Dynamic effects of fixed orthodontic treatment on oral health and oral microbiota: a prospective study
The global prevalence of malocclusion exceeds 56% . With the growing demand for improved aesthetics and functionality, an increasing number of individuals are seeking orthodontic treatment to correct malformations . However, orthodontic appliances can hinder routine oral hygiene, creating conditions that promote the rapid accumulation of plaque . Inadequate oral hygiene during orthodontic treatment can lead to complications such as enamel demineralization, caries, and periodontitis. Research indicates that poor oral hygiene may extend the duration of treatment, compromise treatment outcomes, and, in severe cases, cause irreversible tissue damage . Enamel demineralization and periodontal inflammation are common complications among orthodontic patients, with an incidence rate of 50–70% in those undergoing fixed appliance therapy . Specifically, the incidence of enamel demineralization ranges from 23 to 46% in patients with fixed orthodontic appliances . Many patients develop irreversible white spot lesions around the brackets after orthodontic treatment, which goes against the aesthetic principles of orthodontic treatment . Enamel demineralization, an early stage of caries, occurs when the oral microbial balance shifts from equilibrium to imbalance, with microorganisms playing a central role. Under healthy conditions, a stable ecological balance exists between oral microorganisms and the host, where both sides are interdependent and mutually regulated. However, disruptions in this balance can create conditions favorable for the growth of caries-causing bacteria . Orthodontic appliances can reduce the oral cavity’s natural self-cleaning ability, leading to the accumulation of food residues and plaque between the appliances and teeth. This buildup of pathogenic bacteria disrupts the oral microecological balance. A cross-sectional study comparing the oral microbial diversity of orthodontic patients and healthy individuals revealed that orthodontic patients had greater microbial diversity . Previous studies have monitored the levels of oral cariogenic bacteria in orthodontic patients, and the results show that orthodontics led to an increase in Streptococcus mutans and Lactobacillus, which increased the risk of enamel demineralization . Traditional microbiological identification methods, such as culture enumeration, quantitative polymerase chain reaction, and DNA-DNA hybridization, are limited to detecting specific pathogenic bacteria . With advancements in gene sequencing technology, 16S rRNA sequencing now allows for a more comprehensive and sensitive analysis of the structure, diversity, and abundance of oral bacteria. The impact of orthodontic procedures on the oral microbiome has garnered increasing attention . However, most existing studies on changes in the oral microbiome of orthodontic patients are based on small clinical samples with limited sample selection. Additionally, there are few studies that dynamically monitor changes in the oral microecological environment during orthodontic treatment. This multicenter prospective study aims to address this gap by monitoring the changes in oral hygiene among patients with fixed orthodontic appliances and systematically comparing the microbial diversity in saliva before and six months after treatment using 16S rRNA gene sequencing. 

Study population This study was an observational study conducted in accordance with the STROBE guidelines. Forty-five individuals (15 males and 30 females, aged 10 to 35 years) undergoing fixed orthodontic treatment were included in this study. Participants were recruited from the Department of Orthodontics at Beijing Stomatological Hospital, Capital Medical University; the Department of Stomatology at Beijing Tiantan Hospital, Capital Medical University; and the Department of Stomatology at Beijing Chaoyang Hospital, Capital Medical University. The study received approval from the Ethics Committees of these institutions. Informed consent was obtained from all participants after providing a thorough explanation of the study. For participants under 18 years of age, consent was obtained from their parents or legal guardians. All samples were anonymized after collection. Patients were enrolled according to the following criteria: No periodontal and mucosal diseases, no history of orthodontic treatment. Complete permanent dentition with intact clinical crowns, no defects or fractures, and no dental caries. Full-mouth metal fixed orthodontic treatment as the planned treatment approach. No use of antibiotics or hormones within two months before the start of the study. No pregnancy or systemic diseases. Clinical examination and sample collection Study visits were scheduled at two time points: T0 (before treatment) and T1 (six months after treatment). At each visit, clinical examinations and sample collections were conducted. A questionnaire was used to gather information on the participants’ socio-demographic characteristics, oral hygiene practices, and dietary habits (see Additional file ). The clinical examination comprised two components: oral hygiene and dental status. Oral hygiene assessment included the evaluation of plaque and tartar, while dental status assessment covered tooth eruption, developmental defects, white spot lesions, and caries. Caries were assessed using the International Caries Detection and Assessment System (ICDAS). Six experienced orthodontists, two from each participating sub-center, conducted and recorded the clinical examinations. These orthodontists underwent uniform training and evaluation on the relevant procedures before the study commenced. The consistency among examiners was assessed using the unweighted kappa tests. Kappa values above 0.75 denoted excellent agreement. Participants were instructed to collect samples between 9:30 AM and 11:30 AM and refrain from cleaning their teeth and eating for two hours before sample collection. During collection, participants sat in a comfortable position with their heads slightly tilted forward to allow saliva to flow gradually into a sterile centrifuge tube. A total of 2 ml of unstimulated whole saliva was collected. The samples were temporarily stored in liquid nitrogen and subsequently stored at -80 °C until further analysis. DNA extraction DNA was extracted from the samples using the MagPure Stool DNA KF Kit B (MAGEN, Guangzhou, China) following the manufacturer’s instructions. A 100–200 mg sample was transferred into a centrifuge tube containing grinding beads. One milliliter of Buffer ATL/PVP-10 was added, and the sample was ground using a grinding machine (Shanghai Jingxin Tech, China). The mixture was then incubated at 65 °C for 20 min. After incubation, the sample was centrifuged at 14,000×g for 5 min (Eppendorf, Germany), and the supernatant was transferred to a new tube. Next, 0.6 mL of Buffer PCI was added to the sample, and it was mixed thoroughly by vortexing for 15 s. The mixture was centrifuged at 18,213×g for 10 min, after which the supernatant was transferred to a deep well plate containing a magnetic bead binding solution (600 µL Buffer with magnetic beads, 20 µL Proteinase K, 5 µL RNase A), followed by sequential washes with 700 µL Wash 1, 700 µL Wash 2, and 700 µL Wash 3. Finally, 100 µL of Elution Buffer was added. The deep well plate was placed into the Kingfisher system (Thermo Fisher, USA), and the corresponding program was initiated. Upon completion of the program, the extracted DNA was transferred to a 1.5 mL centrifuge tube for storage. Library preparation and 16S rRNA gene sequencing Library preparation was conducted using the 2× Phanta Max Master Mix kit (VAZYME, China). The microbial 16S rRNA (V3-V4) regions were amplified. A genomic DNA sample of 30 ng, of acceptable quality, was combined with PCR reaction reagents and fusion primers. The library products were then amplified through PCR. Following amplification, the PCR products were purified, and size selection was performed using magnetic beads (Agencourt AMPure XP). The final library products underwent a quality control process, after which those that passed were loaded onto the Illumina MiSeq platform to generate sequencing reads with a PE300 base length. Sequence analysis Quality control of the raw sequencing data was conducted using iTools Fqtools fqcheck (v0.25) . Primer and junction contaminants were removed using Cutadapt (v2.6) to isolate fragments of the target region. Amplicon Sequence Variants (ASVs) were generated by filtering, denoising, merging, and removing chimeras using the DADA2 (Divisive Amplicon Denoising Algorithm) within the QIIME2 platform. Taxonomic classification of ASVs was performed based on the Greengenes database ( https://greengenes.secondgenome.com/ ) using the RDP classifier software. Bioinformatics and statistical analysis Alpha diversity was assessed using the Chao1, ACE, Simpson, and Shannon indices. The Wilcoxon rank-sum test was applied to compare alpha diversity between the two sample groups. Beta diversity was evaluated through Principal Coordinate Analysis (PCoA) based on unweighted UniFrac distance. Differences in community structure were tested using Permutational Multivariate Analysis of Variance (PERMANOVA). Species differences between the two groups were analyzed using the Wilcoxon rank-sum test, with the Kruskal-Wallis test employed for multiple comparisons. The impact of different species on group differences was estimated using Linear Discriminant Analysis (LDA) effect size (LEfSe) analysis, with an LDA score threshold set at 2. A p-value of less than 0.05 was considered statistically significant. 

This study was an observational study conducted in accordance with the STROBE guidelines. Forty-five individuals (15 males and 30 females, aged 10 to 35 years) undergoing fixed orthodontic treatment were included in this study. Participants were recruited from the Department of Orthodontics at Beijing Stomatological Hospital, Capital Medical University; the Department of Stomatology at Beijing Tiantan Hospital, Capital Medical University; and the Department of Stomatology at Beijing Chaoyang Hospital, Capital Medical University. The study received approval from the Ethics Committees of these institutions. Informed consent was obtained from all participants after providing a thorough explanation of the study. For participants under 18 years of age, consent was obtained from their parents or legal guardians. All samples were anonymized after collection. Patients were enrolled according to the following criteria: No periodontal and mucosal diseases, no history of orthodontic treatment. Complete permanent dentition with intact clinical crowns, no defects or fractures, and no dental caries. Full-mouth metal fixed orthodontic treatment as the planned treatment approach. No use of antibiotics or hormones within two months before the start of the study. No pregnancy or systemic diseases. 

Study visits were scheduled at two time points: T0 (before treatment) and T1 (six months after treatment). At each visit, clinical examinations and sample collections were conducted. A questionnaire was used to gather information on the participants’ socio-demographic characteristics, oral hygiene practices, and dietary habits (see Additional file ). The clinical examination comprised two components: oral hygiene and dental status. Oral hygiene assessment included the evaluation of plaque and tartar, while dental status assessment covered tooth eruption, developmental defects, white spot lesions, and caries. Caries were assessed using the International Caries Detection and Assessment System (ICDAS). Six experienced orthodontists, two from each participating sub-center, conducted and recorded the clinical examinations. These orthodontists underwent uniform training and evaluation on the relevant procedures before the study commenced. The consistency among examiners was assessed using the unweighted kappa tests. Kappa values above 0.75 denoted excellent agreement. Participants were instructed to collect samples between 9:30 AM and 11:30 AM and refrain from cleaning their teeth and eating for two hours before sample collection. During collection, participants sat in a comfortable position with their heads slightly tilted forward to allow saliva to flow gradually into a sterile centrifuge tube. A total of 2 ml of unstimulated whole saliva was collected. The samples were temporarily stored in liquid nitrogen and subsequently stored at -80 °C until further analysis. 

DNA was extracted from the samples using the MagPure Stool DNA KF Kit B (MAGEN, Guangzhou, China) following the manufacturer’s instructions. A 100–200 mg sample was transferred into a centrifuge tube containing grinding beads. One milliliter of Buffer ATL/PVP-10 was added, and the sample was ground using a grinding machine (Shanghai Jingxin Tech, China). The mixture was then incubated at 65 °C for 20 min. After incubation, the sample was centrifuged at 14,000×g for 5 min (Eppendorf, Germany), and the supernatant was transferred to a new tube. Next, 0.6 mL of Buffer PCI was added to the sample, and it was mixed thoroughly by vortexing for 15 s. The mixture was centrifuged at 18,213×g for 10 min, after which the supernatant was transferred to a deep well plate containing a magnetic bead binding solution (600 µL Buffer with magnetic beads, 20 µL Proteinase K, 5 µL RNase A), followed by sequential washes with 700 µL Wash 1, 700 µL Wash 2, and 700 µL Wash 3. Finally, 100 µL of Elution Buffer was added. The deep well plate was placed into the Kingfisher system (Thermo Fisher, USA), and the corresponding program was initiated. Upon completion of the program, the extracted DNA was transferred to a 1.5 mL centrifuge tube for storage. 

Library preparation was conducted using the 2× Phanta Max Master Mix kit (VAZYME, China). The microbial 16S rRNA (V3-V4) regions were amplified. A genomic DNA sample of 30 ng, of acceptable quality, was combined with PCR reaction reagents and fusion primers. The library products were then amplified through PCR. Following amplification, the PCR products were purified, and size selection was performed using magnetic beads (Agencourt AMPure XP). The final library products underwent a quality control process, after which those that passed were loaded onto the Illumina MiSeq platform to generate sequencing reads with a PE300 base length. 

Quality control of the raw sequencing data was conducted using iTools Fqtools fqcheck (v0.25) . Primer and junction contaminants were removed using Cutadapt (v2.6) to isolate fragments of the target region. Amplicon Sequence Variants (ASVs) were generated by filtering, denoising, merging, and removing chimeras using the DADA2 (Divisive Amplicon Denoising Algorithm) within the QIIME2 platform. Taxonomic classification of ASVs was performed based on the Greengenes database ( https://greengenes.secondgenome.com/ ) using the RDP classifier software. 

Alpha diversity was assessed using the Chao1, ACE, Simpson, and Shannon indices. The Wilcoxon rank-sum test was applied to compare alpha diversity between the two sample groups. Beta diversity was evaluated through Principal Coordinate Analysis (PCoA) based on unweighted UniFrac distance. Differences in community structure were tested using Permutational Multivariate Analysis of Variance (PERMANOVA). Species differences between the two groups were analyzed using the Wilcoxon rank-sum test, with the Kruskal-Wallis test employed for multiple comparisons. The impact of different species on group differences was estimated using Linear Discriminant Analysis (LDA) effect size (LEfSe) analysis, with an LDA score threshold set at 2. A p-value of less than 0.05 was considered statistically significant. 

Clinical parameters All 45 participants completed the 6-month study. The clinical parameters are summarized in Table . Compared to baseline, patients significantly increased the frequency of tooth brushing per day after six months of orthodontic treatment. While the frequency of dessert consumption per day slightly increased post-treatment, this change was not statistically significant. Additionally, there were no significant changes observed in Plaque Index (PI) and Debris Index (DI). Sequence analysis After sequencing, a total of 6,927,788 raw tags were obtained from 90 saliva samples. Quality filtering yielded 5,428,456 tags, with an average of 60,316 tags per sample. Following quality control and sequence merging, the sequences were denoised using DADA2 via QIIME2, resulting in 3,344,549 high-quality tags. High-throughput sequencing identified a total of 3,662 Amplicon Sequence Variants (ASVs), with an average of 210 ASVs detected per sample. A total of 20 phyla, 30 orders, 8 orders, 142 families, and 338 genera levels were detected in the two groups of samples. To investigate the changes in the oral salivary microbiota, the distribution and relative abundance of microbiota at the phylum and genus levels were analyzed. Alpha and beta diversity Alpha diversity measures the richness and diversity of microbial communities within each set of samples. The Chao1 and ACE indices were used to assess microbial richness, while the Shannon and Simpson indices were employed to evaluate microbial diversity. After six months of orthodontic treatment (T1), both the Chao1 and ACE indices increased compared to the pre-treatment period (T0), indicating a rise in the total number of species and a significant difference in community richness (Fig. a, b). Additionally, the Shannon index was significantly higher at T1 than T0 (Fig. c), and the Simpson index was significantly lower (Fig. d), suggesting an increase in microbial community diversity after six months of treatment compared to the baseline. The beta diversity of microbial community changes between samples was calculated and compared using unweighted UniFrac distance measurements. The analysis revealed no significant difference ( p > 0.5) in the unweighted UniFrac distance values (mean ± standard deviation) between T0 and T1 (Fig. a). However, Principal Coordinate Analysis (PCoA) based on unweighted UniFrac distances indicated a trend of separation between the T0 and T1 ( p < 0.01) (Fig. b). Analysis of microbial community composition The heat map depicting the salivary microbial structure at the phylum level is shown in Fig. a. At both T0 and T1, the dominant phyla were Proteobacteria, Bacteroidota, Firmicutes_C, Firmicutes_D, Actinobacteriota, Fusobacteriota, Firmicutes_A, and Patescibacteria. Figure b illustrates the relative abundance of the top 10 bacterial phyla at T0 and T1, along with the results of the ANOVA test. The abundance of Firmicutes_C was significantly higher at T1 compared to T0 ( p < 0.01), while the abundance of the other phyla did not show significant changes. The bacterial distribution at the genus level in saliva samples, based on relative abundance, is shown in Fig. a, b. At T1, the abundance of Centipeda, Anaeroglobus, Actinomyces, and Corynebacterium was significantly higher, while the abundance of Granulicatella was significantly lower compared to T0 ( P < 0.05). Additionally, the core microbiota showed a decrease in the abundance of Prevotella, Neisseria, Streptococcus, Rothia, and Alloprevotella, while Veillonella_A, Haemophilus_D, Fusobacterium_C, and Leptotrichia_A showed a relative increase, although these changes were not statistically significant. To further investigate the taxonomic composition changes between the two groups, a branching diagram was generated using LEfSe (Fig. c). The analysis revealed significant enrichment of 66 taxonomic units, including Firmicutes, Negativicutes, and Veillonellales at T1. 

All 45 participants completed the 6-month study. The clinical parameters are summarized in Table . Compared to baseline, patients significantly increased the frequency of tooth brushing per day after six months of orthodontic treatment. While the frequency of dessert consumption per day slightly increased post-treatment, this change was not statistically significant. Additionally, there were no significant changes observed in Plaque Index (PI) and Debris Index (DI). 

After sequencing, a total of 6,927,788 raw tags were obtained from 90 saliva samples. Quality filtering yielded 5,428,456 tags, with an average of 60,316 tags per sample. Following quality control and sequence merging, the sequences were denoised using DADA2 via QIIME2, resulting in 3,344,549 high-quality tags. High-throughput sequencing identified a total of 3,662 Amplicon Sequence Variants (ASVs), with an average of 210 ASVs detected per sample. A total of 20 phyla, 30 orders, 8 orders, 142 families, and 338 genera levels were detected in the two groups of samples. To investigate the changes in the oral salivary microbiota, the distribution and relative abundance of microbiota at the phylum and genus levels were analyzed. 

Alpha diversity measures the richness and diversity of microbial communities within each set of samples. The Chao1 and ACE indices were used to assess microbial richness, while the Shannon and Simpson indices were employed to evaluate microbial diversity. After six months of orthodontic treatment (T1), both the Chao1 and ACE indices increased compared to the pre-treatment period (T0), indicating a rise in the total number of species and a significant difference in community richness (Fig. a, b). Additionally, the Shannon index was significantly higher at T1 than T0 (Fig. c), and the Simpson index was significantly lower (Fig. d), suggesting an increase in microbial community diversity after six months of treatment compared to the baseline. The beta diversity of microbial community changes between samples was calculated and compared using unweighted UniFrac distance measurements. The analysis revealed no significant difference ( p > 0.5) in the unweighted UniFrac distance values (mean ± standard deviation) between T0 and T1 (Fig. a). However, Principal Coordinate Analysis (PCoA) based on unweighted UniFrac distances indicated a trend of separation between the T0 and T1 ( p < 0.01) (Fig. b). 

The heat map depicting the salivary microbial structure at the phylum level is shown in Fig. a. At both T0 and T1, the dominant phyla were Proteobacteria, Bacteroidota, Firmicutes_C, Firmicutes_D, Actinobacteriota, Fusobacteriota, Firmicutes_A, and Patescibacteria. Figure b illustrates the relative abundance of the top 10 bacterial phyla at T0 and T1, along with the results of the ANOVA test. The abundance of Firmicutes_C was significantly higher at T1 compared to T0 ( p < 0.01), while the abundance of the other phyla did not show significant changes. The bacterial distribution at the genus level in saliva samples, based on relative abundance, is shown in Fig. a, b. At T1, the abundance of Centipeda, Anaeroglobus, Actinomyces, and Corynebacterium was significantly higher, while the abundance of Granulicatella was significantly lower compared to T0 ( P < 0.05). Additionally, the core microbiota showed a decrease in the abundance of Prevotella, Neisseria, Streptococcus, Rothia, and Alloprevotella, while Veillonella_A, Haemophilus_D, Fusobacterium_C, and Leptotrichia_A showed a relative increase, although these changes were not statistically significant. To further investigate the taxonomic composition changes between the two groups, a branching diagram was generated using LEfSe (Fig. c). The analysis revealed significant enrichment of 66 taxonomic units, including Firmicutes, Negativicutes, and Veillonellales at T1. 

Orthodontic treatment affects oral health by altering the microecological environment, host immunity, oral hygiene practices, and is influenced by factors such as age and gender . Previous studies have suggested that female patients have higher standards for oral hygiene . However, the conclusions regarding the differences in the incidence of enamel demineralization between genders are inconsistent . This study compared the microbiome diversity before and after treatment in male and female patients, and no significant differences were observed. Research indicated that younger patients experience a higher incidence of enamel demineralization during orthodontic treatment . Therefore, in this study, we conducted a differential analysis of microbial diversity between adult and adolescent groups among the subjects. However, no significant difference in microbial diversity was observed between the two populations at two time points. The higher rate of enamel demineralization may be attributed to adolescents’ tendency to consume sugary or sticky foods and their often limited attention to oral hygiene and dietary control. The presence of cariogenic bacteria is a key factor contributing to complications like enamel demineralization and caries. In our study, we observed a downward trend in plaque and soft scale accumulation among patients, although the difference was not statistically significant. This may be attributed to the alleviation of dental crowding during treatment, coupled with improved oral hygiene habits, including increased brushing frequency. Numerous studies have identified Streptococcus mutans and Lactobacillus species as primary cariogenic bacteria due to their acid-producing and acid-resistant properties, which are closely linked to the development of caries . Additionally, some studies have associated these bacteria with enamel demineralization, particularly during orthodontic treatment. Research indicates that levels of Streptococcus pyogenes and Lactobacillus spp. increase significantly during orthodontic procedures, potentially leading to oral health risks such as white spot lesions (WSLs) . However, most previous studies have relied on bacterial culture or PCR techniques to examine a limited number of cariogenic bacteria . The broader changes in oral microecology during fixed orthodontic treatment have not been extensively explored. Previous longitudinal studies using high-throughput sequencing to investigate microecological changes during orthodontic treatment have had relatively small sample sizes, mostly focusing on 10 to 30 individuals . In this study, we recruited orthodontic patients from multiple centers and examined the structure of their salivary microflora using high-throughput 16S rRNA gene sequencing, with a follow-up period of six months. Previous studies have systematically evaluated the impact of fixed orthodontic treatment on oral microflora , but more sensitive microbiological analysis methods are needed to further validate these findings. In our study, we observed a significant increase in the alpha diversity of saliva samples from 45 patients after 6 months of orthodontic treatment, compared to the baseline. This suggests that the salivary microecological environment becomes more diverse following orthodontic treatment, aligning with the results of earlier research . The analysis of relative abundance at both the phylum and genus levels provided a detailed view of bacterial distribution. Our study found a significant increase in the abundance of Firmicutes and Spirochaetota at T1. At the genus level, Centipeda, Anaeroglobus, Actinomyces, and Veillonellales showed a notable rise. While these bacteria typically exist as commensal organisms in a healthy oral environment, they can become pathogenic when oral ecological balance is disrupted, such as during orthodontic treatment. This shift is particularly associated with conditions like periodontal disease, dental caries, and plaque formation. Firmicutes are crucial components of healthy oral microbiota. However, when the quantity of Firmicutes increases excessively, it may lead to dysbiosis, contributing to oral health issues such as periodontal disease . Spirochaetota plays a significant role in the pathogenesis of periodontal disease and can cooperate with other pathogenic bacteria to promote the destruction of periodontal tissues . The increase of Centipeda, Anaeroglobus, Actinomyces, and Veillonellales at T1, all of which are anaerobic bacteria, may contribute to the development of oral health problems in the case of microbial dysbiosis. Anaeroglobus is commonly found in patients with periodontitis . Veillonella species typically thrive in anaerobic environments. While they do not directly cause dental caries, they utilize lactic acid produced by the fermentation of other bacteria, thereby maintaining an acidic environment in the mouth, which indirectly contributes to the development of caries . With the advent of high-throughput sequencing technology, recent studies have suggested that WSLs may be caused by a variety of microorganisms . Jiang et al. identified Actinomyces and Corynebacterium as key players in the early stages of caries, contributing to enamel demineralization . Tanner et al. reported that Streptococcus granulicatellus, Streptococcus vaiveronensis, Streptococcus bifidus, Streptococcus anomalanus, and Streptococcus saprophyticus were involved in the initial stages of caries . Additionally, Bacillaceae, Streptococcus pyogenes, and Scardovia wiggsiae were linked to the development of WSLs in adolescents undergoing fixed orthodontic treatment.The variations in study results can be attributed to several factors. Differences in sample types, collection methods, specific testing time points, and the use of various sequencing platforms significantly influenced the observed outcomes. Additionally, it has been suggested that patients with pre-existing demineralization are more likely to develop new demineralization after beginning orthodontic treatment. This may be related to factors such as the patient’s oral microbiome and hygiene practices, warranting further research to clarify the underlying causes . These changes in the microbial structure of the oral cavity indicate that the placement of fixed orthodontic appliances alters the oral microbiome, with an increase in anaerobic and pathogenic bacteria, potentially leading to a decline in oral hygiene . This shift may contribute to enamel demineralization, periodontal inflammation, and other oral health issues. These findings underscore the importance of reinforcing oral hygiene education for patients during orthodontic treatment and considering the use of antimicrobial materials  to help maintain a balanced oral microbiome and prevent orthodontic complications. The primary limitation of this study is the short observation period. Previous studies on the impact of orthodontic treatment on the oral microbiome had follow-up periods ranging from 1 month to 18 months . Furthermore, it has been noted that complications such as enamel demineralization may occur after 6 months of orthodontic treatment, with the incidence increasing over time . To gain a more comprehensive understanding of the effects of orthodontic treatment on oral health and the oral microbiota, future research should extend the observation period and examine the patients’ oral health in greater detail, including the degree of enamel demineralization and periodontal condition, to further elucidate the underlying mechanisms and their correlations. 

In this prospective clinical study, we examined the impact of fixed orthodontic appliances on oral hygiene and analyzed changes in the oral microflora using high-throughput sequencing technology. After six months of treatment, there was a significant increase in microbial diversity in the patients’ saliva, with notable rises in the abundance of Firmicutes, Centipeda, Anaeroglobus, and Actinomyces. These findings suggest that the placement of fixed orthodontic appliances alters the oral microbiome, particularly increasing the presence of anaerobic and pathogenic bacteria, which may contribute to the deterioration of oral hygiene. Consequently, it is essential to reinforce oral hygiene education for patients undergoing orthodontic treatment and consider the use of antimicrobial materials. 

Below is the link to the electronic supplementary material. Additional file 1: The questionnaire assessing participants’ oral hygiene practices and dietary habits. 

