Identification of Leptotrichia hofstadii as a Post-Treatment Recurrence Biomarker in Severe Early Childhood Caries.

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.