The impact of fixed orthodontic appliances on oral microbiome dynamics in Japanese patients

The oral cavity hosts over 700 bacterial species distributed across diverse niches, and fixed orthodontic appliances, while effective for correcting malocclusion, can complicate hygiene, promote plaque accumulation, and potentially shift the oral microbiome toward dysbiosis. Prior reports link orthodontic treatment with increased risk of white-spot lesions and dental caries and detection of periodontal pathogens around appliances. However, most studies focused on a few cultivable species, leaving the broader microbiome dynamics, including unculturable taxa, unclear. This study asks how fixed orthodontic appliances alter the oral microbiome over time in plaque and saliva, hypothesizing that appliance placement drives a shift toward anaerobic, periodontopathogenic communities indicative of transition from health toward periodontitis.

Conventional cultivation-based studies have shown increased Streptococcus mutans levels and associations with caries during fixed appliance therapy, as well as increased detection of periodontal pathogens (e.g., Porphyromonas, Prevotella, Fusobacterium, Tannerella). Some clinical reports noted periodontal attachment loss during treatment. Next-generation sequencing (NGS) enables comprehensive profiling of both culturable and unculturable bacteria, yet few NGS-based studies assessed orthodontic impacts, and prior NGS work largely focused on plaque and indicated peaks of periodontal pathogens during treatment. Reported changes include decreases in Actinobacteria after appliance placement and variable findings for genera such as Actinomyces and Neisseria across studies. Gaps remain regarding longitudinal dynamics across multiple niches (plaque and saliva) and over extended treatment intervals including after appliance removal.

Design: Longitudinal observational study of Japanese orthodontic patients receiving fixed appliances. Ethics: Approved by the Independent Ethics Committee of Hiroshima University Hospital (No. 1645); informed consent obtained; samples anonymized. Participants: n=71, treated July 2016–April 2018 for anterior crowding. Inclusion: healthy; no antibiotics/medications before sampling; no severe gingivitis (PPD <4 mm) or radiographic bone loss; no fixed restorations/removable dentures; received tooth brushing instruction and maintained plaque control record <20% prior to bracket placement. Orthodontic appliances: Standard edgewise 0.018-inch slot brackets bonded labially; archwires of NiTi or cobalt-chrome ligated with ligature wires. Sampling time points: T0 (immediately before placement), T1 (~6 months after start), T2 (immediately after removal; average 40 months post-placement, range 20–62 months). Plaque: supragingival plaque from upper and lower anterior teeth at T0 and T1 (above gingival margin at T0; between gingival margin and cervical side of brackets at T1) under cotton roll isolation and air drying using sterilized explorer. Saliva: unstimulated saliva into paper cup at T0, T1, T2; standardized by fasting from eating/drinking/brushing for ≥2 h before collection. Samples pooled into microcentrifuge tubes with PBS and stored at −80°C. DNA extraction: MasterPure Complete DNA and RNA Purification Kit (Epicentre) with minor modifications. Library prep and sequencing: PCR amplification of V1–V2 16S rRNA regions using KAPA HiFi HotStart ReadyMix and Nextera XT Index primers; verification by agarose gel; cleanup with AMPure XP; quantification by NanoDrop and Qubit; pooling in equimolar amounts; qPCR quantification (KAPA SYBR FAST). Sequencing on Illumina MiSeq. Data availability: DDBJ SRA DRA010713. Bioinformatics and statistics: CLC Genomics Workbench v9. OTU clustering at 97% similarity against Greengenes. Diversity analyses: alpha (richness/evenness) and beta diversity (PCoA based on Jaccard index). Statistical tests: Mann–Whitney U for two-group comparisons (e.g., T0 vs T1 plaque), Kruskal–Wallis for multi-group comparisons (e.g., T0 vs T1 vs T2 saliva).

Sequencing output: 13,506,556 reads from 144 samples (mean length 300.4 bp); after trimming, 8,284,029 high-quality reads (mean 222.5 bp). Successfully analyzed samples: plaque paired T0/T1 n=44; saliva T0 n=16, T1 n=23, T2 n=17. Total OTUs detected: 983; mean 341 OTUs/sample. OTU counts showed non-significant increases over time: plaque T0 312 vs T1 321; saliva T0 329, T1 366, T2 376. Diversity: Beta diversity (PCoA, Jaccard) showed distinct community structures between plaque and saliva and segregation between T0 and T1 plaque communities; saliva showed no obvious shift across T0–T2. No differences between upper vs lower teeth. Phylum-level shifts:

• Plaque: Proteobacteria decreased 28.74%→23.56% (significant); Actinobacteria decreased 15.11%→9.39% (significant); Bacteroidetes increased 17.78%→23.44% (significant); Saccharibacteria (TM7) increased 2.59%→4.08% (significant).
• Saliva: Actinobacteria increased T1 6.68%→T2 12.07% (significant). TM7 increased T0 0.87%→T1 1.99%→T2 2.77% (time-dependent, significant). Spirochaetes increased 0.08%→0.15%→0.64% (time-dependent trend). Proteobacteria similar at T0 31.22% and T1 35.75%, then decreased at T2 27.13% (significant). Fusobacteria and Bacteroidetes trended upward (not significant). Genus/species-level dynamics:
• Plaque increases (mostly obligate/facultative anaerobes, many periodontopathogenic): Prevotella 3.81%→7.69% (p=0.001), Capnocytophaga 3.63%→8.94% (p=0.001), Campylobacter 2.77%→4.48% (p=0.006), Parvimonas micra 0.01%→0.09% (p=0.001), Selenomonas 2.54%→4.61% (p=0.001), Selenomonas noxia 0.12%→0.25% (p=0.036), Atopobium 0.12%→0.44% (p=0.002), Solobacterium moorei 0.05%→0.18% (p=0.006), Prevotella melaninogenica 0.52%→1.18% (p=0.048). Porphyromonas increased without significance.
• Plaque decreases (aerobes/facultative commensals): Actinomyces 6.40%→3.82% (p=0.007), Neisseria 4.80%→2.51% (p=0.009), Lautropia mirabilis 3.33%→0.72% (p=0.001), Haemophilus parainfluenzae 3.88%→2.35% (p=0.046), Corynebacterium 2.86%→1.80% (p=0.041), Kingella 1.23%→0.67% (p=0.039), Actinobacillus 0.70%→0.01% (p=0.005), Rothia dentocariosa 1.49%→0.77% (p=0.049). Streptococcus decreased 9.47%→7.53% (ns).
• Saliva increases (anaerobes): Fusobacterium 3.25%→4.02%→7.04% (p=0.001), Leptotrichia 2.58%→5.94%→6.03% (p=0.001), Selenomonas 0.26%→1.34%→2.47% (p=0.001), Selenomonas noxia 0.01%→0.10%→0.12% (p=0.004), Prevotella 4.94%→5.56%→7.57% (p=0.032), Prevotella nigrescens 0.04%→0.04%→0.10% (p=0.035), Tannerella 0.08%→0.14%→0.24% (p=0.023), Capnocytophaga ochracea 0.38%→0.53%→1.17% (p=0.002), Campylobacter 1.08%→1.85%→2.32% (p=0.008), Paludibacter 0.04%→0.17%→0.67% (p=0.001), Atopobium 0.28%→0.64%→0.65% (p=0.046).
• Saliva decreases: Granulicatella 5.14%→3.04%→2.05% (p=0.046). Neisseria 9.16%→7.58%→3.62% (ns). Streptococcus 15.97%→12.86%→11.73% (ns). Overall: Fixed appliances shifted both plaque and saliva microbiomes toward increased relative abundance of anaerobic, periodontopathogenic taxa and decreased aerobes/facultative commensals, particularly pronounced in plaque. Patterns align with a microbiome transition from health to gingivitis/periodontitis.

The findings confirm that fixed orthodontic appliances drive measurable restructuring of the oral microbiome. In plaque, beta-diversity separation between T0 and T1 and significant phylum-level shifts (decreased Proteobacteria/Actinobacteria; increased Bacteroidetes/TM7) indicate an appliance-associated ecological transition. Genus-level increases in obligate/facultative anaerobes implicated in periodontal disease (e.g., Prevotella, Selenomonas, Parvimonas micra, Solobacterium moorei) coupled with decreases in commensal aerobes/facultative taxa (e.g., Neisseria, Haemophilus, Lautropia, Actinomyces) support a shift toward an anaerobic, periodontopathogenic state. Saliva exhibited similar trends, including time-dependent increases in TM7 and Spirochaetes and rises in Fusobacterium and Tannerella, consistent with the progression model from health to periodontitis. The increase in Saccharibacteria (TM7), detectable via NGS, may influence community structure and could be partly age-associated at T2. Comparisons with prior literature suggest that fixed appliances, more than removable ones, promote anaerobe enrichment, and that observed taxa overlap with reported gingivitis-associated mediators of health-to-periodontitis transitions. Clinically, the results underscore the importance of periodontal monitoring during orthodontic treatment, as microbial dysbiosis can precede overt gingival inflammation.

Orthodontic treatment with fixed appliances alters the oral microbiome in plaque and saliva, characterized by increased anaerobes and periodontal pathogens and decreased commensal aerobes/facultative anaerobes. The supragingival microbiome particularly reflects a transitional state between health and periodontitis during treatment. Continued monitoring and preventive care are warranted during therapy, and future work integrating metagenomics/metabolomics with clinical parameters and control groups is needed to clarify functional implications and reversibility after appliance removal.

• Methodological scope: 16S rRNA sequencing provides taxonomic, not functional, insights; interspecies metabolic interactions were not assessed.
• Study design: No non-orthodontic control group; uneven sample sizes across saliva time points; limited power for some comparisons.
• Time point differences: T2 occurred at appliance removal (average 40 months) with a different average age, potentially confounding age-related microbiome changes (e.g., Saccharibacteria).
• Clinical correlation: No significant clinical gingival changes were documented; comprehensive periodontal parameters were not systematically monitored, limiting clinicomicrobial correlation.
• Sampling scope: Focused on supragingival plaque and saliva; subgingival niches, which are critical in periodontitis, were not sampled.