. These data show that optimal migration of spirochetes is viscosity-dependent. The results support the hypothesis that such viscosity-dependent locomotion could be a virulence factor that enables oral spirochetes to initiate and sustain periodontal disease.     

Identity of 1:2:1 and 2:4:2 subgingival spirochetes by DNA hybridization

A group of 1:2:1 and 2:4:2 subgingival spirochetes, well characterized by transmission electron microscopy, biochemical tests, cellular fatty acid and carbohydrate analyses, and ribotyping, was recently suggested to represent new treponemal species. The present study used DNA hybridization to examine this possibility. When DNA of a representative strain (no. 16) of the 8 1:2:1 spirochetes examined was labeled by iodination, it showed, after SI nuclease treatment, from 58 to 104% (average 76%) homology with DNA from the 1:2:1 spirochetes. 94% homology with DNA from the type strain of Treponema socranskii and of T. socranskii subsp. socranskii , i.e., ATCC 35536T, and 62% homology with DNA from T. socranskii subsp. buccale , strain ATCC 35534^T. Similarly treated DNA from a representative strain (no. 3) of 8 2:4:2 spirochetes exhibited from 90 to 105% (average 97%) homology with DNA from the 2:4:2 spirochetes, and 85% and 87% homology, respectively, with DNA from Treponema denticola strains ATCC 33520 and FDC T1. There was a negligible degree of homology between the 1:2:1 and 2:4:2 spirochetes. Thus, all the 2:4:2 spirochetes belonged to T. denticola . 1:2:1 strains with DNA homology levels >70% (5 strains) belonged to T. socranskii or T. socranskii subsp. socranskii , while those with homology levels from 58 to 63% (3 strains) most likely belonged to other subspecies of T. socranskii .     

Binding of hemin and Congo red by oral hemolytic spirochetes

Colony-forming units or cells in suspension of oral anaerobic spirochetes ( Treponema denticola, Treponema vincentii and Treponema socranskii ) bind hemin and Congo red. Hemin or Congo red binds to a hydrophobia polypeptide receptor that is located in the outer membrane of the bacterial cells and it has a relative molecular mass of 47 kDa. These oral spirochetes also lyse sheep erythrocytes to produce beta-hemolytic zones around colony-forming units. The oral spirochetes may acquire iron for growth when they lyse erythrocytes and bind heme from which they may sequester and transport iron into the cells.     

Common and specific antigens of several treponemes detected by polyclonal antisera against major cellular proteins

Thirteen polypeptide antigens with molecular weights ranging from 34 kDa to 83 kDa were selected and their antigenic behaviors and distribution were examined in 12 strains of microorganisms including Treponema, Borrelia, Leptospira and Leptonema. Immunoblot analysis demonstrated that 45 kDa and 83 kDa polypeptides of Treponema socranskii subsp. buccale ATCC 35534, 53 kDa antigen of Treponema denticola ATCC 33520 and 44 kDa polypeptide of the strain G7201 were strain-specific. The 34, 62, 66 and 84 kDa polypeptide antigens were detected in all 8 treponemal strains examined. T. denticola ATCC 33520 and ATCC 35404 possessed 38 kDa, 48 kDa, 52 kDa and 72 kDa common polypeptide antigens. All 12 strains possessed the 84 kDa polypeptide antigen. Immunoelectron microscopy demonstrated that the 34 kDa and 38 kDa polypeptide antigens were located on the axial flagella and that other polypeptide antigens were located on the outer envelopes or wall-membrane complexes.     

Population genetic analysis of oral treponemes by multilocus enzyme electrophoresis

Seventeen treponemes recently isolated from necrotic pulps, periodontal and periapical infections and 17 previously well characterized oral treponemal strains were analyzed by multilocus enzyme electrophoresis. Ten genetic loci were characterized on the basis of the electrophoretic mobilities of their enzymatic products. All loci were polymorphic. The average number of alleles per locus was 7.8. The genetic diversity among the electrophoretic types at each locus ranged from 0.624 to 0.836 with a mean genetic diversity per locus of 0.751. The 34 strains represented 34 electrophoretic types, constituting 6 main divisions (I-VI) separated at genetic distances greater than 0.75. Several of the previously characterized treponemes revealed multiple bands of enzyme activity at several loci, indicating that they were not pure. The characterized strains usually clustered within established species, whereas fresh clinical isolates overlapped species borders. There was a large genetic difference between some reference and clinical strains, indicating that the latter may contain undescribed species. Treponema socranskii and Treponema denticola strains clustered in distinct divisions (IV and V, respectively), with the exception of T. denticola strain FDC 51B2 and T. socranskii subsp. paredis strain VPI D46CPE1, both previously well described. This indicated that the taxonomic assignment of these 2 strains should be reconsidered.     

Nutritional requirements for oral Treponema

Oral Treponema is still difficult to cultivate in vitro because of lacking of our knowledge about what they require for growth. The aim of this study was to find what were the nutritional requirements for them. Treponema denticola, T. vincentii, and T. socranskii were cultivated in various broths to seek which fractions of tryptone and rabbit serum, or serum proteins were stimulatory. Treponemal growth was assessed by turbidity readings. It was obvious that a weakly negative-charged peptide isolated from tryptone with molecular weight of about 800 daltons was stimulatory. However, further characterization of this peptide was failed, since the yield of the final fractions was too low to be identified with the conventional methods. The results on rabbit serum fractionated by a liquid chromatograph showed that serum proteins with molecular weight of 150,000 daltons were also stimulatory for T. denticola. Among the commercially available serum proteins, human and bovine ceruloplasmin gave good results. They could substitute for rabbit serum in the growth of T. denticola and T. vincentii.     

Associations between Porphyromonas gingivalis and oral treponemes in subgingival plaque

Colonization and/or proliferation of Treponema denticola may depend on the presence of Porphyromonas gingivalis . The aims of this study were to confirm this synergistic relationship, to determine whether other oral bacteria were similarly associated with P. gingivalis and to relate coinfection to the periodontal status of plaque donors. Subgingival plaque was collected from every tooth except third molars in 106 subjects who were grouped by their worst periodontal condition. In addition to P. gingivalis , monoclonal antibodies were used to identify Campylobacter rectus, Eikenella corrodens, T. denticola, Treponema socranskii and pathogen-related oral spirochetes. Associations of these bacteria with coinfection by P. gingivalis were assessed by estimated odds ratios. The results indicate that coinfection with P. gingivalis is linked to all tested bacteria, but each pair was associated with distinct periodontal conditions. The distribution of coinfected sites suggests biased colonization of facial surfaces over lingual surfaces.