Correlative imaging of extracellular DNA and pH at the microscale in the extracellular matrix of dental biofilms

Extracellular DNA (eDNA) is a ubiquitous component of the extracellular matrix of bacterial biofilms, which has been proposed to act as a proton trap. Locally trapped protons will influence the pH of biofilms on the microscale; therefore, we developed a method for correlative imaging of both pH and eDNA microarchitecture in biofilms. We used multispecies dental biofilms inoculated from pooled saliva as a complex biofilm model where it is known that the local pH and matrix composition can vary substantially. We combined correlative imaging of pH and eDNA with analysis of biofilm composition by 16S rRNA sequencing to investigate the association between local pH and abundance of eDNA, and whether biofilm age, sucrose supplementation during biofilm growth, or microbial composition affected this association. Biofilms grown in the presence of sucrose showed significantly lower pH and higher abundance of eDNA than biofilms grown in the absence of sucrose, although pH and the abundance of eDNA were not correlated at the microscale in similarly treated biofilms. The effect was more pronounced in mature four-day old biofilms compared to younger two-day old biofilms. The abundance of streptococci and lactobacilli increased in more acidic biofilms, and we propose that increases in polysaccharides produced in acidogenic and aciduric biofilms are responsible for the increased abundance of eDNA in the biofilm matrix, which points towards complex interactions linking the biofilm matrix composition to dental caries development.