Molecular analysis of the subgingival microbiota in health and disease

Abstract

This investigation provides molecular analyses of the periodontal microbiota in health and disease. Subgingival samples from 47 volunteers with healthy gingivae or clinically diagnosed chronic periodontitis were characterized by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the V2-V3 region of the eubacterial 16S rRNA gene. A hierarchical dendrogram was constructed from band patterns. All unique PCR amplicons (DGGE bands) were sequenced for identity. Samples were also analyzed for the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis by multiplex PCR. Associations of patient age, gender, and smoking status together with the presence of each unique band and putative periodontal pathogens with disease were assessed by logistic regression. Periodontal pockets were colonized by complex eubacterial communities (10 to 40 distinct DGGE bands) with substantial individual variation in the community profile. Species diversity in health and disease was determined by the Shannon-Weaver index of diversity and compared by the Mann-Whitney U test. Sequence analyses of DGGE amplicons indicated the occurrence of many nontypical oral species and eubacteria previously associated with this environment. With the exception of T. forsythensis, the putative pathogens were not detected by DGGE. Multiplex PCR, however, detected T. forsythensis, A. actinomycetemcomitans, and P. gingivalis in 9% 16%, and 29% of the patients with disease, respectively. The presence of A. actinomycetemcomitans was significantly associated with disease (P < 0.01). Statistical analyses indicated that the presence of Treponema socranskii and Pseudomonas sp. was a significant predictor of disease (P < 0.05) and that there was no significant difference (P > 0.05) in terms of eubacterial species diversity between health and disease.

FIG. 1.

Representative negative DGGE image of six selected lanes showing the positions of all excised gel bands corresponding to the synthetic reference lane. Bands numbered 1 to 52 correspond to the numbered bands in Table 1.

FIG. 2.

UPGMA dendrogram showing the percent DGGE fingerprint matching of 47 samples of periodontally healthy (H) and diseased (D) samples derived from the subgingival crevice.

FIG. 3.

Classification plot derived from logistic regression analysis. The x axis is the predicted probability (Prob), from 0.0 to 1.0, of the dependent being classified “1” (disease). The y axis is frequency, i.e., the number of cases classified. Inside the plot are columns of observed ones and zeros, which stand for disease and health, respectively, with 2.5 cases per symbol.

FIG. 4.

Multiplex PCR of the periodontal pathogens A. actinomycetemcomitans (aa; 360 bp), P. gingivalis (pg; 197 bp), and T. forsythensis (tf; 745 bp). Lane 1, molecular size marker (100 to 1,000 bp). Lanes 2, 3, 4, and 6, amplified DNA from periodontal samples. Lane 5, positive control derived from the type strains A. actinomycetemcomitans NCTC 9710, P. gingivalis NCTC 11834, and T. forsythensis ATCC 43037.