Effect of KappaZinc^TM on Streptococcus mutans biofilms

The Kappacin^TM antimicrobial peptide is derived from bovine milk protein and has in vitro antibacterial activity against both Gram-positive and Gram-negative oral bacteria. Kappacin^TM binds two zinc ions to form the KappaZinc^TM antimicrobial complex that has enhanced antibacterial activity, especially at neutral pH. Streptococcus mutans, the major aetiological agent of human dental caries, forms biofilms in flow cells that can be visualized and quantitated by confocal laser scanning microscopy (CLSM). Objective: To investigate the effect of the KappaZinc^TM antimicrobial complex on S. mutans biofilms using CLSM. Method: A 3 channel flow cell system was inoculated with S. mutans and 25% artificial salivary medium (ASM) fed into the system at a flow rate of 0.2 ml/min. After 16 h, either KappaZinc^TM (10mg/ml Kappacin^TM with 20mM ZnCl₂) or sterile water (positive control) was injected into the system and incubated for 10 min followed by ASM addition at 0.2 ml/min for a further 10 min then LIVE/DEAD BacLight staining. CLSM of the bacterial biofilms was carried out on a Zeiss Meta 510. Horizontal (xy) optodigital sections, each 2 µm thick over the entire thickness of the biofilm (z) were imaged using a 63X objective. Image and volumetric analyses were carried out using COMSTAT software. Results: The KappaZinc^TM complex had a dramatic impact on S. mutans biofilms, reducing total biomass from 9.16 ± 2.14 µm3/µm2 to 1.76 ± 0.53 µm3/µm2, a decrease of 81%. The average thickness of the biofilm also decreased by 80% from 11.88 ± 1.81 µm to 2.30 ± 0.78 µm. Conclusion: KappaZinc^TM disrupts the S. mutans biofilm structure, thus reducing its biomass, indicating its efficacy against oral streptococcal biofilms for future oral health applications.