Microbiome in adult severe caries and cross-kingdom biofilms validation

Adult severe caries (ASC) is a form of rampant caries that develops in adulthood, causing severe impairment of oral function and reducing quality of life. However, the pathogenic mechanism of ASC remains unclear. This study aimed to identify the core microbiota in patients with ASC and preliminarily investigate the microbial interactions and pathogenicity of key ASC-associated core microorganisms.

Saliva samples were collected from 7 adult patients with severe caries and 6 caries-free volunteers for metagenomic analysis. Based on microbiome profiling results, an in vitro cross-kingdom biofilm model composed of Streptococcus mutans (S. mutans), Candida albicans (C. albicans) and Veillonella parvula (V. parvula) was established to simulate a high caries-risk microenvironment. Scanning electron microscopy (SEM), crystal violet (CV) staining, and live/dead bacterial staining were used to evaluate biofilm formation. Acid production assays, acid stress challenge tests, confocal laser scanning microscopy (CLSM) and qRT-PCR were performed to analyze the acidogenicity and synthesis of extracellular polysaccharides (EPS). Additionally, atomic force microscopy (AFM) was used to assess the surface roughness of demineralized dentin slices.

Metagenomic analysis revealed significant enrichment of C. albicans and V. parvula in the saliva of patients with high caries susceptibility. The in vitro cultured cross-kingdom biofilms exhibited enhanced growth and EPS synthesis compared with single-species S. mutans biofilms. Moreover, cross-kingdom biofilms significantly increased surface roughness of demineralized samples, with a stronger effect than single- and dual-species biofilms.

Colonization by C. albicans and V. parvula increases biofilm biomass, enhances microbial survival under stress, and elevates biofilm virulence, which induces demineralization of dentin slices in vitro.

This study demonstrates that the interspecies interactions among caries-related microorganisms in ASC patients confer enhanced virulence and cariogenicity, providing novel insights for the investigation and prevention of high caries susceptibility.