High throughput DNA sequencing to detect differences in the subgingival plaque microbiome in elderly subjects with and without dementia

Abstract

Background: To investigate the potential association between oral health and cognitive function, a pilot study was conducted to evaluate high throughput DNA sequencing of the V3 region of the 16S ribosomal RNA gene for determining the relative abundance of bacterial taxa in subgingival plaque from older adults with or without dementia.

Methods: Subgingival plaque samples were obtained from ten individuals at least 70 years old who participated in a study to assess oral health and cognitive function. DNA was isolated from the samples and a gene segment from the V3 portion of the 16S bacterial ribosomal RNA gene was amplified and sequenced using an Illumina HiSeq1000 DNA sequencer. Bacterial populations found in the subgingival plaque were identified and assessed with respect to the cognitive status and oral health of the participants who provided the samples.

Results: More than two million high quality DNA sequences were obtained from each sample. Individuals differed greatly in the mix of phylotypes, but different sites from different subgingival depths in the same subject were usually similar. No consistent differences were observed in this small sample between subjects separated by levels of oral health, sex, or age; however a consistently higher level of Fusobacteriaceae and a generally lower level of Prevotellaceae was seen in subjects without dementia, although the difference did not reach statistical significance, possibly because of the small sample size.

Conclusions: The results from this pilot study provide suggestive evidence that alterations in the subgingival microbiome are associated with changes in cognitive function, and provide support for an expanded analysis of the role of the oral microbiome in dementia.

Figure 1

Alpha rarefaction plot demonstrating phylotype diversity in subgingival plaque samples. Shown are the numbers of different Operational Taxonomic Units (OTUs) as a function of the numbers of sequences analyzed and generated with QIIME. OTUs that occur less than 150 times/sample are not included. C, cognitively impaired, not dementia; D, dementia; N, normal.

Figure 2

Taxonomic assignments found in subgingival plaque clustered by cognitive status. Counts for each OTU that was identified more than 150 times/sample were included in this analysis. The total height of the bar represents 100% of the assigned sequences after quality filtering, and the size of the colored regions represents proportional contributions of each phylotype shown. For clarity, only major families (>3%) are listed in the color key. OTU, Operational Taxonomic Units.

Figure 3

High number of distinct OTUs assigned to Prevotellaceae from both dementia and non-dementia samples. The number of distinct OTUs that are identified in four families that represent 2/3 of the total sequences were averaged for all samples and shown. Error bars represent standard error. Asterisk represents statistically significant difference by analysis of variance followed by Tukey’s multiple comparison test (P < 0.05). OTU, Operational Taxonomic Units.

Figure 4

Clustering of bacterial taxa by cognitive function. Weighted UniFrac was used to generate a matrix of pairwise distances between communities and a scatterplot was generated from the matrix of distances using Principal Coordinate Analysis in QIIME. Each symbol represents the values of all samples from one participant analyzed collectively. In the left hand panel all OTUs that occur more than 150 times are included in the analysis. In the middle panel, the OTU table is further edited to remove any OTUs assigned to the Fusobacteriaceae or Prevotellaceae. In the right hand panel, only OTUs that were identified as Fusobacteriaceae or Prevotellaceae were analyzed. (green circle) cognitively normal, (red square) cognitively impaired without dementia, and (purple triangle) dementia. OTU, Operational Taxonomic Units.

Figure 5

Comparison of phylotypes found at various pocket probing depths (PPDs). Sample identifications as in Table 1 and PPDs are listed at the base of each bar. For PPD: 1 = 1 to 3 mm, 3 = 3 to 5 mm 5 = > 5 mm. Results show at the phylum and genus levels. DNA samples obtained from various PPDs were sequenced and analyzed separately. The total height of the bar represents 100% of the assigned sequences after quality filtering, and the size of the colored regions represents proportional contributions of each phylotype shown. For clarity, only major genera (>3%) are listed in the color key.