"Checkerboard" DNA-probe analysis and anaerobic culture of initial periodontal lesions.

In this study, we compared a new, rapid DNA-probe assay sample) of each species by using the total anaerobic count for each sample. with anaerobic culture for characterizing the microbiology Five healthy sites and five gingivitis sites from selected of initial periodontal lesions in adults. We used the microbisubjects were sampled for DNA-probe analysis with 13 probes ological data from both assays to identify the microbiota by using the checkerboard hybridization method [2] and a associated with the conversion from gingival health to periMiniblotter 45 apparatus (Immunetics Inc., Cambridge, MA). odontal disease. These sites were all different from those sampled for culture. In previous studies of the microbiota of periodontal infecThe probes were whole chromosomal DNA, random-primer tions, investigators have mainly examined the microbiota labeled with digoxigenin (Genius 2 kit, Boehringer Mannof periodontal pockets in patients with established disease heim, Mannheim, Germany) for health-associated and diseaseand have used anaerobic cultural methods. Little informaassociated periodontal species [5]. Reactions were detected tion is available about the microbiota associated with the by chemiluminescence, and the results were recorded on initiation of periodontitis [1]. An understanding of the initial x-ray film (figure 1). The DNA probe reactions were visually stages of periodontal disease (i.e., when loss of attachment read from the x-ray films and scored relative to quantitation first occurs around a previously healthy tooth) will be imcontrols for each species on the same membrane/film on a portant for risk assessment and prevention of these infecscale of 0–3, where 0 Å õ10, 1 Å 10–õ10, 2 Å 10– tions. By using cultural methods, in which the predominant õ10, and 3 Å §10 cells per sample. species are isolated in pure culture and identified by phenoWe identified 64 species and at least 15 other taxa or typic tests, all isolates can be characterized, but these methgroups amongú3,000 isolates examined with use of anaeroods are labor intensive and time-consuming and therefore bic culture. The predominant microbiota (table 1) was gram limit the number of samples that can be analyzed. positive, and Actinomyces naeslundii, Streptococcus oralis, To facilitate large-scale clinical studies, which are necesand Peptostreptococcus micros were the most frequently sary to assess microbiological risk markers and risk factors cultured species. The major gram-negative species identified for periodontal disease, more-rapid methods for assaying were Camplyobacter gracilis, Bacteroides forsythus, Veillomultiple bacterial species in plaque samples will be renella parvula, and Selenomonas noxia. Selenomonas and quired. We assessed one such method, the ‘‘checkerboard’’ Eubacterium species were cultured from many of the samDNA-probe assay [2], which enables simultaneous analysis ples. DNA probe results showed Streptococcus sanguis, of 28 plaque samples with £40 DNA probes on a single V. parvula, and S. oralis to be the predominant species membrane, in a study of initial periodontitis. We compared (probes to A. naeslundii, C. gracilis, S. noxia, and Eubactethis method with anaerobic culture by using the predominant rium species were not included in the DNA assay). Porphycultivable microbiota method. romonas gingivalis and Actinobacillus actinomycetemcomiThe gums of 56 healthy adults with minimal periodontal tans were detected infrequently. attachment loss were clinically measured at 3-month interComparison of the cultural and DNA-probe assays for vals for 12–18 months to determine periodontal pocket selected species identified by both methods showed similar depth and periodontal attachment level and to detect plaque, results (figure 2). Culture yielded higher levels of B. forredness, and bleeding on probing. Active periodontitis sites sythus and Campylobacter rectus, and the DNA probes gave showed a §1.5-mm increase in periodontal attachment loss higher levels of Prevotella intermedia, Prevotella nigresduring monitoring. Subgingival plaque from active and inaccens, Fusobacterium nucleatum subspecies vincentii, tive sites was sampled for anaerobic culture. Fifty isolates S. oralis, S. sanguis, and V. parvula (figure 2). Levels of from each sample were cultured and identified on the basis B. forsythus and C. rectus were significantly elevated in of gram staining, growth in air, fluorogenic substrate tests active lesions, as determined both by culture and by DNA[3], and SDS-PAGE of whole cell proteins [4]. The cultural probe assay (figure 2). Anaerobic culture also showed that data were expressed as the percentage of total cultivable S. noxia, Fusobacterium alocis, and Eikenella corrodens microbiota represented by each species identified in each were associated with active sites. Inactive sites contained sample. We calculated the absolute level (count of cfu per elevated levels of Streptococcus oralis. In this study, B. forsythus and C. rectus were the principal species associated with active initial periodontal lesions. Pathogens associated with established disease, including Grant support: National Institute of Dental Research (DE 09513). Reprints or correspondence: Dr. M. F. J. Maiden, Department of Periodontal P. gingivalis and A. actinomycetemcomitans, were rarely Microbiology, Forsyth Dental Center, 140 Fenway, Boston, Massachusetts found in these subjects with minimal periodontal attachment 02115. loss. Good agreement with cultural results showed the poClinical Infectious Diseases 1997;25(suppl 2):S230–2 tential usefulness of the DNA-probe method for studying q 1997 by The University of Chicago. All rights reserved. 1058–4838/97/2503–0045$03.00 the microbiology of periodontal infections.