Identification of Seven Treponema Species in Health- and Disease-Associated Dental Plaque by Nested PCR

Abstract

Species-specific nested PCR was used to detect Treponema amylovorum, Treponema denticola, Treponema maltophilum, Treponema medium, Treponema pectinovorum, Treponema socranskii, and Treponema vincentii in dental plaque. Subjects with periodontitis harbored all species, but T. pectinovorum and T. vincentii were not found in plaque from disease-free subjects.

Plaque treponemas are associated with periodontal diseases (6), but detection of individual species has been limited by the availability of specific antibodies and DNA probes and by the lack of selective culture methods (7). Monoclonal antibodies were used to detect Treponema denticola and Treponema socranskii in plaque from healthy and diseased sites (10), but direct microscopic observation probably underestimated cell numbers. A 16S rRNA-based PCR was used to detect T. denticola in plaque from sites of gingivitis and periodontitis (2), but PCR has not been used to identify other treponema species. Recently, dot blot hybridization with species-specific probes and amplified bacterial DNA was used to identify T. denticola, Treponema maltophilum, T. socranskii, and Treponema vincentii in health-associated and disease-associated plaque from subjects with rapidly progressive periodontitis (8). However, the prevalence of Treponema amylovorum (13) and Treponema medium (11) has not been determined, and the distribution of treponema species in plaque from periodontal disease-free subjects has not been described. The purpose of this investigation was to use species-specific nested PCR (nPCR) to identify each of seven treponema species in plaque from young individuals without periodontal disease and from both healthy and diseased sites in older subjects with chronic periodontitis.

Crevicular plaque was collected from a single site in individuals with no detectable gingivitis or periodontitis (HH, n = 10 subjects, mean age ± standard deviation [SD] = 25.4 ± 7.1 years) and from clinically healthy sites (probing depth, <4 mm; relative attachment level, <2 mm) in subjects with chronic periodontitis (HD, n = 10 subjects, 59.6 ± 10.5 years), and subgingival plaque was collected from sites of periodontitis (probing depth, ≥5 mm; relative attachment level, ≥2 mm) (DD, n = 11 subjects, 64.7 ± 11.0 years). Subjects had not received periodontal treatment or taken antibiotics within 3 months prior to sampling. Samples were suspended in 0.5 ml of sterile saline, and spirochetes were counted by dark-field (DF) microscopy at 400×. Suspensions were centrifuged, and pellets were lysed by three cycles of freeze-thawing. One microliter of lysate was resuspended in 9 μl of distilled water, and 1 μl (1.5 μl for HH) was used as target in the universal reaction. One microliter of the universal reaction mixture was used as template for each nested specific reaction. In brief, 25-μl-mixture reactions were carried out in a buffer containing 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl (pH 8.3; Boehringer Mannheim), 25 μM concentrations of each deoxynucleoside triphosphate (Sigma), 0.1 μM concentrations of forward and reverse primers (Table 1), and 1.25 U of Taq polymerase (Boehringer Mannheim) and overlaid with 1 drop of mineral oil (Sigma). After initial denaturation at 97°C for 1 min, 26 cycles were performed as follows: denaturation for 45 s at 97°C, annealing for 45 s (temperatures listed in Table 1), extension for 1 min at 72°C, and a final extension at 72°C for 4 min. Eight microliters of each PCR product was electrophoresed on a 1% agarose gel in 0.5× Tris-borate-EDTA buffer at 4 V/cm for 1 h, stained for 20 min with ethidium bromide, destained for 10 min in distilled water, and photographed. Positive reactions were determined by the presence of bands of the appropriate size (Table 1).

TABLE 1.

Primer sequences, product size (base pairs), and temperature conditions for nPCR

Target	PCR primer (5′-3′)	Position (bp)	Tm (°C)
Universal 16S ribosomal DNA	AGA GTT TGA TCC TGG CTC AG	8–1,513 (1,505)	55
	ACG GCT ACC TTG TTA CGA CTT
T. amylovorum 16S	AGA GTT TGA TCC TGG CTC AG	8–211 (193)	54
	CTC ACG CCT TTA TTC CGT GAG
T. denticola 16S	TAA TAC CGT ATG TGC TCA TTT ACA T	193–508 (316)	60
	TCA AAG AAG CAT TCC CTC TTC TTC TTA
T. maltophilum 16S	AGA GTT TGA TCC TGG CTC AG	8–446 (438)	54
	CCT ATT GTG CTT ATT CAT CAG GC
T. medium 16S	AGA GTT TGA TCC TGG CTC AG	8–200 (192)	54
	CCT TAT GAA GCA CTG AGT GTA TTC
T. pectinovorum 16S	AGA GTT TGA TCC TGG CTC AG	8–205 (194)	53
	ATA TAT CTC CAA CTT ATA TGA CCT
T. socranskii 16S	GAT CAC TGT ATA CGG AAG GTA GAC A	179–468 (285)	53
	TAC ACT TAT TCC TCG GAC AG
T. vincentii 16S	AGA GTT TGA TCC TGG CTC AG	8–201 (193)	56
	AAT ACT TCT TAT GAA CAT TGA GAC

The following bacteria were used for specificity tests and as nPCR controls: Actinobacillus actinomycetemcomitans (American Type Culture Collection, Manassas, Va.) ATCC 33384, Bacteroides forsythus ATCC 43037, Borrelia burgdorferi ATCC 35210, Eikenella corrodens ATCC 23834, Porphyromonas gingivalis ATCC 33277, Prevotella intermedia ATCC 25611, T. amylovorum ATCC 700233 (Christopher Wyss, University of Zurich, Zurich, Switzerland), T. denticola ATCC 35405, T. denticola ATCC 33521, T. denticola ATCC 35404, T. denticola GM-1 (Denée Thomas, University of Texas, San Antonio), T. maltophilum ATCC 51939 (C. Wyss), T. medium G7201 (Toshihiko Umemoto, Asahi University, Gifu, Japan), Treponema pallidum (D. Thomas), Treponema pectinovorum ATCC 33768, Treponema phagedenis ATCC 51274, Treponema socranskii subsp. buccale ATCC 35534, Treponema socranskii subsp. paredis ATCC 35535, Treponema socranskii subsp. socranskii ATCC 35536, and T. vincentii ATCC 35580 and ATCC 700013.

Universal primers (4, 13) anneal at conserved regions near the 5′ and 3′ ends of 16S rRNA, giving a nearly full-length 16S product. Primers for T. denticola (2) and T. socranskii (9) have specific forward and reverse primers. Primers for T. amylovorum, T. maltophilum, T. medium, T. pectinovorum, and T. vincentii utilize the universal forward primer (positions 8 to 27 of Escherichia coli 16S rRNA) (5) with a specific reverse primer designed by us. EMBL accession numbers used for primer design and comparisons are as follows: E. coli 16S rRNA gene (GenBank accession no. J01859); T. amylovorum, Y09959; T. denticola, M71236 and D85438; T. maltophilum, X87140; T. medium, D85437; T. pectinovorum, M71237; T. socranskii, AF033305, AF033306, and AF033307; and T. vincentii, AF033309 and AF033310.

Primers were tested against all control bacteria, and no bands of the predicted size were produced with nontarget DNA. BLAST (1) searches revealed no likely cross-reactivity with other oral bacteria. Sensitivity, estimated from serial dilutions of pure treponemas, was 10 to 100 cells for all nPCRs. Experiments with spiked plaque indicated that some plaque samples raised the detection threshold as much as 10-fold for target species (unpublished observations).

nPCR was more sensitive than DF microscopy, particularly for samples with low numbers of spirochetes (Tables 2 to 4). nPCR detected treponemas in five more HH samples and four more HD samples (P = 0.035 and P = 0.043, respectively; Fisher exact probability test) than did DF microscopy, suggesting that the prevalence of treponemas in health-associated plaque may be greater than expected from microscopy-based estimates (8, 10). DF microscopy analysis detected as many spirochete-positive samples as did PCR among DD specimens. There were no significant differences between groups for mean DF microscopy counts (P > 0.05 for HH versus HD and for HH versus DD; t test).

TABLE 2.

Spirochete counts with DF microscopy and identification of treponema species by nPCR in plaque from subjects without periodontal diseases (group HH)

Individual plaque sample	1	2	3	4	5	6	7	8	9	10	No. of samples/species
DF resulta	0	0	0	0	0	0	380	10	540	0
nPCR amplification result for spirochete
T. amylovorum	−	−	−	+	−	−	+	−	+	−	3
T. denticola	−	+	+	+	+	+	+	−	−	−	6
T. maltophilum	−	−	−	+	+	−	+	+	+	−	5
T. medium	−	−	−	+	−	−	+	−	+	−	3
T. pectinovorum	−	−	−	−	−	−	−	−	−	−	0b
T. socranskii	−	−	−	+	+	+	+	+	+	−	6
T. vincentii	−	−	−	−	−	−	−	−	−	−	0
No. of species/sample	0	1	1	5	3	2	5	2	4	0

^aProportion of DF microscopy-positive samples was significantly less than proportion of PCR-positive samples (P = 0.035; Fisher exact probability test). Results are shown as 10³ treponemas per sample. The mean count ± SD was (310 ± 270) × 10³ treponemas (n = 3). There was no significant difference in mean DF microscopy counts (t test) between HH and either HD (Table 3) or DD (Table 4). 

^bProportion of HH subjects with T. pectinovorum was significantly less than that of HD (P = 0.029; Fisher exact test). 

TABLE 4.

Spirochete counts by DF microscopy and identification of treponema species in plaque samples by nPCR in disease-associated plaque from subjects with periodontitis (group DD)

Individual plaque sample	1	2	3	4	5	6	7	8	9	10	11	No. of samples/species
DF resulta	0	50	560	3,100	860	1,500	700	4,400	900	250	51
nPCR amplification result for spirochete
T. amylovorum	−	+	−	−	+	+	+	+	+	+	−	7
T. denticola	−	+	+	+	+	+	+	+	+	+	−	9
T. maltophilum	−	+	+	+	+	+	+	+	+	+	+	10
T. medium	+	−	−	+	+	+	+	+	+	+	−	8
T. pectinovorum	+	−	−	−	−	−	+	−	−	+	−	3
T. socranskii	−	+	+	+	+	+	+	+	+	+	−	9
T. vincentii	+	+	+	+	−	+	+	−	+	+	+	9b
No. of species/sample	3	5	4	5	5	6	7	5	6	7	2

^aResults are shown as 10³ treponemas per sample. The mean count ± SD was (1,200 ± 1,400) × 10³ treponemas (n = 10). 

^bProportion of DD subjects with T. vincentii was significantly greater than that of either HH or HD subjects (P = 0.024; Fisher exact probability test). 

The number of treponema species detected ranged from zero to five species/sample of HH plaque to two to seven species/sample of HD and DD plaque. However, there were no significant differences in mean numbers of species detected between groups (t test). These findings indicate that treponema diversity varies from sample to sample. Although samples of health-associated plaque contained multiple treponema species, diversity is likely to be less than that reported for diseased sites (3, 8).

T. amylovorum, T. denticola, T. maltophilum, T. medium, and T. socranskii were detected in plaque from every group, and there were no significant differences between groups for proportions of subjects with these treponemas. Neither T. pectinovorum nor T. vincentii was detected in HH plaque, and in contrast to a previous report (8), both T. pectinovorum and T. vincentii were detected in health-associated plaque from subjects with periodontitis. There were significantly fewer HH subjects with T. pectinovorum than HD or DD subjects (P = 0.029; Fisher exact test), and there were significantly more DD subjects with T. vincentii than either HH or HD subjects (P = 0.024; Fisher exact test). Differences in detection may exist because HH subjects were significantly younger than subjects with periodontitis (P < 0.05; t test), because nPCR is more sensitive than dot blot hybridization used previously (8), or because treponema distribution differs between subjects with chronic periodontitis in this report and subjects who had rapidly progressive periodontitis (8).

Although the current subject population was small, the data provide some potentially important new information. For example, these preliminary observations suggest that even young adults with healthy periodontal tissues may harbor several oral treponema species. Furthermore, whether T. pectinovorum and/or T. vincentii is detected in health-associated plaque may correlate with the relative risk for disease on a site-specific basis. Further studies are needed to verify these findings and to determine whether differences in prevalence correlate with disease susceptibility or disease activity.

TABLE 3.

Spirochete counts by DF microscopy and identification of treponema species by nPCR in health-associated plaque from subjects with periodontitis (group HD)

Individual plaque sample	1	2	3	4	5	6	7	8	9	10	No. of samples/species
DF resulta	0	0	0	610	8	15	2,000	760	120	0
nPCR amplification result for spirochete
T. amylovorum	−	−	−	+	+	+	+	+	+	−	6
T. denticola	−	−	+	+	−	+	+	+	+	+	7
T. maltophilum	−	−	−	+	+	+	+	+	+	+	7
T. medium	−	−	+	+	+	+	+	+	+	−	7
T. pectinovorum	−	+	−	+	+	+	+	−	−	−	5
T. socranskii	+	+	−	+	+	+	+	+	+	+	9
T. vincentii	+	+	−	+	−	−	−	−	−	−	3b
No. of species/sample	2	3	2	7	5	6	6	5	5	3

^aProportion of DF microscopy-positive samples was significantly less than proportion of PCR-positive samples (P = 0.043; Fisher exact probability test). Results are shown as 10³ treponemas per sample. The mean count ± SD was (590 ± 760) × 10³ treponemas (n = 6). 

^bProportion of HD subjects with T. vincentii was significantly less than that of DD subjects (Table 4).