Are putative periodontal pathogens reliable diagnostic markers?

Abstract

Periodontitis is one of the most common chronic inflammatory diseases. A number of putative bacterial pathogens have been associated with the disease and are used as diagnostic markers. In the present study, we compared the prevalence of oral bacterial species in the subgingival biofilm of generalized aggressive periodontitis (GAP) (n = 44) and chronic periodontitis (CP) (n = 46) patients with that of a periodontitis-resistant control group (PR) (n = 21). The control group consisted of subjects at least 65 years of age with only minimal or no periodontitis and no history of periodontal treatment. A total of 555 samples from 111 subjects were included in this study. The samples were analyzed by PCR of 16S rRNA gene fragments and subsequent dot blot hybridization using oligonucleotide probes specific for Aggregatibacter (Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, a Treponema denticola-like phylogroup (Treponema phylogroup II), Treponema lecithinolyticum, Campylobacter rectus, Fusobacterium spp., and Fusobacterium nucleatum, as well as Capnocytophaga ochracea. Our data confirm a high prevalence of the putative periodontal pathogens P. gingivalis, P. intermedia, and T. forsythia in the periodontitis groups. However, these species were also frequently detected in the PR group. For most of the species tested, the prevalence was more associated with increased probing depth than with the subject group. T. lecithinolyticum was the only periodontopathogenic species showing significant differences both between GAP and CP patients and between GAP patients and PR subjects. C. ochracea was associated with the PR subjects, regardless of the probing depth. These results indicate that T. lecithinolyticum may be a diagnostic marker for GAP and C. ochracea for periodontal health. They also suggest that current presumptions of the association of specific bacteria with periodontal health and disease require further evaluation.

FIG. 1.

Dot blot hybridizations of identical membranes with probes for P. gingivalis and C. ochracea. In columns 1 to 9, PCR products of the following strains were applied as controls: Actinobacillus actinomycetemcomitans ATCC 43718 (A1); Actinobacillus actinomycetemcomitans ATCC 33384 (A2); Actinobacillus actinomycetemcomitans serotype a (A3); Leptotrichia buccalis MCCM 00448 (A4); Pasteurella haemolytica ATCC 33396 (A5); Haemophilus influenzae ATCC 33391 (A6); Haemophilus influenzae clinical isolate (A7); Haemophilus aphrophilus NCTC 55906 (A8); Haemophilus paraphrophilus ATCC 29241 (A9); Porphyromonas gingivalis ATCC 33277 (B1); Prevotella intermedia ATCC 25611 (B2); Porphyromonas assacharolyticus ATCC 25260 (B3); Prevotella nigrescens NCTC 9336 (B4); Prevotella oralis MCCM 00684 (B5); Prevotella buccalis ATCC 33690 (B6); Capnocytophaga ochracea ATCC 27872 (B7); Capnocytophaga sputigena ATCC 33612 (B8); Capnocytophaga gingivalis ATCC 33624 (B9); Campylobacter rectus ATCC 33238 (C1); Campylobacter concisus ATCC 33237 (C2); Bacteroides gracilis ATCC 33236 (C3); Bacteroides fragilis ATCC 25285 (C4); Eikenella corrodens CCUG 2138 (C5); Kingella kingae ATCC 23330 (C6); Veillonella parvula ATCC 10790 (C7); Veillonella dispar ATCC 17748 (C8); Klebsiella pneumoniae ATCC 23357 (C9); Fusobacterium nucleatum ATCC 25586 (D1); Flavobacterium odoratum MCCM 02932 (D2); Neisseria lactamica ATCC 23970 (D3); Streptococcus mutans ATCC 35668 (D4); Streptococcus intermedius ATCC 27335 (D5); Actinomyces viscosus ATCC 15987 (D6); Actinomyces israelii ATCC 10048 (D7); Eubacterium lentum ATCC 25559 (D8); Selenomonas sp. clinical strain (D9); Fusobacterium simiae CCUG 16798 (E4); Fusobacterium periodonticum CCUG 14345 (E5); and Fusobacterium necrophorum NCTC 25286 (E6). Asterisks indicate empty fields without PCR product. In columns 10 to 14, 15 to 19, and 20 to 24, PCR products from subgingival plaque samples of PR subjects, CP patients, and GAP patients (five patients each) were applied, respectively. Each column represents four deep pockets plus one control site from a single patient. For an accurate analysis, the samples from each patient were spotted on the membrane in a random order. Samples were considered positive if the dot was clearly visible above the background level of the negative controls.

FIG. 2.

Prevalence of target species in the GAP patients, CP patients, and PR subjects as determined by dot blot hybridizations using oligonucleotide probes. A patient was regarded as positive if at least one sample was positive. Numbers in parentheses indicate statistical significances between the groups: 1, GAP versus CP; 2, GAP versus PR; and 3, CP versus PR, as determined by chi-square analysis with Bonferroni's correction for multiple comparisons. Empty parentheses (), as for A. actinomycetemcomitans, indicate an overall significance but no significant differences between the groups in the post hoc test results.

FIG. 3.

Percentage of patients with 1, 2, 3, 4, or 5 of the sites colonized by target bacteria as revealed by dot blot analysis. Numbers in parentheses indicate statistical significances between the groups: 1, GAP versus CP; 2, GAP versus PR; and 3, CP versus PR, as determined with the Mann-Whitney U test. P values were adjusted for multiple comparisons (Bonferroni's correction).

FIG. 4.

Percentage of positive samples at different probing depths. According to the inclusion criteria, the four deepest pockets showed a probing depth of ≥6 mm in the GAP group and <6 mm in the PR group. Therefore, only the CP patient group and the PR group were compared at probing depths of 4 to 5 mm, and only the two periodontitis groups were compared at probing depths of 6 to 8 mm and ≥9 mm. Asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001) indicate significant differences as determined by analysis of variance. The mean percentages of positive sites were derived by averaging the positive sites of each species within a subject and then across subjects in the clinical groups.