Abstract
The exopolysaccharides (EPS) produced by Streptococcus mutans-derived glucosyltransferases (Gtfs) are essential virulence factors associated with the initiation of cariogenic biofilms. EPS forms the core of the biofilm matrix-scaffold, providing mechanical stability while facilitating the creation of localized acidic microenvironments. Cranberry flavonoids, such as A-type proanthocyanidins (PACs) and myricetin, have been shown to inhibit the activity of Gtfs and EPS-mediated bacterial adhesion without killing the organisms. Here, we investigated whether a combination of cranberry flavonoids disrupts EPS accumulation and S. mutans survival using a mixed-species biofilm model under cariogenic conditions. We also assessed the impact of cranberry flavonoids on mechanical stability and the in situ pH at the biofilm-apatite interface. Topical application of an optimized combination of PACs oligomers (100–300 μM) with myricetin (2 mM) twice daily was used to simulate treatment regimen experienced clinically. Treatments with cranberry flavonoids effectively reduced the insoluble EPS content (>80% reduction vs. vehicle-control; p<0.001), while hindering S. mutans outgrowth within mixed-species biofilms. As a result, the 3D architecture of cranberry-treated biofilms was severely compromised, showing a defective EPS-matrix and failure to develop microcolonies on the saliva-coated hydroxyapatite (sHA) surface. Furthermore, topical applications of cranberry flavonoids significantly weaken the mechanical stability of the biofilms; nearly 90% of the biofilm was removed from sHA surface after exposure to a shear stress of 0.449 N/m2 (vs. 36% removal in vehicle-treated biofilms). Importantly, in situ pH measurements in cranberry-treated biofilms showed significantly higher pH values (5.2 ± 0.1) at the biofilm-apatite interface vs. vehicle-treated biofilms (4.6 ± 0.1). Altogether, the data provide important insights on how cranberry flavonoids treatments modulate virulence properties by disrupting the biochemical and ecological changes associated with cariogenic biofilm development, which could lead to new alternative or adjunctive antibiofilm/anticaries chemotherapeutic formulations.
Highlights
Biofilms are the prevailing microbial lifestyle in natural niches, causing many infectious diseases in humans [1]
Preliminary experiments have shown that the combination of these flavonoids (CranFlav) is more effective in disrupting EPS synthesis by GtfB and S. mutans accumulation within biofilms than either compound alone (S2 Fig)
Chemotherapeutic agents that interfere with the biofilm assembly process by virulent species and compromise its physical integrity under topical exposure regimen could effectively control pathogenic biofilms without indiscriminately killing commensal bacteria in the mouth [25]
Summary
Biofilms are the prevailing microbial lifestyle in natural niches, causing many infectious diseases in humans [1]. Cariogenic biofilms develop as pathogens accumulate on tooth surfaces, forming highly structured microbial communities that are tightly adherent and enmeshed in an extracellular matrix [3]. Exopolysaccharides (EPS), e.g. glucans, are key components in the cariogenic biofilm matrix, and are recognized virulence factors involved in the pathogenesis of dental caries [4,5,6]. Within the complex oral microbiome, Streptococcus mutans is not always the most abundant organism. This bacterium can rapidly orchestrate the formation of cariogenic biofilms when exposed to sucrose via EPS synthesis by S. mutans-derived glucosyltransferases (Gtfs) present on the tooth-pellicle and bacterial surfaces [4]. Sugars are fermented by bacteria within the biofilm matrix, creating highly acidic microenvironments [7,8,9,10]
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