Selective antivirulence and antibiofilm effects of pomegranate-derived compounds punicalagin and punicalin in a dual-species experimental oral biofilm model

Dental caries is a biofilm-mediated disease resulting from an ecological imbalance within the oral microbiome, characterised by increased dominance of acidogenic and extracellular polysaccharide producing bacteria, particularly Streptococcus mutans (S. mutans). Although conventional antimicrobial agents such as chlorhexidine remain widely used for caries control, their non-selective activity may disrupt the commensal oral microbiota and compromise long-term ecological stability. Therefore, strategies that selectively modulate cariogenic biofilms without eliminating health-associated species are increasingly recognised as a priority in contemporary ecology-based caries management. Accordingly, this thesis aimed to systematically evaluate pomegranate -derived compounds (PDCs) as selective antibiofilm agents capable of attenuating cariogenic biofilms while preserving health-associated oral bacteria. A structured two-phase experimental framework was employed. In Phase I, seven PDCs, namely, punicalagin (PUG), punicalin (PUN), corilagin, ellagic acid, gallocatechin, kaempferol glycoside, and rutin, were comparatively screened using antimicrobial assays, biofilm inhibition and eradication assays, acidogenicity assessment, and preliminary toxicity evaluation. Among these candidates, PUG and PUN emerged as the leading candidates, demonstrating effective inhibition of early biofilm formation at sub-minimum inhibitory concentrations, attenuation of S. mutans virulence traits, and favourable safety profiles in preliminary toxicity screening. Phase II focused on mechanistic characterisation using optimised dual-species biofilm models comprising S. mutans and the commensal Streptococcus gordonii (S. gordonii). Biofilm inhibition assays revealed selective suppression of S. mutans biofilm biomass and viability, while largely preserving the viability of S. gordonii. Transcriptional analyses of S. mutans demonstrated significant downregulation of genes associated with extracellular polysaccharide synthesis and adhesion (gtfB, gtfC, gbpB, gbpD), quorum sensing (comDE), and biofilm maturation (brpA), with minimal effects on core glycolytic energy metabolism (ldh, atpD). In contrast, S. gordonii exhibited limited transcriptional perturbation, with only modest, transient changes in adhesion- and biofilm-associated genes (gtfG, abpA, cshA). Biofilm eradication assays targeting preformed dual-species biofilms demonstrated time-dependent destabilisation of extracellular polysaccharide rich biofilm structures without extensive bacterial killing, supported by upregulation of autolysis- and cell wall remodelling–associated genes (atlA, lytF) in S. mutans. Scanning electron microscopy further corroborated progressive structural destabilisation of biofilms following compound treatment, particularly with PUG. Collectively, these findings demonstrate that PUG and PUN selectively modulate biofilm behaviour within a dual-species oral biofilm model through coordinated antibiofilm and antivirulence mechanisms, reducing S. mutans dominance while preserving commensal species. This work provides evidence supporting the development of plant-derived antivirulence strategies aligned with ecology-based approaches for sustainable caries prevention.