Antimicrobial and Potent Anti‐Biofilm Properties of Rationally Designed <i>α</i>‐Helix Antimicrobial Peptides

The antimicrobial resistance (AMR) crisis represents a significant global threat. Unlike traditional antibiotics, antimicrobial peptides offer a promising pathway because of their primary mechanisms. This study aimed to evaluate and rationally design novel AMPs based on tobacco nectar's AMP (Pep 6) to combat antibiotic resistance issues. Substitution and truncation of some amino acids were applied. Four peptides, KF19, KF16, LK16, and LR16, were designed with enhanced net charge hydrophobicity. They were evaluated for their in vitro antibacterial activity. However, only promising AMPs were further evaluated for their hemolytic activity, time-killing kinetics, mode of action, and anti-biofilm properties. The results showed that only KF19 and LR16 have potent activity against Staphylococcus aureus ATCC25923 and resistant isolates with MIC values from 7.81 to 15.62 μg/mL. Hemolysis ratios were 2.38% and 2.24% at 125 μg/mL for KF19 and LR16, respectively. Both peptides were able to kill S. aureus ATCC25923 within 2 h. SEM results showed their ability to target the cell membrane. Both peptides destroyed the S. aureus biofilms significantly at 62.5 and 125 μg/mL (**p < 0.01, ***p < 0.001, ****p < 0.0001). This study supported rational design in developing new antibacterial agents and demonstrated the therapeutic potency of novel peptides that could solve the resistance issues.