The Toothbrush Microbiome: Impact of User Age, Period of Use and Bristle Material on the Microbial Communities of Toothbrushes

Abstract

Toothbrushes play a central role in oral hygiene and must be considered one of the most common articles of daily use. We analysed the bacterial colonization of used toothbrushes by next generation sequencing (NGS) and by cultivation on different media. Furthermore, we determined the occurrence of antibiotic resistance genes (ARGs) and the impact of different bristle materials on microbial growth and survival. NGS data revealed that Enterobacteriaceae, Micrococcaceae, Actinomycetaceae, and Streptococcaceae comprise major parts of the toothbrush microbiome. The composition of the microbiome differed depending on the period of use or user age. While higher fractions of Actinomycetales, Lactobacillales, and Enterobacterales were found after shorter periods, Micrococcales dominated on both toothbrushes used for more than four weeks and on toothbrushes of older users, while in-vitro tests revealed increasing counts of Micrococcus on all bristle materials as well. Compared to other environments, we found a rather low frequency of ARGs. We determined bacterial counts between 1.42 × 10⁶ and 1.19 × 10⁷ cfu/toothbrush on used toothbrushes and no significant effect of different bristles materials on bacterial survival or growth. Our study illustrates that toothbrushes harbor various microorganisms and that both period of use and user age might affect the microbial composition.

Keywords: antibiotic resistance; microbial contamination; oral microbiome; toothbrush.

Figure 1

Taxonomic composition of the toothbrush microbiome based on next generation sequencing of 23 toothbrush samples. To ameliorate presentation, only results ≥1% are given.

Figure 2

Principal component analysis (PCA) of toothbrush samples (n = 23) based on weighted UniFrac distance analysis. Unit variance scaling was applied to rows and NIPALS (Nonlinear Iterative Partial Least Squares) PCA was used to calculate principal components. X- and Y-axis show principal component 1 and principal component 2 that explain 57.8% and 24.9% of the total variance, respectively. Period of use: <2 weeks (n = 6), 2–4 weeks (n = 6), 4–>12 weeks (n = 11)) and user age: (10–20 years (n = 5), 20–60 years (n = 14), >60 years (n = 4)).

Figure 3

Taxonomic composition on order-level for all analysed toothbrushes grouped by period of use (<2 weeks (n = 6), 2–4 weeks (n = 6), 4–>12 weeks (n = 11)) and user age (10–20 years (n = 5), 20–60 years (n = 14), >60 years (n = 4)) based on NGS. For samples TB6 and TB7, the generation of amplicons failed and therefore these samples were not sequenced. Due to better presentation, only results ≥1% are given.

Figure 4

Microbial count of toothbrushes grouped by period of use (a) and user age (b) on different culture media. Columns show the mean and standard error of mean (n = 25). Different letters indicate significant differences at p ≤ 0.05 (multiple t-test) between groups. Where no letters are shown, no significant differences were detected.

Figure 5

Absolute abundance of total ARGs (a,b) and intI1 (c,d) of toothbrush samples grouped by age (10–20 years (n = 5), 20–60 years (n = 16), >60 years (n = 4)) and period of use (<2 weeks (n = 6), 2–4 weeks (n = 6), 4–>12 weeks (n = 13)). Analyses were performed in duplicate and non-parametric Kruskal-Wallis test (p ≤ 0.05) revealed no significant differences between sample groups.

Figure 6

Logarithmic microbial counts of different bristle types after one week of artificial contamination. The following strains were used: Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Micrococcus luteus (M. luteus), Bacillus subtilis (B. subtilis), Streptococcus mutans (S. mutans), and Streptococcus anginosus (S. anginosus) (n = 3). Different letters indicate significant differences at p ≤ 0.05 between groups. Where no letters are shown, no significant differences were detected.