Use of randomly cloned DMA fragments for the identification of oral spirochetes

DNA probes were produced for the detection and identification of 4 cultivable species of oral spirochetes, Treponema denticola, Treponema socranskii, Treponema vincentii and Treponema pectinovorum. To obtain probe sequences, chromosomal DNA, isolated from representative strains within each species, was cloned in Escherichia coli K-12. Cloned DNA fragments were screened for the ability to hybridize to DNA only from homologous strains. Several such fragments were identified and shown to be specific when tested against a series of DNAs from gram-negative and gram-positive oral bacteria. The selected probe sequences were semi-conserved within strains of T. denticola and T. socranskii such that restriction fragment length polymorphism (RFLP) was observed. In the case of T. socranskii, RFLP was useful in distinguishing between the 3 known subspecies. Chromosomal DNA fragments from 2 strains of T. vincentii failed to cross-hybridize, under stringent conditions, to genomic DNA from each of these strains. The hybridization probes were suitable for the identification of clinical isolates of T. denticola and could be used to detect the presence of individual Treponema species in mixed cultures. On this basis, the probes were used successfully to detect T. denticola in uncultured plaque samples.     

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.     

Serum antibodies in young adult humans reactive with periodontitis associated treponemes

The present study was undertaken to determine whether young adults with generalized severe periodontitis (SP) or the more localized juvenile periodontitis (JP) have serum antibody reactive with a panel of periodontitis associated treponemes more frequently or at higher titer than a group of young adults with a healthy periodontium (HP). The human subjects consisted of 52 SPs, 47 JPs, and 52 HPs. The panel of treponemes included three strains of Treponema denticola and several strains of newly described treponemes, Treponema socranskii ss. socranskii, Treponema socranskii ss. buccale , and Treponema socranskii ss. paredis . The frequency and magnitude of serum antibody titers were determined using a sensitive radioimmunoassay. Subjects with JP were more frequently seropositive than were HP subjects to several strains of the treponemes tested (p<0.01 for Treponema socranskii ss. buccale [D40PE2], Treponema denticola [D3A9], and Treponema socranskii ss. paredis [D28C3]). However, the most surprising result was the low level and frequent lack of antibody in SP sera reactive with these periodontitis-associated treponemes. Excluding data where only two points above the cut off were required for seropositivity, neither the frequency of seropositivity nor the magnitude of antibody titers of SP subjects exceeded those respective values for HP subjects. The frequency of seropositivity in SP subjects to T. socranskii ss. socranskii (D43BR1) was significantly depressed as compared to HP subjects (p<0.01). In short, it appeared that SP subjects either failed to mount a significant anti-treponemal antibody response or those responses were specifically suppressed.     

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.     

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 .     

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.     

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.     

Treponema socranskii, Treponema denticola, and Porphyromonas gingivalis are associated with severity of periodontal tissue destruction.

BACKGROUND: The aim of the present study was to identify Treponema socranskii in addition to Treponema denticola and Porphyromonas gingivalis by polymerase chain reaction (PCR), and to clarify the relationship between the presence of these microorganisms and the severity of clinical periodontal parameters. METHODS: Saliva and subgingival plaque collected from 123 subjects (38 aggressive periodontitis patients, 65 chronic periodontitis patients, 20 healthy patients) were subjected to PCR to detect the aforementioned 3 microorganisms. RESULTS: Detection frequencies of T. socranskii, T. denticola, and P. gingivalis in plaque samples from aggressive periodontitis patients (71.1%, 73.7%, 84.2%, respectively) and chronic periodontitis patients (89.2%, 93.8%, 95.3%) were much higher than those from healthy subjects (30%, 5.0%, 10.0%). In aggressive and chronic periodontitis patients, these 3 species of bacteria were detected frequently at sites that showed deep periodontal pockets and severe attachment loss. The percentage of these bacteria-positive sites increased as the gingival index score of chronic periodontitis patients increased. T. socranskii was frequently detected together with T. denticola or P. gingivalis at the same sites, and coexistence of these microorganisms was frequently observed in deep periodontal pockets of aggressive periodontitis patients. CONCLUSIONS: T. socranskii, T. denticola, and P. gingivalis were frequently detected in periodontitis patients by PCR. The prevalence of these 3 microorganisms was correlated with various clinical parameters. Taken together, our findings suggest that T. socranskii, T. denticola, and P. gingivalis are associated with the severity of periodontal tissue destruction.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

Ribotyping on small-sized spirochetes isolated from subgingival plaque

In the present study DNA restriction patterns and corresponding ribotypes of 17 subgingival small-sized spirochetes (1:2:1 and 2:4:2 isolates), 2 Treponema socranskii strains and two Treponema denticola strains were examined. Purified chromosomal DNA was digested by Bam HI, Hin dIII, Pst I and Cla I. The DNA fragments were separated in a horizontal slab of 0.7% agarose containing ethidium bromide and transferred by nylon membranes. Hybridization was carried out with digoxigenin-labelled copy DNA of 16S and 23S ribosomal RNA from Escherichia coli . Depending on the restriction endonuclease used, up to 4 distinct bands were observed for the 2:4:2 isolates and the T. denticola strains. For each of the endonucleases used, identical band patterns were always observed for this group of isolates, and these patterns differed persistently from the T. denticola strains. For the 1:2:1 strains, up to 11 distinct bands were observed after digestion with Hindlll, whereas a maximum of 6 bands were observed when Pst I or Cla I was used. By using Clal, the examined 1:2:1 isolates were separated into 8 groups, whereas Pst I and Hin dIII separated these isolates into 5 groups. The ribotyping showed that the tested 1:2:1 spirochetes were more heterogeneous than the 2:4:2 spirochetes examined.     

Prostaglandin E(2) is a main mediator in receptor activator of nuclear factor-kappaB ligand-dependent osteoclastogenesis induced by Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii

BACKGROUND: Periodontitis is an inflammatory disease that often leads to destruction of alveolar bone; a number of bacteria in subgingival plaque are associated with bone destruction in periodontitis. To understand the mechanism of how periodontopathogens induce osteoclastogenesis, we determined which mediators are involved in the osteoclastogenesis. METHODS: We investigated effects of sonicates from three periodontopathic bacteria, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, on osteoclast formation in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. The osteoclast formation was determined by tartrate resistant acid phosphatase (TRAP) staining. The expression of the receptor activator of nuclear factor-kappa B ligand (RANKL), prostaglandin E(2) (PGE(2)) and osteoprotegerin (OPG) in mouse calvaria-derived osteoblasts was determined by immunoassay. RESULTS: Each bacterial sonicate induced the osteoclast formation in the co-culture system. These bacterial sonicates increased the expression of RANKL and PGE(2), and decreased the expression of OPG in osteoblasts. The addition of OPG, an inhibitor of RANKL, in the co-culture completely suppressed the osteoclastogenesis that was stimulated by each bacterial sonicate. Indomethacin, which is an inhibitor of PGE(2) synthesis, reduced more than 88% of the osteoclast formation induced by each bacterial sonicate. Indomethacin inhibited more than 80% of RANKL expression in osteoblasts induced by T. denticola and T. socranskii, and 59% by P. gingivalis. Indomethacin completely recovered the depression of OPG expression in osteoblasts by T. denticola and T. socranskii to the level of the untreated osteoblasts. Indomethacin recovered the reduction of OPG expression by P. gingivalis to 67%. CONCLUSION: These findings suggest that the osteoclastogenesis by P. gingivalis, T. denticola, and T. socranskii is mediated by a RANKL-dependent pathway and that PGE(2) is a main factor in the pathway by the enhancing of RANKL expression and the depression of osteoprotegerin, a RANKL inhibitor.     

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.     

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.     

Identification of oral spirochetes at the species level and their association with other bacteria in endodontic infections

OBJECTIVES: Recent molecular approaches have revealed that fastidious organisms such as Bacteroides forsythus and oral treponemes were frequently found in root canals with apical periodontitis. The purpose of this study was to identify the isolates of oral spirochetes at the species level in endodontic infections and to determine their association with B forsythus and Porphyromonas gingivalis. STUDY DESIGN: Seventy-nine teeth with apical periodontitis were selected for this study. After sampling from the root canals aseptically, polymerase chain reaction amplification for the 16S rRNA gene was performed with eubacterial universal primers. Subsequently, dot-blot hybridization was performed with 8 species-specific oligonucleotide probes. The microbial associations were analyzed by using the odds ratio. RESULTS: The most frequently found species was P gingivalis (27.4%), followed by Treponema maltophilum (26%), B forsythus (16.4%), and Treponema socranskii (2.7%). Other treponemes, including Treponema denticola, were not detected in our samples. Significant microbial associations were identified between T maltophilum, B forsythus, and P gingivalis by performing analysis with the odds ratio. CONCLUSION: Our results indicate that T maltophilum should be included in etiologic studies of endodontic diseases.