Metagenomic analysis of oral and intestinal microbiome of patients during the initial stage of orthodontic treatment

Introduction

This prospective study analyzed changes in the oral and intestinal microbiomes in patients before and after fixed orthodontic treatment, elucidating the impacts of fixed orthodontic treatment on patient health and metabolism.

Methods

Metagenomic analysis was conducted on stool, dental plaque, and saliva samples from 10 fixed orthodontic patients. All the samples were sequenced with Illumina NovaSeq 6000 with a paired-end sequencing length of 150 bp. Identification of taxa in metagenomes and functional annotation of genes of the microbiota were performed using the data after quality control. Clinical periodontal parameters, including the gingiva index, plaque index, and pocket probing depth, were examined at each time point in triplicates. Patients also received a table to record their oral hygiene habits of brushing, flossing, and dessert consumption frequency over 1 month.

Results

The brushing and flossing times per day of patients were significantly increased after treatment compared with baseline. The number of times a patient ate dessert daily was also fewer after treatment than at baseline. In addition, the plaque index decreased significantly, whereas the pH value of saliva, gingiva index, and pocket probing depth did not change. No significant differences were observed between the participants before and after orthodontic treatment regarding alpha-diversity analysis of the gut, dental plaque, or saliva microbiota. However, on closer analysis, periodontal disease-associated bacteria levels in the oral cavity remain elevated. Alterations in gut microbiota were also observed after orthodontic treatment.

Conclusions

The richness and diversity of the microbiome did not change significantly during the initial stage of fixed orthodontic treatment. However, the levels of periodontal disease-associated bacteria increased.

Highlights

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Good oral hygiene did not prevent periodontal bacteria rise during orthodontic start.
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First metagenomic study on post-orthodontic gut microbiota changes.
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Twenty-two gut microbiota taxa increased significantly after orthodontic treatment.

Malocclusion is 1 of the 3 major oral diseases, mainly referring to abnormal deviation in skeletal and/or dental form. It could impair chewing efficiency, increase the incidence of caries, affect esthetics, cause psychological discomfort, endanger health, and reduce the quality of life. Fixed orthodontic treatment is the most popular and effective method favored by orthodontists and patients. However, fixed appliances are considered to alter the oral microenvironment because they benefit the colonization, growth, and accumulation of microorganisms (bacteria, fungi).

The oral cavity harbors a diverse microbiota comprising >700 distinctive bacterial species. The microbiota plays a significant role in maintaining oral homeostasis and ecological changes; for example, poor quality of oral hygiene or the placement of brackets and archwires can induce structural and functional alterations of local oral biofilms. Related studies have noted that orthodontic treatment can disrupt the ecological balance of oral microorganisms, leading to changes in the oral status of patients. For instance, the abundance of Pseudomonas and obligate anaerobe, especially periodontal pathogens, was found to be increased in the saliva of a healthy oral population. ^, In addition, levels of Streptococcus mutans and Lactobacillus acidophilus , recognized as cariogenic microorganisms, increased after orthodontic treatment.

Few studies have demonstrated alterations in the intestinal microbiome after fixed orthodontic treatment. As the primary entry point to the human body, the oral cavity boasts the second largest and most diverse microbiota after the gut, accommodating >770 bacterial species. The oral microbiome can colonize the intestinal tract, subsequently impacting the structure and functionality of the intestinal microbiome. Inversely, the gut microbiome could directly or indirectly influence the composition of the oral microbiome by influencing host immunity. For example, research has reported the detection of identical strains of Fusobacterium nucleatum in both the saliva and colonic tumors of patients, suggesting that the colonization of F. nucleatum in colonic tumors stems from the oral microbiota. Therefore, the interaction between oral and intestinal microbiome have attracted high attention.

In the past, the effects of orthodontic treatment on oral microorganisms of patients were mostly limited to the application of 16S rRNA sequencing technology, which can only reflect the community species composition and evolutionary relationship. Metagenomic sequencing can be used to annotate species composition more accurately and investigate microbial gene function and metabolic pathways. Therefore, this study used metagenomic sequencing technology to conduct metagenomic sequencing on stool, dental plaque, and saliva samples of orthodontic patients and compare the microbial differences before and after fixed orthodontic treatment to provide theoretical reference for the health of orthodontic patients.

Material and methods

This study incorporated a cohort of 10 patients (4 females, 6 males) slated to undergo fixed orthodontic treatment at the Department of Orthodontics of West China Hospital of Stomatology between August 2021 and February 2022. The age range of the participants was 18-34 years, with an average age of 21 years. After recruitment, written informed consent was obtained from each participant before the study performance. Ethical approval for the study was granted by the Ethics Committee of West China Hospital of Stomatology (no. WCHSIRB-CT-2021-423).

The following inclusion criteria were applied: (1) aged ≥18 years, (2) no missing permanent teeth except third molars, (3) adequate oral hygiene, (4) good compliance, and (5) no antibiotics taken during the preceding 3 months. The exclusion criteria encompassed patients with preexisting conditions such as gingivitis, severe periodontal diseases, generalized caries, systemic diseases, and those with medical contraindications, smoking habits, or irregular attendance.

Before the experiment, donors were asked to answer a questionnaire, including 23 questions about their health, dietary, and hygiene habits ( Supplementary Table ). All the subjects were treated with passive self-ligating brackets (Damon Q; Ormco, Calif), and 0.014-in nickel-titanium archwires (Damon Q) were engaged in the brackets after the brackets were placed. Unstimulated whole saliva (1.5 mL), supragingival plaque, and fecal samples of each participant were collected at 2-time points (before the treatment and 1 month after the treatment started) and were transiently placed in liquid nitrogen and then stored at −80°C for further analysis. The pH of each donor’s saliva was measured before sample collection. Before the visit, the participant was instructed to refrain from drinking or eating at least 1 hour before saliva collection. Samples were collected before (Group A) and 1 month after the treatment (Group B).

Clinical periodontal parameters, including gingiva index (GI), plaque index (PI), and pocket probing depth (PPD), were examined at each time point in triplicate. The measurements were conducted at 6 sites per tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual), excluding third molars, and were performed by a single examiner using a manual probe.

All subjects received standardized oral hygiene instructions from the same dental hygienist (J.L. and J.F.). Oral hygiene instructions included a detailed protocol of the Bass brushing technique and the use of floss. Patients also received a table to record their oral hygiene habits of brushing, flossing, and dessert consumption frequency over 1 month.

Total DNA was extracted using a Tiangen DNA Stool Mini Kit (Tiangen Biotech, Beijing, China). All the samples were sequenced with Illumina NovaSeq 6000 (Novogene Co, Ltd, Tianjin, China) with a paired-end sequencing length of 150 bp; then, we filtered the adapters and low-quality reads by Trimmomatic. Potential human sequences were removed by Bowtie2 on the basis of the NCBI reference genome.

The genes were then translated into amino acid sequences to be aligned to the Carbohydrate-Active enZYmes (CAZy) database using DIAMOND. Antibiotic resistance genes (ARGs) were quantified using ShortBRED. The shortbred_identify.py script used ARG sets from the comprehensive antibiotic resistance database (CARD) as proteins of interest and UniRef90 sequences as reference proteins. The shortbred_quantify.py script quantified the abundance of ARGs in metagenomes. Gene families and microbial metabolic pathways were assessed using HUMAnN3 on the basis of the ChocoPhlAn database, and UniRef90 EC filtered database and was normalized by count per million.

The taxonomic labels of metagenomic sequences were assigned using kraken2 with the use-mpa-style option. The abundances of taxa were normalized by relative abundance.

Alpha-diversity analysis was done by R statistical software (version 4.3; R Core Team, Vienna, Austria). The stacked column chart was completed using the Wekemo Bioincloud ( https://www.bioincloud.tech ). Differences in abundances of taxa, functional genes, and metabolic pathways were identified using LEfSe, completed by the script on https://pypi.org/project/lefse/#files . We used the principal coordinates analysis on the basis of the Bray-Curtis metric. Furthermore, we performed a permutational multivariate analysis of variance test on each principal coordinates analysis using the adonis function of R software to ensure significant separation of different groups. We used paired t test, or Wilcoxon signed rank test to see whether the differences of relative abundance of taxa were significant before and after orthodontic treatment. The taxon whose relative abundance of anybody among the groups is above 0.01% and the P value is <0.05 was selected to draw the boxplot by Python.

Results

Ten volunteers who participated in the trial all completed the 1-month study. The clinical parameters are illustrated in the Table . The brushing and flossing times per day of patients were significantly increased after treatment ( P <0.05). The number of times a patient ate dessert daily was also fewer after treatment. In addition, the PI decreased significantly ( P <0.05), whereas the pH value of saliva, GI, and PPD did not change.

Table

Clinical parameters of the subjects

Variables	Brushing	Flossing	Dessert	pH value of saliva	GI	PI	PPD
Before treatment	2.08 ± 0.51 ^∗	0.33 ± 0.49 ^∗	2.75 ± 2.67 ^∗	7.08	1.33 ± 0.89	2.08 ± 0.90 ^∗	1.92 ± 0.67
1-mo after treatment	2.50 ± 0.52 ^∗	2.25 ± 0.86 ^∗	1.16 ± 0.93 ^∗	7.04	1.08 ± 0.67	0.75 ± 0.62 ^∗	1.75 ± 0.45

Note: Values are presented as mean ± standard deviation. Brushing: How many times does a patient brush their teeth daily. Flossing: How many times does a patient floss daily. Dessert: How many times does a patient eat dessert weekly.

∗ Statistically significant ( P <0.05; paired t test).

All the samples were sequenced with Illumina NovaSeq 6000. A total of 2.20 × 10 ⁵ clean reads were obtained from 60 samples, with an average of 3.66 × 10 ⁷ sequences per sample. The average Q30 was 92.3%, indicating data were ready for the following analysis. Based on these sequences, 82 phyla, 2341 genera, and 7199 species for all samples were detected.

We calculated the Shannon, Simpson, chao1, ACE, observed species, and goods_coverage index at the species level for each sample ( Supplementary Fig 1 ). There were no significant differences between the participants before and after orthodontics based on the alpha-diversity analysis in the gut, dental plaque, or saliva microbiota, which indicated that wearing braces did not change the richness or diversity of the microbiome in the initial stage.

A principal coordinate analysis plot based on Bray-Curtis distance also showed no obvious separation of the gut, dental plaque, or saliva microbiota between the participants before and after orthodontics ( Supplementary Fig 2 , A-C ).

At the phyla level, Actinobacteria (2.84%-68.74%), Proteobacteria (4.52%-57.81%), Bacteroidetes (3.55%-30.35%), and Firmicutes (5.05%-37.50%) were enriched in the dental plaque of participants ( Fig 1 , A ). After orthodontic treatment, the relative abundance of Firmicutes was significantly decreased in the dental plaque of participants ( Fig 1 , B ; P <0.05)

The dental plaques of participants were dominated by Actinomyces (2.06-46.08%), Corynebacterium (0.29%-40.25%), Neisseria (0.45%-38.98%), and Capnocytophaga (1.62%-27.57%) at the genus level ( Fig 1 , C ). We compared the relative abundances of dental plaque microbes at the genus level between participants before and after orthodontic treatment using LEfSe ( Fig 1 , D ). Rothia and Cardiobacterium elevated in the dental plaques of patients with braces, whereas Veillonella decreased.

The relative abundances of 34 species became significantly greater after orthodontic treatment, whereas 7 species decreased. Rothia dentocariosa and Cardiobacterium hominis are oral pathogens significantly elevated in the dental plaques of patients with braces ( Fig 1 , E ; P <0.05).

CE1 , GT85 , and GT20 of the CAZy enzyme family were significantly upregulated in Group B, whereas GT41 and GH117 were significantly upregulated in Group A ( Fig 1 , F ). K03536 was significantly upregulated in Group B, whereas K02907 was significantly upregulated in Group A ( Supplementary Fig 2 , D ). For GO enrichment analysis, upregulated genes in Group B were enriched in GO:0004526, whereas downregulated genes were enriched in GO:0019867 ( Supplementary Fig 2 , E ). For microbial metabolic pathway enrichment analysis ( Fig 1 , G ), anaerobic energy metabolism invertebrates cytosol and so on were enriched in Group B, whereas heme biosynthesis and so on were enriched in Group A.

The saliva of participants was dominated by Firmicutes (16.59%-41.44%), Proteobacteria (5.93%-42.96%), Bacteroidetes (10.88%-52.32%), and Actinobacteria (1.95%- 25.61%), which was similar to the dental plaque microbial composition ( Fig 2 , A ).

Prevotella (4.44%-49.77%), Neisseria (1.05%-31.51%), Veillonella (5.40%-24.23%), Streptococcus (4.67%-31.10%), and Haemophilus (1.77%-24.07%) were enriched in the saliva of participants at the genus level ( Fig 2 , B ). After wearing dental braces for a month, Fusobacterium , Aggregatibacter , Cardiobacterium, and Actinobacillus elevated in the saliva ( Fig 2 , C ).

The relative abundances of 14 species increased after taking dental braces, whereas 8 species decreased. Pathogens such as Aggregatibacter actinomycetemcomitans and Cardiobacterium hominis increased after participants took dental braces for a month ( Fig 2 , D ; P <0.01). At the same time, Faecalibacterium prausnitzii is a probiotic whose relative abundance significantly decreased ( Fig 2 , D ; P <0.05).

In Group A, GH6, GT2, and GH27 were significantly upregulated ( Supplementary Fig 2 , F ). For KEGG analysis, K02899 and K02078 were significantly upregulated in Group B, whereas K02952 and K03664 were significantly downregulated ( Supplementary Fig 2 , G ). For GO enrichment analysis, upregulated genes in Group B were enriched in GO:0005887 , whereas GO:0003824 was significantly enriched in Group A ( Supplementary Fig 2 , H ). Mixed acid fermentation and super pathway of fatty acids biosynthesis were significantly upregulated in Group B, whereas peptidoglycan biosynthesis was significantly upregulated in Group A ( Fig 2 , E ).

There were 4 dominant phyla whose average relative abundances were >1%, namely Firmicutes (9.72%-95.80%), Bacteroidetes (0.20%-73.57%), Actinobacteria (0.28%-25.54%), Proteobacteria (0.97%-35.65%) ( Fig 3 , A ). After orthodontic treatment, the relative abundances of Proteobacteria and Ignavibacteriae in the guts of participants were significantly greater ( Fig 3 , B ; P <0.05).