Salivary microbiome and periodontal status of patients with periodontitis during the initial stage of orthodontic treatment


Patients with severe periodontitis typically present with pathologic tooth migration. To improve esthetics and masticatory function, orthodontic treatment is required. Research on periodontal orthodontic treatment has been sparse, particularly from the microbial perspective. Hence, we analyzed the microbial and clinical changes in patients with well-controlled periodontitis in the early stage of orthodontic treatment.


Ten patients with well-controlled periodontitis were asked to collect saliva before and 1 and 3 months after appliance placement (T0, T1, and T2, respectively) and underwent clinical examinations before and 1, 3, and 6 months after appliance placement (T0, T1, T2, and T3, respectively). The microbial community of saliva was analyzed by 16S rRNA gene sequencing. Gingival index, the plaque index, and the probing pocket depth were clinically assessed.


The plaque index significantly increased from T0 to T1 and decreased at T2 and T3. The probing pocket depth and gingival index increased slightly at T2, but not significantly, in both the high-risk site and low-risk site. The alpha and beta diversity increased at T1. The microbial community structure was similar at T0 and T2. The relative abundance of core genera and periodontal pathogens was stable during the initial 3 months of orthodontic treatment.


The orthodontic appliance promoted plaque accumulation and altered the microbial community of patients with well-controlled periodontitis during the first month of orthodontic treatment. The microbial community returned to the basal composition at 3 months after appliance placement, and the periodontal inflammation during the 6-months orthodontic treatment was under control.


  • In patients with periodontitis, microbial diversity increased after appliance placement.

  • Probing pocket depth and gingival index were stable during the first 6 months.

  • The plaque index increased transiently but returned to baseline at 3 months.

  • Host-microorganism balance was reestablished 3 months after appliance placement.

Periodontitis is a common disease among adults, and its incidence increases with age. Patients with severe periodontitis typically present with occlusal interference, irregular spacing, proclined incisor, deepbite, and deep jet. These pathologic changes in tooth position necessitate combined periodontal orthodontic treatment. Severe periodontitis is no longer considered a contraindication for orthodontic treatment. With adequate plaque control and proper force application, a tooth with decreased support by the alveolar bone can undergo orthodontic treatment without jeopardizing periodontal health. Most clinical studies have shown no periodontal tissue destruction after placement of an orthodontic appliance. , ,

Periodontitis has high rates of progression and relapse. The stagnant area around the fixed appliance is difficult to clean, and new occlusal interference might occur during the tooth movement. Therefore, patients with severe periodontitis receiving orthodontic treatment are at risk of the relapse of periodontal inflammation. If inflammation is not fully controlled during orthodontic treatment, tooth movement can cause additional attachment loss. Hence, efficient periodontal screening should be performed during orthodontic treatment because clinical and radiographic examinations have limited ability to evaluate the risk for periodontitis.

Microbial infection plays an important role in the mechanism of periodontitis. Changes in the microbiome of subgingival plaque precede clinical signs of periodontitis, such as gingival swelling, attachment loss, deep pockets, and bone loss. The salivary microbiota is positively correlated with subgingival microbiota , Indeed, the presence of periodontal pathogens in the subgingival plaque can be reflected in saliva. Thus, because saliva sampling is noninvasive and convenient, changes in the microbial composition of saliva could be an indicator of periodontal health.

Our previous systematic review indicated that the relative abundance of periodontal pathogens varies in patients over time. In addition, 16S rRNA gene sequencing has shown that disruption of the structure and composition of the microbiome is associated with periodontal inflammation. In this study, we evaluated the composition and diversity of the microbiome. We also analyzed the microbial and clinical parameters of patients with well-controlled periodontitis in the early stage of orthodontic treatment.

Material and methods

Ten patients with well-controlled periodontitis (6 women and 4 men; age range, 30-45 years; mean age, 36.4 years; standard deviation [SD], 2.1) were analyzed. After completing periodontal treatment, the 10 patients were referred by their periodontist for orthodontic treatment. The ethics committee approved the study protocol, and all of the patients provided written informed consent. Patients with the following conditions were included in this study: aged 30-50 years; periodontitis stage III or IV and grade C (attachment loss of 30% of teeth of more than 5 mm, and radiographs showing alveolar bone loss around at least half the length of the root ); systematic periodontal treatment within 3 months and a stable periodontal condition (no pocket with >4 mm probing pocket depth [PPD], plaque index [PLI] <30%, gingival index [GI] <30%, and no occlusal trauma); mild tooth crowding or spacing; good oral hygiene behavior and no smoking; no crowns, implants, or fixed bridges; no diabetes or other systemic disease; no pregnancy; and had not taken antibiotics or hormones within 3 months.

All of the patients underwent fixed appliance treatment for both maxillary and mandibular arch at the fourth-week visit. Metal brackets (Shinye, Hangzhou, China), a nickel-titanium archwire (Shinye), and stainless steel archwires were used for orthodontic treatment. Bands and excess cement residues were avoided. Light force (50-100 g of force) was applied. During the first 6 months of treatment, oral hygiene instructions, including modified bass brushing and flossing using an interdental brush, were given for each including patient at each visit. Periodontal treatments and antibacterial mouthwashes were prohibited.

The patients provided saliva before appliance placement (T0) and 1 (T1) and 3 (T2) months after appliance placement and underwent clinical examinations at T0, T1, T2, and 6 months after appliance placement (T3). Saliva sampling was performed before the clinical examination. Unstimulated saliva samples were collected in the morning, and the patients were asked to avoid eating and brushing their teeth for at least 8 hours. The patients were instructed to pool the saliva into a sterile tube for 5 minutes. Saliva samples were stored at −80°C.

After saliva sampling, GI, PLI, and PPD were clinically assessed. The GI was assessed using the following classification: (0) healthy gingiva, no bleeding; (1) edema, change in color without bleeding; (2) bleeding without flow along the gingival margin; (3) bleeding with the flow along the gingival margin; (4) copious bleeding; and (5) severe inflammation with a tendency to spontaneous bleeding. The PLI was assessed using the following classification: (0) no plaque, (1) flecks of plaque at the gingival margin, (2) definite line of plaque at the gingival margin, (3) plaque covering less than one-third of the tooth surface, (4) plaque covering more than one-third and less than two-thirds of the tooth surface, and (5) plaque covering more than two-thirds of the tooth surface. Six sites (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, distolingual) of each tooth were recorded by an experienced periodontist. First, the overall periodontal status is presented as the full mouth means on the basis of the 6 measurements of each tooth. Second, the sites were divided into 2 subgroups: high-risk site (attachment loss ≥5 mm at T0) and low-risk site (attachment loss <5 mm at T0). PPD and GI were also assessed in 2 subgroups. According to periodontal risk assessment, the suggested recall interval is 3 months. Hence, the PPD and GI were assessed every 3 months and were not evaluated at T1.

Genomic DNA was isolated from saliva samples using Tiangen Bacteria DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The DNA concentration was measured using the NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass). The v3-v4 regions of the bacterial 16S rDNA gene were amplified by polymerase chain reaction, and the products were deep sequenced on the MiSeq Platform at the Auwigene Institute (Beijing, China). Next, image analysis, base calling, and error estimation were performed using Illumina Sequencing Analysis Pipeline (version 2.6; Genome Analyzer, Illumina, Inc, San Diego, Calif). The sequence data obtained in our study have been deposited in the National Center for Biotechnology Information Sequence Read Archive database under Accession No. SUB5553353.

The Mothur software package and the Quantitative Insights Into Microbial Ecology pipeline were used to analyze the sequencing data further. Low-quality sequences were trimmed and filtered. The operational taxonomic units (OTUs) clustered at 97% similarity for the remaining high-quality sequences, and their taxonomy was assigned on the basis of the Human Oral Microbiome Database. The Shannon, Chao1, observed species, and phylogenetic diversity (PD) whole-tree indexes of alpha diversity were calculated to estimate the microbial community diversity. Beta diversity and principal coordinate analysis (PCoA) plots were generated to assess the microbial community composition. The relative abundances at each taxonomic level were calculated. To compare the microbial communities among the time points, we generated the microbial distribution at the genus and phylum levels. Statistical analyses of clinical parameters were done using SPSS software (version 20.0; IBM Corp, Armonk, NY). The significance of differences among time points were evaluated using repeated-measures analysis of variance.


The overall mean PPD was 2.49 mm at T0, 2.77 mm at T2, and 2.75 mm at T3. The overall mean GI was 1.68 at T0, 1.98 at T2, and 1.75 at T3. The PPD and GI increased slightly, but not significantly, at T2. After appliance placement, the PLI significantly increased between T0 and T1 (1.1 and 2.1, respectively), followed by a decrease from T2 to T3 (1.3 and 1.0, respectively) ( Fig 1 ). In subgroup analyses, the PPD and GI of both high-risk sites and low-risk sites showed a slight increase at T2 without statistically significant difference ( Fig 2 ). The mean PPD at the high-risk site was 2.78 mm at T0, 2.99 mm at T2, and 2.98 mm at T3, whereas the corresponding mean PPD at the low-risk site was 2.30 mm, 2.62 mm, and 2.60 mm, respectively. The mean GI at the high-risk site was 1.72 at T0, 1.93 at T2, and 1.75 at T3, compared with 1.65 at T0, 1.95 at T2, and 1.76 at T3 in the low-risk sites.

Fig 1
The overall periodontal status of patients with periodontitis during orthodontic treatment. The PLI was significantly greater at T1 than at the other 3 time points. The GI and PPD were not significantly different among the time points. Data are represented as mean ± SD. P <0.05 was considered statistically significant using the repeated-measures analysis of variance.

Jun 12, 2021 | Posted by in Orthodontics | Comments Off on Salivary microbiome and periodontal status of patients with periodontitis during the initial stage of orthodontic treatment
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