Evaluation of the Synergistic Effect of LL-37 and Oncorhyncin II Recombinant Proteins on Staphylococcus aureus Under In Vitro Conditions

Background: The treatment of Staphylococcus aureus infections has become a public health crisis due to the extensive development of antimicrobial resistance. Antimicrobial peptides (AMPs) have been introduced as promising naturally-derived antimicrobial alternatives to antibiotics. LL-37 and oncorhyncin II are 2 AMPs with notable proven antibacterial effects. Objectives: This study aimed to produce recombinant LL-37 and oncorhyncin II and investigate their synergistic effects on S. aureus (ATCC25923). Methods: The synthetic genes of LL-37 and oncorhyncin II were individually ligated into the pET32a expression vector. Transformed pET32a was introduced into Escherichia coli BL21 as an expression host. The protein expression and purification steps were optimized, and the biological effectiveness of the peptides was evaluated by assessing the minimum inhibitory concentration (MIC), time-kill, and growth kinetic tests against S. aureus. Results: The MIC assay confirmed the effective antibacterial performances of LL-37 and oncorhyncin II against S. aureus at 30.6 and 47.93 µg/mL, respectively. The peptides’ synergistic activity was validated by the checkerboard method. A combination of LL-37 and oncorhyncin II at 2 × MIC showed a sharp decline of the viable cells with over 3-time reductions in log 10 colony-forming units (CFU)/mL within the first 5 hours. The growth kinetic results confirmed the high effectiveness of the peptides’ combination in eliminating the bacterial inoculum turbidity by 50% reduction during the first hour of exposure. Conclusions: The produced recombinant LL-37 and oncorhyncin II showed effective antimicrobial function against S. aureus. The synergistic performance of the peptides was repeatedly confirmed through checkerboard, time-kill, and growth kinetic assays.