Aims Most studies of biofilm effects on dental materials use single-species biofilms, or consortia. Microcosm biofilms grown directly from saliva or plaque are much more diverse, but difficult to characterize. We used the Human Oral Microbial Identification Microarray (HOMIM) to validate a reproducible oral microcosm model. Methods and Results Saliva and dental plaque were collected from adults and children. Hydroxyapatite and dental composite disks were inoculated with either saliva or plaque, and microcosm biofilms were grown in a CDC biofilm reactor. In later experiments, the reactor was pulsed with sucrose. DNA from inoculums and microcosms were analyzed by HOMIM for 272 species. Microcosms included about 60% of species from the original inoculum. Biofilms grown on hydroxyapatite and composites were extremely similar. Sucrose-pulsing decreased diversity and pH, but increased the abundance of Streptococcus and Veilonella. Biofilms from the same donor, grown at different times, clustered together. Conclusions This model produced reproducible microcosm biofilms that were representative of the oral microbiota. Sucrose induced changes associated with dental caries. Significance and Impact of the Study This is the first use of HOMIM to validate an oral microcosm model that can be used to study the effects of complex biofilms on dental materials.

Previously, we used in situ hybridization and confocal microscopy to detect the periodontal pathogens Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis within buccal epithelial cells taken directly from the mouth. This study tested the hypothesis that the intracellular flora of buccal cells is polymicrobial. Mixtures containing a red fluorescent universal probe paired with green fluorescent versions of either A. actinomycetemcomitans-, P. gingivalis-, or T. forsythensis-specific probes were hybridized with buccal cells collected from each of 38 healthy humans. We verified co-localization of probe pairs within cells by generating three-dimensional reconstructions. Intracellular bacteria were detected in every subject. Each cell that was labeled with a species-specific probe also contained bacteria recognized only by the universal probe. Bacteria labeled with specific probes often occupied smaller regions within larger masses of bacteria. Those findings suggest that future studies of invasion by oral bacteria may need to include microbial consortia.

Oral biofilms can degrade the components in dental resin-based composite restorations, thus compromising marginal integrity and leading to secondary caries. In this study, we investigated the mechanical integrity of the dentin-composite interface challenged with multi-species oral biofilms. While most studies used single-species biofilms, we used a more realistic, diverse biofilm model produced directly from plaques collected from donors with a history of early childhood caries. Dentin–composite disks were made using bovine incisor roots filled with Z100™ or Filtek™ LS (3M ESPE). The disks were incubated for 72hr in paired CDC biofilm reactors, using a previously published protocol. One reactor was pulsed with sucrose, and the other was not. A sterile saliva-only control group was run with sucrose pulsing. The disks were fractured under diametral compression to evaluate their interfacial bond strength. Surface deformation of the disks was mapped using digital image correlation (DIC) to ascertain fracture origin. Fracture surfaces were examined using SEM/EDS to assess demineralization and interfacial degradation. Dentin demineralization was greater under sucrose-pulsed biofilms, as the pH dropped below 5.5 during pulsing, with LS and Z100 specimens suffering similar degrees of surface mineral loss. Biofilm growth with sucrose pulsing also caused preferential degradation of the composite-dentin interface, depending on the composite/adhesive system used. Specifically, Z100 specimens showed greater bond strength reduction and more frequent cohesive failure in the adhesive layer. This was attributed to the inferior dentin coverage by Z100 adhesive which possibly led to a higher level of chemical and enzymatic degradation. The results suggested that factors other than dentin demineralization were also responsible for interfacial degradation. We have thus developed a clinically relevant in vitro biofilm model which would allow us to effectively assess the degradation of the dentin-composite interface subjected to multi-species biofilm challenge.

Previously, we reported that intracellular Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis were present within buccal epithelial cells from human subjects, as lesser components of a polymicrobial flora. In this study, we further characterized that intracellular flora by using the same double-labeling techniques to identify Fusobacterium nucleatum, Prevotella intermedia, oral Campylobacter species, Eikenella corrodens, Treponema denticola, Gemella haemolysans, Granulicatella adiacens, and total streptococci within buccal epithelial cells. All those species were found within buccal cells. In every case, species recognized by green-labeled species-specific probes were accompanied by other bacteria recognized only by a red-labeled universal probe. Streptococci appeared to be a major component of the polymicrobial intracellular flora, being present at a level from one to two logs greater than the next most common species (G. adiacens). This is similar to what is observed in oral biofilms, where diverse species interact in complex communities that often are dominated by streptococci.

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