Microbial differences between dental plaque and historic dental calculus are related to oral biofilm maturation stage

Abstract

Background: Dental calculus, calcified oral plaque biofilm, contains microbial and host biomolecules that can be used to study historic microbiome communities and host responses. Dental calculus does not typically accumulate as much today as historically, and clinical oral microbiome research studies focus primarily on living dental plaque biofilm. However, plaque and calculus reflect different conditions of the oral biofilm, and the differences in microbial characteristics between the sample types have not yet been systematically explored. Here, we compare the microbial profiles of modern dental plaque, modern dental calculus, and historic dental calculus to establish expected differences between these substrates.

Results: Metagenomic data was generated from modern and historic calculus samples, and dental plaque metagenomic data was downloaded from the Human Microbiome Project. Microbial composition and functional profile were assessed. Metaproteomic data was obtained from a subset of historic calculus samples. Comparisons between microbial, protein, and metabolomic profiles revealed distinct taxonomic and metabolic functional profiles between plaque, modern calculus, and historic calculus, but not between calculus collected from healthy teeth and periodontal disease-affected teeth. Species co-exclusion was related to biofilm environment. Proteomic profiling revealed that healthy tooth samples contain low levels of bacterial virulence proteins and a robust innate immune response. Correlations between proteomic and metabolomic profiles suggest co-preservation of bacterial lipid membranes and membrane-associated proteins.

Conclusions: Overall, we find that there are systematic microbial differences between plaque and calculus related to biofilm physiology, and recognizing these differences is important for accurate data interpretation in studies comparing dental plaque and calculus.

Keywords: Ancient dental calculus; Metagenomics; Metaproteomics; Oral microbiome; Periodontal disease.

Fig. 1

Dental plaque and dental calculus contain distinct microbial communities. a Principal component analysis (PCA) clusters modern dental plaque distinctly from modern and historic dental calculus, while calculus samples do not separate by time period. b PCA clustering of calculus samples is not related to health status

Fig. 2

Microbial profile differences between plaque and calculus of differing health status. a Principal component analysis (PCA) clusters plaque and healthy site calculus distinctly. b Distinct species are significantly more abundant in plaque and healthy site calculus. c Microbial profile differences between plaque and all healthy site calculus are sufficient for discrimination of sample types by sparse partial least squares-discriminant analysis (sPLS-DA). Ellipses indicate 95% confidence intervals. d PCA clusters plaque and disease site calculus distinctly. e Distinct species are significantly more abundant in plaque and disease site calculus. f Microbial profile differences between plaque and all disease site calculus are sufficient for discrimination of sample types by sPLS-DA. Ellipses indicate 95% confidence intervals

Fig. 3

Healthy site calculus microbial profile differs from plaque independent of sample age. a Principal component analysis (PCA) of plaque and historic healthy site calculus cluster samples by type. b Distinct species are significantly more abundant in plaque and historic healthy site calculus. c Microbial profile differences between plaque and historic healthy site calculus are sufficient for discrimination of sample types by sparse partial least squares-discriminant analysis (sPLS-DA). Ellipses indicate 95% confidence intervals. d PCA clusters plaque and modern healthy site calculus distinctly. e Microbial profile differences between plaque and modern healthy site calculus are sufficient for discrimination of sample types by sPLS-DA. Ellipses indicate 95% confidence intervals

Fig. 4

Modern and historic calculus microbial community profiles overlap. a Principal component analysis (PCA) does not cluster modern and historic healthy site calculus distinctly. b Historic and modern healthy site calculus contain sufficient discriminatory taxa for distinct clustering by sparse partial least squares-discriminant analysis (sPLS-DA). c PCA does not cluster modern and historic disease site calculus distinctly. d Historic and modern disease site calculus contain sufficient discriminatory taxa for distinct clustering by sPLS-DA. e Modern healthy and disease site calculus microbial profiles are not distinctly different and are not separated by PCA. f Modern healthy and disease site calculus contain sufficient discriminatory taxa for distinct clustering by sPLS-DA. g Historic healthy and disease site calculus microbial profiles are not distinctly different and are not separated by PCA. h Modern healthy and disease site calculus do not contain sufficient discriminatory taxa for distinct clustering by sPLS-DA. Ellipses indicate 95% confidence intervals

Fig. 5

Microbial co-exclusion patterns reflect oxygen use and carbon source. Network graphs presenting species as nodes and co-exclusion between species as edges, where darker lines indicate stronger co-exclusions. Nodes are colored based on oxygen tolerance (left) and carbon source (right). Identical networks with labels indicating the bacterial species are presented in Additional file 1: Figure S10A,B. Historic calculus has more co-exclusions between species with different oxygen tolerance and carbon source than does plaque or modern calculus

Fig. 6

Microbial species differences between plaque, modern calculus, and historic calculus. a The majority of taxa detected are shared between plaque, modern calculus, and historic calculus. b Relative abundance of Socransky’s bacterial complexes in plaque and calculus, presented by age and health status. *p < 0.05 vs. modern plaque, ^xp < 0.05 vs. modern healthy site calculus

Fig. 7

Potential metabolic functional profiles differ by sample type but not health status. a SEED metabolic functional category profiles separate plaque and historic calculus in a principal component analysis. b SEED profiles of plaque and historic calculus are sufficiently discriminatory to cluster samples by type in the sparse partial least squares-discriminant analysis (sPLS-DA). c Healthy and periodontal disease site historic calculus SEED profiles overlap and are not distinctly separated by PCA. d Historic healthy and disease site calculus SEED profiles overlap and are not sufficiently discriminatory for distinct clustering by sPLS-DA

Fig. 8

Historic calculus protein profiles reflect host homeostasis. Filled area charts presenting relative abundance in each sample of a host protein categories, b human proteins detected in at least 50% of the samples, c bacterial protein categories, and d bacterial virulence protein categories

Fig. 9

Canonical correlations between historic sample proteins and metabolites. The strongest canonical correlations in historic calculus are presented as network graphs where the nodes are proteins/metabolites and edges represent canonical correlations, with darker, thinner lines lower values and thicker, lighter lines higher values. Nodes are sized by the number of edges they have and are colored based protein on metabolite category. a Bacterial and human protein correlations ≥ 0.9. b All protein and metabolite correlations ≥ 0.9. Graphs with node names are included in Additional file 1: Figures S18-S19