Studies using molecular techniques have demonstrated that a culture-based approach can severely underestimate the bacterial diversity in most environments. One of the molecular techniques that has been applied in microbial ecology is denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to investigate differences in the microbiota of plaque, using a number of analysis techniques, from children without gingivitis (n ‫؍‬ 30) and from those with gingivitis (n ‫؍‬ 30). Extracted DNA from gingival margin plaque was subjected to PCR targeting the 16S rRNA gene using universal primers. DGGE profiles were analyzed in three ways. (i) Bacterial diversity was compared between cohorts by using the Shannon-Wiener index (also known as the Shannon-Weaver index). (ii) A hierarchical cluster analysis of the banding patterns was calculated and expressed as a dendrogram. (iii) Individual DGGE bands and their intensities for both cohorts were compared using a logistic regression analysis. The Shannon-Wiener indices demonstrated a greater bacterial diversity associated with no-gingivitis plaque (P ‫؍‬ 0.009). Dendrograms demonstrated that seven clades associated with gingivitis and five clades associated with no gingivitis. The logistic regression demonstrated that one band was significantly associated with no gingivitis (P ‫؍‬ 0.001), while two bands were significantly associated with gingivitis (P ‫؍‬ 0.005 and P ‫؍‬ 0.042). In conclusion, this study demonstrates that the development of gingivitis might be accompanied by a decrease in bacterial diversity. Furthermore, we have demonstrated that logistic regression is a good statistical method for analyzing and characterizing DGGE profiles.

Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Tannerella forsythensis have been implicated as the main etiological agents of periodontal disease. The purpose of this work was to estimate the prevalence of these organisms in plaque from children without gingivitis (group 1; n ‫؍‬ 65) and from those with gingivitis (group 2; n ‫؍‬ 53). Extracted DNA from plaque was subjected to two rounds of PCR targeting the 16S rRNA gene using both universal primers and species-specific primers. The results were as follows: group 1, P. gingivalis, 49%; A. actinomycetemcomitans, 55%; and T. forsythensis, 65%; group 2, P. gingivalis, 47%; A. actinomycetemcomitans, 59%; and T. forsythensis, 45%. T. forsythensis was detected more frequently in children with no gingivitis than in those with gingivitis (P ‫؍‬ 0.03). There was no significant difference between the two groups with respect to the presence of P. gingivalis or A. actinomycetemcomitans in either group (P > 0.05). Logistic regression analysis revealed that the odds of a patient having gingivitis were 2.3 times greater in the absence of T. forsythensis. In conclusion, the results of this study have shown that the three pathogens can be detected in the dental plaque of healthy children and of those with gingivitis and that T. forsythensis is associated with dental plaque at sites with no gingivitis.

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Recent molecular approaches for the study of microbial communities such as PCR-cloning have enabled the detection and identification of as-yet-unculturable taxa. Cloning and sequencing of multiple samples is extremely laborious and expensive to perform thoroughly due to the large diversity involved. For this purpose, techniques such as denaturing gradient gel electrophoresis (DGGE) may be better suited. There is increasing evidence suggesting that DGGE of complex polymicrobial communities may be limited by co-migration of different sequences. In this study, we attempt to address this limitation by excising individual bands and running them through a shorter denaturant gradient, a process we have termed "denaturing gradient gel electrophoresis gel expansion" (DGGEGE).

Aims:  To develop a perfusion biofilm system to model tongue biofilm microflora and their physiological response to sulfur‐containing substrates (S‐substrates) in terms of volatile sulfide compound (VSC) production. Methods and Results:  Tongue‐scrape inocula were used to establish in vitro perfusion biofilms which were examined in terms of ecological composition using culture‐dependent and independent (PCR‐DGGE) approaches. VSC‐specific activity of cells was measured by a cell suspension assay, using a portable industrial sulfide monitor which was also used to monitor VSC production from biofilms in situ. Quasi steady states were achieved by 48 h and continued to 96 h. The mean (±SEM) growth rate for 72‐h biofilms (n = 4) was μ = 0·014 h−1 (±0·005 h−1). Comparison of biofilms, perfusate and original inoculum showed their ecological composition to be similar (Pearson coefficient > 0·64). Perfusate and biofilm cells derived from the same condition (co‐sampled) were equivalent with regard to VSC‐specific activities which were up‐regulated in the presence of S‐substrates. Conclusions:  The model maintained a stable tongue microcosm suitable for studying VSC production; biofilm growth in the presence of S‐substrates up‐regulated VSC activity. Significance and Impact of the Study:  The method is apt for studying ecological and physiological aspects of oral biofilms and could be useful for screening inhibitory agents.