Mixed aerobic-anaerobic incubation conditions induce proteolytic activity from in vitro salivary biofilms

Oral biofilms have not been studied using both metabolome and protein profiling concurrently. Bacteria produce proteases that lead to degradation of functional salivary proteins. The novel protocol described here allows for complete characterisation of in vitro oral biofilms, including proteolytic, metabolic, and microbiome analysis. Biofilms were grown on hydroxyapatite discs from whole mouth saliva, using sterilised saliva as a growth-medium, in different growth environments. Salivary protein degradation was assessed from spent saliva growth-medium using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and metabolic activity by nuclear magnetic resonance (NMR). Discs were assessed for depth and coverage of biofilms by confocal laser scanning microscopy (CLSM), and biofilms were collected at the end of the experiment for 16S rRNA gene sequence analysis. There was a significant difference in biofilm viability, salivary protein degradation, and metabolites identified between biofilms grown aerobically and biofilms exposed to an anaerobic environment. Bacterial 16S rRNA gene sequencing showed the predominant genus in the 7-day aerobic biofilms was Streptococcus, in aerobic-anaerobic and anaerobic 7-day biofilms Porphyromonas, and in aerobic-anaerobic and anaerobic 13-day biofilms Fusobacterium. This data suggests new growth requirements and capabilities for analysing salivary biofilms in vitro, which can be used to benefit future research into oral bacterial biofilms.