Exopolysaccharides Matrix Degradation Enhances Antimicrobial Peptide Efficacy Against Biofilms

Objectives: Cariogenic biofilms are characterized by bacterial cells enmeshed in an extracellular matrix rich in exopolysaccharides (EPS). Treating cariogenic oral biofilms is challenging as antimicrobials often fail to kill the pathogens protected by the EPS-rich matrix. We hypothesize that EPS-degrading enzymes such as dextranase can facilitate bacterial killing by an antimicrobial peptide (AMP) to enhance biofilm disruption efficacy.
Methods: A well-characterized EPS-matrix producing oral pathogen, Streptococcus mutans UA159, was used to form biofilms on saliva-coated hydroxyapatite disc surfaces. Alexa Fluor 647-tagged dextran conjugate was used to label the EPS-matrix, while live/dead (Syto 9/PI) fluorescent stains were used to evaluate cell viability within intact 3D biofilm architecture. Using time-lapse high-resolution confocal microscopy and computational analysis, we examined the spatiotemporal changes of the matrix structure and in situ cell viability following biofilm treatment with 1) dextranase, 2) AMP, or 3) AMP+dextranase for up to 30 minutes.
Results: We first optimized the concentration of dextranase required for EPS-matrix disruption without affecting the cell viability. Dextranase (50-100U) was capable of effectively digesting the EPS, reducing both the matrix density and volume throughout the biofilm 3D architecture without antibacterial activity. Quantitative fluorescence imaging analyses showed that >60% of the EPS-matrix was digested by dextranase, resulting in drastic alterations in the 3D structure of the matrix. On the other hand, AMP treatments were incapable of disrupting the EPS-matrix, while killing the bacterial cells located only in the peripheral regions of the biofilm. Excitingly, the combination of dextranase and AMP degraded the EPS-matrix and at the same time enhanced the antimicrobial efficacy by 3-fold.
Conclusions: Our findings suggest a potential synergistic approach using EPS-degrading enzymes combined with AMPs to disrupt the matrix structure and thereby facilitate the access of antimicrobials into biofilms to augment killing efficacy of the embedded bacteria.