IADR Abstract Archives
Examining the Role of Extracellular DNA in Oral Biofilm Formation
Objectives: Extracellular DNA (eDNA) is an integral component of dental plaque-associated biofilm, with potential to be targeted for the therapeutic manipulation of plaque development to promote oral health. However, this approach is currently hindered by a lack of reliable methods for the quantification of biofilm eDNA networks in situ. Here, we detail a high-throughput imaging analysis technique to address this gap. This is applied to characterisation of eDNA networks within Streptococcus gordonii mono- and poly-microbial biofilms and of the potential impact of S. gordonii DNase, SsnA.
Methods: S. gordonii wild-type or ssnA monospecies biofilms or polymicrobial biofilms with addition of P. gingivalis, F. nucleatum ± Prevotella nigrescens, Actinomyces oris and Veillonella dispar were formed on salivary pellicle in 24-well plates over 5 h. Networks of eDNA were immunolabeled, visualised by widefield microscopy and quantified using the modular image analysis plugin on Fiji. Biofilm and eDNA composition were assessed via species-specific qPCR. Biomass was quantified by crystal violet staining.
Results: S. gordonii produced significant eDNA networks during early biofilm formation. SsnA modulated these eDNA networks and this activity was impaired by glucose. The capacity to produce eDNA networks was seen for several oral microbes and was found to be species-specific. Overall eDNA network levels were significantly influenced by biofilm community composition. Within polymicrobial biofilms, there was evidence that SsnA could modulate biofilm community development and potentially affect eDNA networks produced by other bacterial species.
Conclusions: These studies have demonstrated that a microscopy-based approach can be used to assess the interplay of eDNA networks and DNase activity in situ within oral biofilm communities. This approach can now be used to better understand these dynamics within dental plaque and how they may be exploited to promote the maintenance of an oral community predisposed to health.
Methods: S. gordonii wild-type or ssnA monospecies biofilms or polymicrobial biofilms with addition of P. gingivalis, F. nucleatum ± Prevotella nigrescens, Actinomyces oris and Veillonella dispar were formed on salivary pellicle in 24-well plates over 5 h. Networks of eDNA were immunolabeled, visualised by widefield microscopy and quantified using the modular image analysis plugin on Fiji. Biofilm and eDNA composition were assessed via species-specific qPCR. Biomass was quantified by crystal violet staining.
Results: S. gordonii produced significant eDNA networks during early biofilm formation. SsnA modulated these eDNA networks and this activity was impaired by glucose. The capacity to produce eDNA networks was seen for several oral microbes and was found to be species-specific. Overall eDNA network levels were significantly influenced by biofilm community composition. Within polymicrobial biofilms, there was evidence that SsnA could modulate biofilm community development and potentially affect eDNA networks produced by other bacterial species.
Conclusions: These studies have demonstrated that a microscopy-based approach can be used to assess the interplay of eDNA networks and DNase activity in situ within oral biofilm communities. This approach can now be used to better understand these dynamics within dental plaque and how they may be exploited to promote the maintenance of an oral community predisposed to health.