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.

Background: Despite previous anatomical studies on PMFMs, detailed classification and comprehensive measurements remain limited, and the association between crown and root morphology has not been systematically investigated. This study aims to reconstruct three-dimensional anatomical models of PMFMs from intact CBCT images obtained from children in Beijing, China, to characterize variations in crown and root morphology and their relationships, and to conduct detailed quantitative and statistical analyses based on refined classifications. Methods: Three-dimensional (3D) anatomical models of 67 intact PMFMs were reconstructed from CBCT scans using a semi-automatic threshold-based region segmentation method. Key morphological parameters of the crown and root were measured. Samples were primarily classified based on the ratio of mesiodistal to buccolingual crown diameter (R) and the number of roots (N), respectively, and their association was explored. A secondary classification was conducted by incorporating the number of root canals (n). For subgroups with sufficient sample sizes, detailed morphological parameters were measured and compared among types. Results: Two crown variants were identified. R > 1 was observed in 59 teeth (88.1%), with a mean value of 1.11 ± 0.06, while R < 1 was observed in 8 teeth (11.9%), with a mean of 0.94 ± 0.03. A significant difference was found between the R values ( p < 0.05). Two root number variants were identified. Fifty-eight teeth (86.6%) had two mesiodistally aligned roots, all with R > 1. Nine teeth (13.4%) had three roots, in which 8 (11.9%) had R < 1; and 1 (1.5%) had R > 1. A significant negative correlation was found between R and N ( r = -0.61, p < 0.05), and ROC curve analysis confirmed that R = 1.00 was the optimal cutoff for distinguishing two-rooted from three-rooted PMFMs (AUC = 1.000). Besides, a secondary classification identified seven types, among which the mesiobuccal canal was consistently the longest ( p < 0.05). Conclusion: PMFMs exhibit considerable morphological variations. The ratio of mesiodistal to buccolingual crown diameter (R) is a useful predictor of root number, and the mesiobuccal canal is significantly longer than the other canals. These features may aid clinicians in preoperative planning.