Eradication of Dual-Species Cariogenic Biofilms by Punicalin and Punicalagin: Microbiological and Gene Expression Analyses

Abstract

This in vitro study evaluated the biofilm eradication potential of two pomegranate-derived compounds, punicalin (PUN) and punicalagin (PUG), against dual-species biofilms comprising Streptococcus mutans and Streptococcus gordonii.  Antimicrobial activity was assessed through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, while the minimum biofilm eradication concentration (MBEC) was determined using the crystal violet assay. At 100 µg/mL (MBEC₅₀), PUN and PUG were further evaluated in a glass beads biofilm model for their effects on bacterial viability, biomass, biofilm morphology (via SEM) and gene expression (via RT-qPCR). Both compounds significantly reduced biofilm biomass and S. mutans population, indicating selective targeting of the cariogenic species. PUN exerted its effects predominantly through downregulation of gtf and gbp genes, which are involved in EPS production and biofilm stability. In contrast, PUG demonstrated greater biofilm disruption and S. mutans reduction, accompanied by significant upregulation of sodA and lytF, suggesting a dual mechanism involving both oxidative stress and autolysin-mediated dispersal. SEM analysis further supported these findings, showing more extensive matrix disintegration in PUG-treated biofilms. These results highlight the therapeutic potential of PUN and PUG as selective antibiofilm agents for managing cariogenic biofilms, with PUG exhibiting both intrinsic and extrinsic modes of action.