Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

oral-biofilm-sampling-for-microbiome-analysis-in-healthy-children

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Medical University of Graz

Despite the input of microbiome research, a group of 20 bacteria continues to be the focus of periodontal diagnostics and therapy. The aim of this study was to compare three commercial kits and laboratory-developed primer pairs for effectiveness in detecting such periodontopathogens.

Accuracy of commercial kits and published primer pairs for the detection of periodontopathogens.

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.

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

Background: Oral microbiota are considered major players in the development of periodontal diseases. Thorough knowledge of intact subgingival microbiomes is required to elucidate microbial shifts from health to disease. Aims: This comparative study investigated the subgingival microbiome of healthy children, possible inter- and intra-individual effects of modified sampling, and basic comparability of subgingival microprints. Methods: In five 10-year-old children, biofilm was collected from the upper first premolars and first molars using sterilized, UV-treated paper-points inserted into the subgingival sulcus at eight sites. After supragingival cleaning using an electric toothbrush and water, sampling was performed, firstly, excluding (Mode A) and, secondly, including (Mode B) cleansing with sterile cotton pellets. DNA was extracted from the pooled samples, and primers targeting 16S rRNA hypervariable regions V5 and V6 were used for 454-pyrosequencing. Wilcoxon signed rank test and t-test were applied to compare sampling modes. Principal coordinate analysis (PCoA) and average agglomerative hierarchical clustering were calculated with unweighted UniFrac distance matrices. Sample grouping was tested with permutational MANOVA (Adonis). Results: Data filtering and quality control yielded 67,218 sequences with an average sequence length of 243bp (SD 6.52; range 231-255). Actinobacteria (2.8-24.6%), Bacteroidetes (9.2-25.1%), Proteobacteria (4.9-50.6%), Firmicutes (16.5-57.4%), and Fusobacteria (2.2-17.1%) were the five major phyla found in all samples. Differences in microbial abundances between sampling modes were not evident. High sampling numbers are needed to achieve significance for rare bacterial phyla. Samples taken from one individual using different sampling modes were more similar to each other than to other individuals' samples. PCoA and hierarchical clustering showed a grouping of the paired samples. Permutational MANOVA did not reveal sample grouping by sampling modes (p = 0.914 by R2 = 0.09). Conclusion: A slight modification of sampling mode has minor effects corresponding to a natural variability in the microbiome profiles of healthy children. The inter-individual variability in subgingival microprints is greater than intra-individual differences. Statistical analyses of microbial populations should consider this baseline variability and move beyond mere quantification with input from visual analytics. Comparative results are difficult to summarize as methods for studying huge datasets are still evolving. Advanced approaches are needed for sample size calculations in clinical settings.

Elisabeth Santigli

Department of Orthodontics and Maxillofacial Orthopedics,
Division of Oral Surgery and Orthodontics,
Department of Dental Medicine and Oral Health,
Division of Oral Surgery and Orthodontics, Department of Dental Medicine and Oral Health

Medical University of Graz

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children