Synergistic antibacterial activity of human salivary peptide Histatin-5 and Phytosphingosine against Streptococcus mutans biofilm

Objective or Background: Dental caries is still one of the most significant dental problems worldwide, with prevalence rates up to 90% among children and adults. Cariogenic bacteria, especially Streptococcus mutans, is the primary microorganism involved in the pathogenesis through carbohydrate metabolism and biofilm formation, which are challenging to eradicate. Histatin-5 (HST-5), a human salivary antimicrobial peptide, has demonstrated antimicrobial activity against various fungal and bacterial pathogens. Phytosphingosine (PHS), an endogenous bioactive sphingolipid found in fungi, plants, and humans, also shows antimicrobial properties. This study aimed to evaluate the killing activity of HST-5 alone and in combination with PHS against S. mutans under biofilm-stimulating conditions. Materials and Methods: Antimicrobial activity against a planktonic culture of S. mutans was evaluated using a time-kill assay, and biofilm-forming capacity was confirmed by crystal violet staining assay. The killing ability against 24h pre-formed biofilm was determined using Transferable Solid Phase (TSP) pin lid model. Synergistic activity between HST-5 and PHS was evaluated using the checkerboard technique. Additionally, the cytotoxicity of the tested agent on human gingival fibroblast cells (hGFs) was assessed after 1 h of incubation using an MTT assay. Results: A time-kill assay revealed that both HST-5 and PHS exhibit time- and concentration-dependent activity against the planktonic form of S. mutans. PHS achieved over 90% killing activity within 15 min at 5 μg/ml, whereas HST-5 required 30 min to reach 90% killing at 20 μM. The biofilm formation capacity of S. mutans was confirmed. The inhibitory concentrations (IC50) of HST-5 and PHS against S. mutans biofilm were 25 μM and 13.5 μg/ml, respectively. A synergistic interaction between HST-5 and PHS, with IC50 values reduced by 8-fold and 16-fold, respectively. No cytotoxic effects were observed in hGFs cells at the concentration of the synergistic interaction. Conclusions: Therefore, the combination of HST-5 and PHS may enhance the effectiveness of anti-infective agents against S. mutans biofilm, potentially preventing the development of dental caries.

Dental caries is still one of the most significant dental problems worldwide, with prevalence rates up to 90% among children and adults. Cariogenic bacteria, especially Streptococcus mutans, is the primary microorganism involved in the pathogenesis through carbohydrate metabolism and biofilm formation, which are challenging to eradicate. Traditional preventions like brushing, while helpful, struggle to fully eradicate this resilient bacteria’s biofilm, as once it is formed, simple brushing is not enough.