Profiling of subgingival plaque biofilm microbiota in female adult patients with clear aligners: a three-month prospective study

Abstract

Background: Clear aligners are well known for facilitating oral hygiene maintenance and decreasing susceptibility to periodontal diseases as compared to conventional fixed appliances. However, few research studies focus on the subgingival microbial community during clear aligner treatment (CAT). Hence, this study investigates changes of the subgingival microbial community and its association with clinical characteristics during the first three months of CAT.

Methods: Ten female patients with clear aligners were enrolled in this study. Subgingival plaque samples were obtained at three time points: before orthodontic treatment (T0), one month after orthodontic treatment (T1) and three months after orthodontic treatment (T2). DNA was then extracted from plaque samples and analyzed by 16S rRNA gene sequencing. Periodontal examinations, including plaque index (PI) and gingival bleeding index (GBI) measurements were also recorded.

Results: The plaque indices (PIs) and gingival bleeding indices (GBIs) were slightly increased at T1 and T2, but no statistically significant difference was found. The alpha diversity indices, including the ACE, Chao1, Shannon indices, all showed a declining trend without significance, and a rising trend in the Simpson diversity index was observed. The weighted UniFrac distance was significantly higher at T1 and T2 compared with T0. Principal Coordinates Analysis (PCoA) demonstrated that the communities at T0 tended to cluster apart from the communities at T1 and T2. The relative abundance of the phylum Firmicutes and genus Mycoplasma was significantly increased at T0 compared with T2. There was no significant difference in the relative abundance of periodontal pathogens at the genus and species levels or core microorganisms at the genus level.

Conclusion: A slightly decreasing microbial diversity with a significant change of microbial structure was found during the first three-month clear aligner treatment (CAT). However, subjects receiving clear aligner treatment were free from periodontal diseases with relatively stable levels of periodontal microorganisms and core microorganisms. Thus, our preliminary findings indicated that clear aligners induced nonpathogenic changes of the subgingival microbiome in the first three-month treatment.

Keywords: 16S rRNA gene sequencing; Clear aligners; Microbial community.

Figure 1. Alpha diversity indices of subgingival plaque at T0, T1 and T2.

The Chao1 (A), ACE index (B) and Shannon index (D) showed a slightly decreasing trend. The Simpson index (C) at T2 was greater than that at T0 and T1 without statistical significance. All data are expressed as mean ± standard deviation.

Figure 2. Beta diversity (the average weighted UniFrac distance value) at three different time points.

Beta diversities were significantly higher at T1 and T2 compared with T0. ^∗P < 0.05 by ANOVA.

Figure 3. Principal Coordinates Analysis (PCoA) based on the relative abundance of OTUs identified in the subgingival plaque at T0, T1 and T2.

The communities at T0 tended to cluster apart from the communities at T1 and T2 (P < 0.05).

Figure 4. Analysis of the change of microbial structure at the phylum level.

(A) Heatmap of the relative abundance of subgingival bacteria at the phylum level at three different time points. (B) Phylum-level taxon distribution at T0, T1 and T2.

Figure 5. The relative abundance of the top 10 phyla.

Firmicutes and Tenericutes exhibited a higher abundance at T0 compared with T2. All data are expressed as mean ± standard deviation. ^∗P < 0.05 by ANOVA.

Figure 6. Analysis of the change of microbial structure at the genus level.

(A) Heatmap of relative abundance of subgingival bacteria at genus level at three different time points. (B) Genus-level taxon distribution at T0, T1 and T2.

Figure 7. Analysis of the relative abundance of core microbiome, significant genus and periodontal pathogens.

(A) The relative abundance of the core microbiome at the genus level. (B) The relative abundance of the significant microbiota at the genus level within three different time points. (C, D) The relative abundance of eight periodontal pathogens at the genus level and five periodontal pathogens at the species level within three different time points. All data are expressed as mean ± standard deviation. ^∗P < 0.05 by ANOVA.