Four-Week Evaluation of the Interaction Pattern Among Saccharibacteria, Nitrate-Reducing Bacteria, and Periodontopathogens in Orthodontic Miniscrew Implants

<b>Background/Objective</b>: Orthodontic mini-implants (MI) create new niches that may alter the oral microbiota and modulate host immune responses. While clinical inflammation is not always evident, microbial and molecular changes may precede visible signs of peri-implant infection. This study investigated microbial shifts and inflammatory responses following MI placement, with a focus on Saccharibacteria, nitrate-reducing bacteria (NRB), and periodontopathogens. <b>Methods</b>: Saliva and peri mini-implant crevicular fluid (PMICF) samples were collected from eight orthodontic patients at baseline (T0), one week (T1), and one month (T2) after mini-implant placement. DNA was extracted from each saliva and PMICF sample and pooled across the eight patients for each time point. The pooled DNA were then subjected to 16S rRNA gene sequencing using the Oxford Nanopore MinION platform. Statistical analysis was performed to determine shifts in bacterial abundance, diversity, and co-occurrence patterns across the different sample types (saliva vs. PMICF) and time points. <b>Results</b>: Alpha diversity decreased in PMICF at T2, while it remained stable in saliva samples. Periodontopathogens (<i>Porphyromonas gingivalis</i>, <i>Treponema denticola</i>, <i>Fusobacterium nucleatum</i>) increased in PMICF at T2, while NRB and Saccharibacteria, along with a representative host bacterium (<i>Schaalia odontolytica</i>), remained relatively stable. Co-occurrence analysis showed antagonistic relationships between Saccahribacteria/NRB and periodontopathogens. IL-6 significantly decreased from T1 to T2, while CRP showed a non-significant downward trend. The expression of nitrate reductase genes <i>narG</i> and <i>napA</i> remained stable across time intervals. <b>Conclusions</b>: Despite no clinical inflammation, MI placement led to localized microbial shift and mild inflammatory responses. NRB and Saccharibacteria's stability and antagonistic relationship to periodontopathogens may indicate that they could be involved in maintaining microbial homeostasis. These findings highlight possible early biomarkers and ecological strategies to support oral health in MI patients.