[Design, screening and antibacterial activity evaluation of the novel antibacterial peptide KR-1]

To design novel antimicrobial peptides with high activity and low toxicity and evaluate their effect against Streptococcus mutans and other oral bacteria for prevention and treatment of dental caries.

We synthesized two antimicrobial peptides (KR-1 and KR-2) using Dhvar4 (a histatins5 mimic) as the template. The antimicrobial peptides with high activity and low toxicity were screened using minimal inhibitory concentration (MIC) test, hemolysis test, and CCK-8 assay. Streptococcus mutans biofilms cultured in 96-well plates were divided into experimental group (KR-1) and positive control group (CHX) and treated with concentration gradients (0.6×, 0.8×, 1× and 2× MICs) of KR-1 and CHX, respectively. Crystal violet staining was used for quantitative analysis of the changes of the biofilms after the treatments. The structural changes of the biofilms were observed with laser confocal microscopy after KR-1 treatment at 10 × MIC. The antimicrobial activity of KR-1 against oral Streptococcus was analyzed based on the time required for sterilization after KR-1 treatment.

The MIC of KR-1 and KR-2 for S. mutans was 3.2 μmol/L and 12.8 μmol/L, respectively. Under the effective concentration, KR-1 and KR-2 resulted in hemolysis rates of 0.35% and 48.8% in rabbit red blood cells and lowered the survival rates of gingival fibroblasts to 88.7% and 21.94%, respectively. KR-1 treatment significantly reduced biofilm formation with a minimum biofilm inhibition concentration (MBIC₅₀) lower than 1.92 μmol/L, and showed an even stronger antimicrobial than CHX at the concentration of 2.56 μmol/L (P=0.001). Confocal laser scanning microscopy revealed that the biofilm structure became loosened after KR-1 treatment, which was capable of killing about 90% of the bacteria within 5 min.

The antimicrobial peptide KR-1 has a stronger antibacterial activity and a low toxicity with a good inhibitory effect against S. mutans biofilm.