Characterization of treponemes isolated from human and non-human primate periodontal pockets

Treponemes were isolated from ligature induced periodontal pockets of non-human primates, and from humans with periodontitis. Approximately 39% of the microscopic count were spirochetes from humans, while 65% of the microscopic microbiota was accounted for by spirochetes from non-human primates. Metabolically, the human treponemal isolates grew on trypticase-yeast extract based media while the non-human primate isolates grew only on pectin, glucuronic or galacturonic acids. The end-products of glucose fermentation by the human treponemes were acetate and propionate, while acetate was produced by the non-human primate treponemes from pectin. All of the human isolates were indole positive, hemolyzed blood, required serum for growth, but did not require thiamine pyrophosphate (TPP). The non-human primate treponemes were indole negative, were inhibited in their growth by blood, grew in the absence of serum, and required TPP for growth. PAGE analysis of whole cell proteins revealed three categories of treponemal isolates: (i) human isolates similar to Treponema denticola ; (ii) human isolates of small size; and (iii) non-human primate isolates similar to the pectinolytic treponemes. Serologically, the human treponemal isolates were similar to T. denticola , while the non-human primate isolates were similar to the pectinolytic treponemes. Four human, 4 non-human primate, and 4 reference treponemes exhibited a Mol% G + C of their DNA of 40.0-43.1. The metabolic differences between the human and non-human primate treponemal isolates may be a reflection of ecological differences in the periodontium of the pathological entities which exist in human and non-human primates.     

Detection frequencies and the colony-forming unit recovery of oral treponemes by different cultivation methods

Selected media were compared for primary isolation and detection of oral treponemes from clinical samples. Forty-eight subgingival plaque samples from 45 patients suffering from periodontitis were anaerobically cultivated for 2 weeks at 37°C. Of the 9 media studied, Medium 10 (M10), which was supplemented with 10% rabbit serum and incubated using the plate-in-bottle method, supported the highest colony-forming units of the anaerobes. The treponemal colonies were detected at least on one medium from 83% of the subgingival plaque samples. The new oral spirochete medium in an anaerobic chamber supported the highest detection frequency of the oral treponemes (64% of samples); however, M10 in the plate-in-bottle was found to produce the highest colony-forming unit recovery of the oral treponemes (median 3.6% of the total colony-forming units). This study suggests that M10 in the plate-in-bottle and new oral spirochete medium in the anaerobic chamber are essential in cultivating oral treponemes.     

Enumeration of viable oral spirochetes from periodontal pockets

We recently developed a successful method for quantifying oral anaerobic spirochetes in pure culture by a viable count. New oral spirochete medium was used with low temperature-gelling agarose in polystyrene tissue-culture flasks. We have extended the use of this method to determine the viable count of spirochetes from periodontal pockets. Sixteen subgingival plaque samples were obtained by insertion of sterile paper points into deep periodontal pockets. The points were placed into reduced transport medium at chairside, vortexed in the microbiology laboratory and aliquots of the medium inoculated into molten new oral spirochete-agarose medium (37°C) containing rifampin (20 μg/ml) in a flask. Subsequent dilutions were made from this initial flask to other flasks containing selective medium in sequence. All flasks were incubated anaerobically. Most other subgingival bacteria were selectively inhibited by rifampin. Spirochete colonies were typically spherical and were either dense or cottony. Their identities were checked by darkfield examination. Counts of colony-forming units of cultivable spirochetes ranged from 12.5% to 28.2% of the total cultivable anaerobic flora by the method described.     

Nutritional analysis and an enriched medium for fermentative treponemes isolated from subgingival plaque

In order to cultivate fermentative treponemes isolated on Medium 10 with the plate-in-bottle method from subgingival plaque, we developed the enriched Medium 10. Enriched Medium 10 broth consisted mainly of Trypticase peptone (2 g/1), yeast extract (10 g/1), glucose (5 g/1) and volatile fatty acids mixture (3.1 ml/1). The growth curves in enriched Medium 10 broth showed that clinical isolates of Treponema socranskii reached the early stationary phase on the 3rd–5th day, and the doubling times averaged 22.2 h. By using enriched Medium 10, the nutritional analysis of oral treponemes was examined for each compound of the volatile fatty acid and various sera. Clinical isolates were found to require iso-butyric acid, 2-methylbutyric acids and/or iso-caproic acid independently for their growth. However, the growth of these fermentative treponemes was inhibited by addition of various sera to this medium.     

Sulfate-reducing bacteria in periodontal pockets and in healthy oral sites

The aim of this study was to monitor the presence of sulfate-reducing bacteria (SRB) at different sites in the mouths of both healthy individuals and periodontitis patients. In 20 healthy subjects and 21 periodontitis patients, samples were taken from the palate, vestibulum, dorsum of the tongue, supragingival plaque, and periodontal pockets. In order to demonstrate growth of SRB, samples were incubated in an anoxic chamber in a reduced growth-medium for SRB, with an iron-indicator for sulfide production. The SRB were detected throughout the oral cavity. They were found on the mucosa in 10% of both healthy subjects and periodontitis patients. On the tongue and in supragingival plaque, the frequency of detection was slightly higher (22% of the subjects). In contrast, 86% of the periodontitis patients harbored SRB in one or more pockets. In 1/3 of the patients, SRB were present in all 3 pockets that were sampled. The data indicated that SRB belong to the normal oral microbiota, and have a preference for periodontal pockets.     

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.     

Reproducibility of microbiological samples from periodontal pockets

Duplicate microbiological samples, were taken 1 week apart using the paper point technique from a total of 112 untreated periodontal pockets greater than 6 mm deep in 16 adult periodontal patients. Duplicate samples were also obtained from these sites 6 months following a therapy of oral hygiene instruction and supra- and subgingival debridement. The reproducibility of the total viable counts and the reproducibility of the proportions of various groups or species of microorganisms were studied from these duplicate samples. The results demonstrated an acceptable degree of reproducibility for the recovery of Actinobacillus actinomycetemcomitans and Bacteroides gingivalis. For the total viable counts and for the other investigated bacterial groups, including Bacteroides intermedius, unacceptable levels of reproducibility were observed.     

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.     

The effect of supragingival plaque control on the composition of the subgingival flora in periodontal pockets

The effect of mechanical supragingival plaque control on the composition of the subgingival microflora in untreated 4-6 mm deep periodontal pockets was investigated. 13 subjects with chronic periodontitis were recruited for the study. Periodontally-diseased sites were subjected to professional plaque control 3 x weekly for a period of 3 weeks. Contralateral sites received no prophylaxis and served as controls. No instructions in oral hygiene procedures were given to the patients who maintained their habitual oral hygiene regime during the observation period. Clinical examination and darkfield microscopic analysis of bacterial samples were performed every week. The PlI scores for the experimental sites were reduced markedly, while those for the control sites remained stable throughout the observation period. No changes in the other clinical parameters were detected during the study. The composition of the subgingival microflora at the control sites did not change during the experimental period. In contrast, at the test sites, the proportion of spirochetes+motile rods decreased continuously. This decrease reached statistical significance at the end of the experiment. The results indicate that at periodontally diseased sites with an established subgingival ecosystem, supragingival plaque removal may influence the composition of the subgingival microflora.     

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.     

Recolonization of a subgingival microbiota following scaling in deep pockets

The present investigation was carried out to study some aspects of the recolonization of a subgingival microbiota following subgingival instrumentation in sites with deep pockets. 16 patients were recruited for the study. From each patient 4 inflamed gingival sites with deep pockets were selected. These sites were examined for plaque, overt gingivitis, bleeding on probing and probing depth. Samples of the subgingival microbiota were obtained and examined in the darkfield microscope and in a Neubauer chamber. Following the Baseline examination the teeth of all 4 jaw quadrants were carefully scaled and planed. Subgingival instrumentation was carried out under local anesthesia and required between 2-4 appointments. The patients were subsequently divided into 2 groups (Groups A and B) consisting of 9 and 7 subjects, respectively. During the first 16 weeks of maintenance the patients of Group A were not supervised regarding their self-performed plaque control measures and they accumulated supragingival plaque. The patients of Group B, however, were during these 16 weeks recalled once every 2 weeks for professional tooth cleaning. In addition they rinsed twice daily with a 0.2% solution of chlorhexidine digluconate. Reexaminations including assessments of the same parameters as those studied at Baseline were performed after 2, 4, 8, 12 and 16 weeks. After the 16-week examination the patients of Group A received a new sequence of subgingival scaling and root planing. During the subsequent 16 weeks the patients of Group A were also recalled for professional tooth cleaning. They were reexamined 18, 20, 24, 28 and 32 weeks after the Baseline examination. Subgingival scaling followed by carefully supervised oral hygiene measures resulted in a marked improvement of periodontal conditions. This improvement was accompanied by a pronounced and sustained reduction in the motile segments of the subgingival microbiota. In the presence of supragingival plaque (Group A), however, a subgingival microbiota containing large numbers of spirochetes and motile rods was soon (4-8 weeks) reestablished. A small number of sites with deep pockets (greater than or equal to 8 mm) was not substantially reduced in depth following subgingival instrumentation. In these sites which were kept free from supragingival deposits a subgingival microbiota with a large proportion of motile bacteria soon recurred.     

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.     

Effects of oral hygiene measures on clinical and microbiological parameters of periodontal disease

The effects of a 12-week period of oral hygiene alone on gingival conditions and subgingival microflora in 15 patients with severe periodontitis were investigated. Clinical measurements and plaque samples from selected sites were taken at week 0 (baseline), week 6, and week 12. Plaque samples were also taken at week 13, that is, 1 week following debridement. At week 0, the patients were instructed in supragingival plaque control and at week 6, the hygiene regimen was supplemented with the subgingival use of a toothpick device. At week 12, the patients received a full mouth supra- and subgingival debridement under local anesthesia. In those patients who complied with oral hygiene instructions (subgroup A), the gingival condition improved moderately while no improvement was found in less compliant patients (subgroup B). No significant changes were noted in the subgingival microflora in either subgroups A or B throughout the 12-week period of oral hygiene alone. However, significant reductions for all microbial parameters were found 1 week after debridement. Therefore, while moderate clinical improvements followed oral hygiene alone, no measurable changes in the subgingival microflora were observed concomitantly.     

An inexpensive solid medium for obtaining colony-forming units of oral spirochetes

A method for the enumeration of colony-forming units of oral anaerobic spirochetes in new oral spirochete agarose (NOS-A) medium was described recently. However, the high cost of agarose limits the extent to which large assays can be carried out. Accordingly, a search for an inexpensive gelling agent that remains molten at 37°C and gels at 25°C was undertaken. Varying amounts of Noble agar or Bacto agar (0.5 to 1.5%, w/v) were mixed with varying amounts of gelatin (0.5 to 1.0%, w/v) in NOS medium. NOS medium containing 0.5% gelatin-0.5% Noble agar (NOS-GN) or 0.5% gelatin-0.5% Bacto agar (NOS-GB) met the above criteria. NOS-GN and NOS-GB media yielded higher colony-forming units with Treponema denticola than NOS-A medium in that order. However, all three media, NOS-GN, NOS-GB and NOS-A, performed equally well in the recovery of viable counts of T. vincentii. The NOS-GN medium was not liquefied by subgingival bacteria or two gelatinase-producing species of bacteria, Bacillus subtilis and Staphylococcus aureus. Thus NOS-GN medium is the recommended medium both in cost and performance for obtaining colony counts of spirochetes.     

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.     

Prevalence of beta-lactamase-producing strains among 149 anaerobic gram-negative rods isolated from periodontal pockets

In a prospective study, 47 adults presenting a rapidly progressive periodontitis were selected in order to evaluate the prevalence of beta-lactamase-producing strains among oral anaerobic gram-negative rods. Predominant anaerobes were identified from two of the deepest periodontal pockets. beta-Lactamase-positive strains fulfilled to at least two of three criteria: positive nitrocefin test, penicillin Etest minimal inhibitory concentration > 1 microgram/ml, and disk diffusion synergy between amoxycillin and clavulanic acid > 10 mm. At least one beta-lactamase-producing strain was found in 53.2% of patients and 39.4% of the periodontal pockets investigated. Prominent beta-lactamase-positive species were Prevotella buccae and Prevotella intermedia (respectively 16 of 38: 42% and 18 of 52: 35% positive strains), followed by Prevotella bivia, Prevotella disiens, Prevotella denticola and Fusobacterium nucleatum (respectively 1 of 6: 17%, 1 of 10: 10%, 1 of 10: 10%, and 1 of 13: 8% positive strains). No beta-lactamase producer could be evidenced in Porphyromonas gingivalis (10 strains tested). All the beta-lactamase-positive strains with the nitrocefin test had penicillin minimal inhibitory concentrations > 1 microgram/ml with the Etest, and a strong synergy between amoxicillin and clavulanic acid was always observed.     

Genome size of human oral Treponema species by pulsed-field gel electrophoresis

The genome sizes of seven strains of oral treponemes were determined using pulsed-field gel electrophoresis (PFGE). These strains represent members from six of the currently known cultivable oral treponeme groups. The PFGE fragments were digitally recorded and then quantitated using GIMP v 1.2, an image manipulation program. The results show that the six oral treponeme genomes are comparable in size, ranging from approximately 2.2 to 2.5 Mbp. The genome sizes of these strains are 20-25% smaller than Treponema denticola strains, which have genome sizes of approximately 2.8-3.0 Mbp.     

The effect of subgingival debridement on periodontal disease parameters and the subgingival microbiota

Abstract The aim of the present investigation was to analyse the effect of subgingival scaling and root planing in subjects who prior to treatment exercised meticulous supragingival plaque control. 300 subjects were examined at baseline and after 1 and 2 years without treatment. After the year 2 examination, 62 subjects were randomly selected for therapy. They were given detailed instruction in proper self-performed toothcleaning measures and were carefully monitored during the subsequent 2 years. Following the year-4 examination, 2 quadrants, 1 maxillary and 1 mandibular in each subject, were randomly selected for additional therapy. The teeth in the selected quadrants were exposed to subgingival scaling and root planing. The subgingival therapy was repeated until a site no longer bled on gentle probing. This basic therapy was completed within a 2-month period. All subjects were re-examined after another 12-month interval. The examinations at year 4 and 5 included assessment of plaque, gingivitis, probing pocket depth and analysis of samples obtained from the subgingival microbiota at 134 selected sites. The findings from the present study demonstrated: (i) that subgingival scaling and root planing were effective in eliminating subgingival plaque and gingivitis; (ii) that professional therapy resulted in a pronounced reduction of probing depth at sites which at year 4 had a probing depth >3 mm; (iii) that in non-scaled quadrants, the extension of self-performed plaque control resulted in a continued improvement of the periodontal conditions at sites which at year 4 were < 5 mm deep.     

Clinical and microbiological effects of root debridement in periodontal furcation pockets

The aim of the present study was to investigate longitudinally over 52 weeks the clinical and microbiological effects of plaque control and root debridement at molar furcation sites. The results were compared with changes at non-molar sites. 24 non-molar sites and 31 grade II molar furcation sites with probing depth greater than or equal to 5.0 mm were monitored in 11 patients. Clinical measurements consisted of plaque scores, probing depths, and changes in probing attachment level. Microbiological monitoring was carried out with phase-contrast microscopy and anaerobic culturing. The debridement resulted in improvement in probing measurements and microbiological counts for both groups of sites. A slightly less favorable clinical response was noted for molar furcation sites. Higher post-operative microbiological counts were found throughout the 52-week observation period for molar furcation sites. Sites with probing attachment loss showed higher microbial counts and higher proportions of spirochetes, black pigmented colony forming units (CFU), and Bacteroides gingivalis CFU than sites with probing attachment gain. Individual site analysis, however, demonstrated marked variations of the microbiological counts at the different postoperative time points. In the few available sites undergoing probing attachment loss, no apparent association between target micro-organisms and periodontal deterioration was observed.