From Mouth to Model: Combining in vivo and in vitro Oral Biofilm Growth

Background: Oral biofilm studies based on simplified experimental setups are difficult to interpret. Models are limited mostly by the number of bacterial species observed and the insufficiency of artificial media. Few studies have attempted to overcome these limitations and to cultivate native oral biofilm. Aims: This study aimed to grow oral biofilm in vivo before transfer to a biofilm reactor for ex situ incubation. The in vitro survival of this oral biofilm and the changes in bacterial composition over time were observed. Methods: Six human enamel-dentin slabs embedded buccally in dental splints were used as biofilm carriers. Fitted individually to the upper jaw of 25 non-smoking male volunteers, the splints were worn continuously for 48 h. During this time, tooth-brushing and alcohol-consumption were not permitted. The biofilm was then transferred on slabs into a biofilm reactor and incubated there for 48 h while being nourished in BHI medium. Live/dead staining and confocal laser scanning microscopy were used to observe bacterial survival over four points in time: directly after removal (T0) and after 1 (T1), 24 (T2), and 48 h (T3) of incubation. Bacterial diversity at T0 and T3 was compared with 454-pyrosequencing. Fluorescence in situ hybridization (FISH) was performed to show specific taxa. Survival curves were calculated with a specially designed MATLAB script. Acacia and QIIME 1.9.1 were used to process pyrosequencing data. SPSS 21.0 and R 3.3.1 were used for statistical analysis. Results: After initial fluctuations at T1, survival curves mostly showed approximation of the bacterial numbers to the initial level at T3. Pyrosequencing analysis resulted in 117 OTUs common to all samples. The genera Streptococcus and Veillonella (both Firmicutes) dominated at T0 and T3. They make up two thirds of the biofilm. Genera with lower relative abundance had grown significantly at T3. FISH analysis confirmed the pyrosequencing results, i.e., the predominant staining of Firmicutes. Conclusion: We demonstrate the in vitro survival of native primary oral biofilm in its natural complexity over 48 h. Our results offer a baseline for cultivation studies of native oral biofilms in (phyto-) pharmacological and dental materials research. Further investigations and validation of culturing conditions could also facilitate the study of biofilm-induced diseases.