Periodontitis and caries are infectious diseases of the oral cavity in which oral biofilms play a causative role. Moreover, oral biofilms are widely studied as model systems for bacterial adhesion, biofilm development, and biofilm resistance to antibiotics, due to their widespread presence and accessibility. Despite descriptions of initial plaque formation on the tooth surface, studies on mature plaque and plaque structure below the gum are limited to landmark studies from the 1970s, without appreciating the breadth of microbial diversity in the plaque. We used fluorescent in situ hybridization to localize in vivo the most abundant species from different phyla and species associated with periodontitis on seven embedded teeth obtained from four different subjects. The data showed convincingly the dominance of Actinomyces sp., Tannerella forsythia, Fusobacterium nucleatum, Spirochaetes, and Synergistetes in subgingival plaque. The latter proved to be new with a possibly important role in host-pathogen interaction due to its localization in close proximity to immune cells. The present study identified for the first time in vivo that Lactobacillus sp. are the central cells of bacterial aggregates in subgingival plaque, and that Streptococcus sp. and the yeast Candida albicans form corncob structures in supragingival plaque. Finally, periodontal pathogens colonize already formed biofilms and form microcolonies therein. These in vivo observations on oral biofilms provide a clear vision on biofilm architecture and the spatial distribution of predominant species.

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BackgroundMicrobial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria.ResultsWe describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes.ConclusionWe developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

The aim of this study was to examine the diffusion of macromolecules through an in vitro biofilm model of supragingival plaque. Polyspecies biofilms containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, Veillonella dispar, and Candida albicans were formed on sintered hydroxyapatite disks and then incubated at room temperature for defined periods with fluorescent markers with molecular weights ranging from 3,000 to 900,000. Subsequent examination by confocal laser scanning microscopy revealed that the mean square penetration depths for all tested macromolecules except immunoglobulin M increased linearly with time, diffusion coefficients being linearly proportional to the cube roots of the molecular weights of the probes (range, 10,000 to 240,000). Compared to diffusion in bulk water, diffusion in the biofilms was markedly slower. The rate of diffusion for each probe appeared to be constant and not a function of biofilm depth. Analysis of diffusion phenomena through the biofilms suggested tortuosity as the most probable explanation for retarded diffusion. Selective binding of probes to receptors present in the biofilms could not explain the observed extent of retardation of diffusion. These results are relevant to oral health, as selective attenuated diffusion of fermentable carbohydrates and acids produced within dental plaque is thought to be essential for the development of carious lesions.The structure of microbial biofilms, consisting of single cells, cell aggregates, and microcolonies embedded in an exocellular polymeric hydrogel, has been the subject of intense experimental and theoretical scrutiny over the past decade (10,20,38,46). Biofilm anatomy and the physiological status of the cells contained within biofilms have profound consequences for clinical, industrial, and environmental microbiology. The exocellular space constitutes a primitive microcirculatory system that enables two-way transport between biofilm constituents and their surroundings and that facilitates intrabiofilm communication.Knowledge of the kinetics of mass transport within biofilms is essential for understanding how they achieve their characteristic architectures and for optimizing strategies to control or eradicate biofilms. Transport phenomena in biofilms have been studied by using microelectrodes (3, 41, 42) fiberoptic microsensors (2, 43), nuclear magnetic resonance spectroscopy (1, 52), infrared spectroscopy combined with Raman microscopy (47), fluorescence recovery after photobleaching (4, 5), fluorescence correlation spectroscopy (FCS) (18), and confocal laser scanning microscopy (CLSM) (11,25,26). Indirect methods predicated upon nuclear magnetic resonance spectroscopy, infrared spectroscopy-Raman microscopy, fluorescence recovery after photobleaching, FCS, or CLSM are preferred for measuring mass transport in biofilms, since invasive procedures (e.g., those involving microelectrodes or microsensors) are likely to compromise structural integrity and therefore perturb the del...

The aim of this investigation was to study the topographic distribution of Actinobacillus actinomycetemcomitans in patients with adult periodontitis before and after mechanical periodontal treatment (repeated oral hygiene instructions, systematic deep scaling, and root planing). In 10 A. actinomycetemcomitans‐positive patients, subgingival microbial samples were obtained from the mesial and distal aspect of every tooth (38 to 56 sites per patient, 479 sites in total) before and one month after treatment. The samples were cultured on TSBV agar. A. actinomycetemcomitans was identified based on phenotypical and serological criteria. A. actinomycetemcomitans was present in 40% of the samples taken before and in 23% of the samples taken after treatment. Before treatment, the frequency of A. actinomycetemcomitans‐positive samples per patient was wide spread and ranged from 7 to 90%. After treatment, two patterns of A. actinomycetemcomitans distribution could be recognized: the majority of the patients showed only a limited percentage of positive samples and yielded less than 105 A. actinomycetemcomitans. In three subjects, however, relatively high numbers of positive sites were still present, and many of these positive sites showed high A. actinomycetemcomitans counts. Logistic multiple regression showed the presence of A. actinomycetemcomitans before treatment depended strongly on the individual and was significantly associated with probing depth (P <0.001) and bleeding upon sampling (P = 0.07). The highest chance of detecting A. actinomycetemcomitans existed in deep pockets which bled upon sampling. After treatment, there was a strong individual influence and an influence of probing depth (P <0.001). The highest chance of detecting A. actinomycetemcomitans existed in residual pockets in the range of 5 mm. J Periodontol 1994; 65:820–826.

The prevalence of Bacteroides forsythus and Bacteroides gingivalis in subgingival plaque of patients on short recall was analyzed in relation to the probing depth of the test sites. The subjects had excellent oral hygiene and therefore were unlikely to suffer from active periodontal destruction. Sixty-four subgingival plaque samples, taken from gingival or periodontal pockets with probing depths ranging from 1 to 8 mm, were quantitatively assessed for the presence of the two species using species-specific monoclonal antibodies in conjunction with a very sensitive indirect immunofluorescence technique. Both organisms were encountered in probes from sites as shallow as 2 mm, but the percentage of positive samples clearly rose in relation to the probing depth of the test sites. Overall, B. forsythus was found to colonize lesions earlier, that is at smaller probing depths, than B. gingivalis. Interestingly, whenever a sample was found to be positive for B. gingivalis it was also positive for B. forsythus. The numbers of B. forsythus and B. gingivalis and the total bacterial cell number found in the pockets were significantly correlated to the probing depth. However, with advancing probing depth the increase of the total cell numbers of the two Bacteroides species was considerably more pronounced than the increase of the total subgingival plaque cell number. The recall interval neither affected the frequency of sites positive for B. forsythus or B. gingivalis nor influenced significantly the proportions of the two species in subgingival plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of the present study was to examine the Actinobacillus actinomycetemcomitans carrier rate in Chinese subjects, and to determine serotype distribution, presence of the leukotoxin gene lktA and the structure of the lktA-promoter region. Subgingival microbiological samples were obtained from 31 Chinese subjects with moderate to advanced adult periodontitis, 73 young factory workers, and 81 adult residents of a rural area. Bacterial isolates phenotypically identified as A. actinomycetemcomitans were found in 116 of the 185 subjects (detection frequency over-all: 63%). Presence of the leukotoxin gene lktA was demonstrated for all 115 isolates that could be subcultured. The PCR analysis of the lktA-promoter region showed that none of these strains had the deletion in the promoter region known to enhance expression of lktA. No significant difference in the frequency of A. actinomycetemcomitans could be observed between the subjects of the 3 study groups. Analysis by logistic multiple regression indicated a homogeneous distribution of A. actinomycetemcomitans in the 3 cohorts and a lack of significant influence of subject gender or age. Serotype a was found in 21 subjects, serotype b was found in 9, serotype c in 67 and serotype e in 11 individuals. Serotype d was not detected in any subject. Nontypeable isolates, lacking serotype a, b, c, d, or e antigens, were found in 9 individuals. A high prevalence irrespective of gender, age, and cohort suggests that A. actinomycetemcomitans is a common constituent of the normal flora in the Chinese subjects of this study and suggests differences in the microbiological composition of subgingival plaque may exist for this population group as compared to north American and European populations.

The term biofilm is increasingly replacing ‘plaque’ in the literature, but concepts and existing paradigms are changing much more slowly. There is little doubt that biofilm research will lead to more realistic perception and interpretation of the physiology and pathogenicity of microorganisms colonizing plaques in the oral cavity. There is clear evidence that the genotypic and phenotypic expression profiles of biofilm and planktonic bacteria are different. Several techniques are available today to study multispecies biofilms of oral bacteria, each having its particular advantages and weaknesses. We describe a biofilm model developed in Zürich and demonstrate a number of applications with direct or indirect impact on prophylactic dentistry: spatial arrangement and associative behavior of various species in biofilms; multiplex fluorescent in situ hybridization analysis of oral bacteria in biofilms; use of the biofilm model to predict in vivo efficacy of antimicrobials reliably; mass transport in biofilms; de- and remineralization of enamel exposed to biofilms in vitro. The potential of biofilm experimentation in oral biology has certainly not yet been fully exploited and dozens of possible interesting applications could be investigated. The overall physiological parameters of multispecies biofilms can be measured quite accurately, but it is still impossible to assess in toto the multitude of interactions taking place in such complex systems. What can and should be done is to test hypotheses stemming from experiments with planktonic cells in monospecies cultures. In particular, it will be interesting to investigate the relevance to biofilm composition and metabolism of specific gene products by using appropriate bacterial mutants.