Differential analysis of culturable and unculturable subgingival target microorganisms according to the stages of periodontitis

Abstract

Objectives: Culturable and unculturable microorganisms have been associated with periodontitis. Their differential proportions and composition have not been evaluated by their severity and complexity defined by stages in the 2018 AAP-EEP classification.

Methods: One hundred eighty subgingival biofilm samples were collected in Spain and Colombia from subjects categorized as health/gingivitis: periodontitis stages I/II periodontitis stages III/IV. Target culturable microorganisms (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Eubacterium nodatum) and target unculturable microorganisms (Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis) were evaluated by quantitative PCR analysis. In addition, their differences and association with periodontal status were analyzed by ANCOVA and logistic regression models once adjusted to age, current smoking, and country.

Results: P. gingivalis was significantly associated with periodontitis stages I/II, OR 2.44 (CI 95% 1.08-5.47) and stages III/V, OR 6.43 (CI 95% 2.43-16.9). T forsythia, OR 7.53 (CI 95% 2.07-27.4); D. oralis, OR 5.99 (CI 95% 2.71-13.23); F. alocis, OR 10.9 (CI 95% 4.56-23.2); E. brachy, 3.57 (CI 95% 1.40-9.11); and E. saphenum, 4.85 (CI 95% 1.99-11.7) were significantly associated only with stages III/IV periodontitis. P. gingivalis evidenced significant differences with the increase in the severity of the periodontal lesion: 2.97 colony forming unit (CFU)/μL (CI 95% 2.32-3.54) health/gingivitis, and 4.66 CFU/μL (CI 95% 4.03-5.30) and 5.90 CFU/μL (CI 95% 5.20-6.48) in stages I/II and III/IV respectively (p < 0.0001). Unculturable microorganisms only evidenced differences in concentration in stages III/IV compared with health-gingivitis (p ≤ 0.001).

Conclusion: Culturable and unculturable are strongly associated with stages III/IV periodontitis. Classic culturable microorganisms are more sensitive to differentiate between stages of periodontitis in the quantitative analysis.

Clinical relevance: Future interventional studies of periodontal disease should include Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, and Desulfobulbus oralis as possible markers of therapy response and as indicators of progressive disease.

Keywords: Bacteria; Biofilm; Diagnosis; Periodontitis; Quantitative PCR.