The differences in microbiota between periodontitis and health have been extensively studied; however, knowledge about how the microbiota shifts from shallow to deep periodontal pockets remains limited despite its clinical importance in disease progres-sion and management. Patients diagnosed with stage III periodontitis commonly pre-sent varied probing depths (PD) within the same oral cavity, reflecting localized disease severity. This study aims to analyze the microbiome of subgingival plaques at various PDs in periodontitis patients. Subgingival plaques were collected from sixteen healthy subjects (health group) and periodontal pockets of sixteen stage III periodontitis pa-tients (PD 0–3 mm, PD 4–5 mm and PD 6–9 mm groups). A total of 64 subgingival plaque samples underwent 16S rRNA gene sequencing. The PD 6–9 mm group exhib-ited significantly higher alpha diversity than the health group, and distinct subgingival microbial community structures were observed in periodontitis patients, regardless of probing depth. The relative abundance of specific genera differed notably between health and periodontitis states; Corynebacterium and Cardiobacterium decreased, whereas Schaalia increased in shallow pockets (PD 0–3 mm) of periodontitis relative to the health group. Co-occurrence network analysis on the species level revealed that the PD 4–5 mm group had the most complex interspecies interactions, followed by the PD 6–9 mm and PD 0–3 mm groups. These findings indicate significant variations in mi-crobial diversity, composition, and interspecies interactions associated with periodon-tal health and periodontitis severity, highlighting their potential relevance for clinical diagnosis and targeted therapeutic strategies.

Recurrence remains a significant challenge following the treatment of Severe Early Childhood Caries (S-ECC). This study aimed to identify candidate recurrence-related biomarkers for S-ECC and elucidate their potential pathogenic mechanisms. Through metagenomic sequencing of supragingival plaque from 32 children at one month post-treatment, we identified Leptotrichia hofstadii as one of the potential biomarkers for S-ECC recurrence (AUC = 0.8438 for the sequencing set and AUC = 0.75 for the validation set). In vitro dual-species biofilm assays using crystal violet staining and Confocal Laser Scanning Microscopy (CLSM) demonstrated that L. hofstadii promotes early-stage S. mutans colonization and extracellular polysaccharide (EPS) formation through contact-dependent synergistic interactions. Scanning electron microscopy revealed that L. hofstadii may function as a spatial scaffold within dual-species biofilm. Furthermore, this synergy significantly accelerates environmental acidification, leading to earlier attainment of the critical demineralization threshold (pH 5.5). At the transcriptional level, carbohydrate metabolism-related pathways were upregulated in dual-species biofilm, including starch and sucrose metabolism, PTS and ABC transporters. Additionally, the fruA gene, which degrades fructan in EPS was downregulated in the dual-species biofilm compared with S. mutans monoculture. These findings suggest that L. hofstadii facilitates a cariogenic microenvironment by enhancing the metabolic activity of S. mutans biofilms. Collectively, this study identifies L. hofstadii as a potential biomarker for S-ECC recurrence prediction and provides preliminary insights into possible interspecies mechanisms, offering valuable clues for future research into targeted preventive strategies.