Combined DNase and Proteinase Treatment Interferes with Composition and Structural Integrity of Multispecies Oral Biofilms

Modification of oral biofilms adhering to dental hard tissues could lead to new treatment approaches in cariology and periodontology. In this study the impact of DNase I and/or proteinase K on the formation of a simulated supragingival biofilm was investigated in vitro. Six-species biofilms were grown anaerobically in the presence of DNase I and proteinase K. After 64 h biofilms were either harvested and quantified by culture analysis or proceeded to staining followed by confocal laser scanning microscopy. Microbial cells were stained using DNA-dyes or fluorescent in situ hybridization. Exopolysaccharides, eDNA and exoproteins were stained with Calcofluor, anti-DNA-antibody, and Sypro(TM) Ruby, respectively. Overall, results showed that neither DNase I nor proteinase K had an impact on total colony-forming units (CFUs) compared to the control without enzymes. However, DNase I significantly suppressed the growth of Actinomyces oris, Fusobacterium nucleatum, Streptococcus mutans, Streptococcus oralis and Candida albicans. Proteinase K treatment induced significant increase in S. mutans and S. oralis CFUs (p < 0.001), whereas C. albicans and V. dispar showed lower CFUs compared to the control. Interestingly, confocal images visualized the biofilm degradation caused by DNase I and proteinase K. Thus, enzymatic treatment should be combined with conventional antimicrobial agents aiming at both bactericidal effectiveness and biofilm dispersal.