Temporal oral microbiome changes with brushing in children with cleft lip and palate

Abstract

This cohort study aimed to characterize the oral microbiome of children with CLP, from two different age groups, and evaluate the effect of supervised or unsupervised toothbrushing on the microbiome of the cleft over time. Swab samples were collected from the cleft area at three different time points (A; no brushing, B; after 15 days and C; after 30 days) and were analyzed using next-generation sequencing to determine the microbial composition and diversity in these time points. Overall, brushing significantly decreased the abundance of the genera Alloprevotella and Leptotrichia in the two age groups examined, and for Alloprevotella this decrease was more evident for children (2-6 years old). In the preteen group (7-12 years old), a significant relative increase of the genus Rothia was observed after brushing. In this study, the systematic brushing over a period of thirty days also resulted in differences at the intra-individual bacterial richness.

Keywords: Alloprevotella; CLP; Children and preteen; Leptotrichia; Microbiome; Next-generation sequencing; Rothia.

Figure 1

Rarefaction curves. Rarefaction curves of calculated for observed richness (top) and H (bottom) at 1 to 20000 sequencing reads, separated for child (left) and Tween (right), with samples grouped by timepoint with bars indicating standard deviations.

Figure 2

Phylum level microbiome composition at timepoint A, per patient. Bar plot showing the relative abundance of all phyla with a mean abundance above 1% and remaining phyla group as Other.

Figure 3

Genus level microbiome composition at time point A, per patient. Bar plot showing the relative abundance of all genera with a mean abundance above 4% and remaining genera group as “Other”.

Figure 4

Boxplot of alpha diversity at time point A for children and preteen determined by observed richness and Shannon Diversity Index (H). Line in box indicates median value, box covers 95% confidence intervals, vertical lines reaches to furthest samples within 1.5 x box height and any outliers are indicated as points.

Figure 5

Heatmap of 10 most abundant genera at time point B and C. The two top rows indicate the age group and time point of each sample. The abundances are shown as percentages with the blue-yellow-red color scale.

Figure 6

Boxplot of the alpha diversity for children and preteen. Boxplots are colored according to brushing (Baseline: red, Brushing: turquois), measured as both observed richness (left) and Shannon Diversity Index (H) (right). Line in box indicates median value, box covers 95% confidence intervals, vertical lines reaches to furthest samples within 1.5 x box height and any outliers are indicated as points.

Figure 7

Boxplots of the relative abundance change (RAC) and abundance of significantly changing genera. A. Boxplot of RAC, per group (child: yellow, preteen blue), for any genus with RAC significantly different from 0 in any of the 3 timespans (Significant change, yes: Black edge, no: green edge), or which differs significantly between groups in any of the timespans (Significant difference, yes: solid fill, no: pale fill). B. Boxplot of the genus abundance of the relevant genera, edge color indicates time point (A (day 0): red, B (day 15): green, C (day 30): blue). Line in box indicates median value, box covers 95% confidence intervals, vertical lines reaches to furthest samples within 1.5 x box height and any outliers are indicated as points.