Biological Characterization of Antimicrobial Peptides Isolated from the Anuran Pithecopus rohdei

Antimicrobial resistance is a global public health threat. Antibiotic misuse promotes the selection and spread of resistant bacteria. The ESKAPEE group comprises a set of clinically relevant pathogens whose acronym is derived from their scientific names: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli. This study compared the antibacterial activity, cytotoxicity, and hemolytic activity of Dermaseptin-2PR (DRS-2PR) and Dermaseptin-3PR (DRS-3PR), two peptides previously identified in the skin secretion of Pithecopus rohdei. Synthetic peptides were tested by broth microdilution against four antibiotic-resistant clinical isolates. Cell viability was assessed by MTT after 24 h of exposure in HeLa cervical carcinoma cells and HaCaT human keratinocytes. Hemolytic activity was evaluated using human erythrocytes. DRS-3PR was at least as active as DRS-2PR against every strain tested. The minimum inhibitory concentrations (MICs) for DRS-2PR and DRS-3PR, respectively, were 4 and 2 µM against carbapenemase-producing Klebsiella pneumoniae, 2 µM for both peptides against carbapenem-resistant Acinetobacter baumannii, 16 and 2 µM against methicillin-resistant Staphylococcus aureus, and >128 and 16 µM against multidrug-resistant Pseudomonas aeruginosa. The cytotoxicity assay demonstrated that both peptides exerted cytotoxic effects on the two cell models; however, neither showed selectivity for tumor cells. The IC₅₀ values for DRS-2PR were 31.5 µM and 24.1 µM for HeLa and HaCaT cells, respectively. For DRS-3PR, the IC₅₀ values were 14.7 µM and 8.1 µM for HeLa and HaCaT cells, respectively. The hemolysis assay showed that both peptides exhibited low hemolytic activity at their respective MICs. DRS-3PR displayed greater hemolytic activity than DRS-2PR, with HC₅₀ values of >128 µM and 118.1 µM for DRS-2PR and DRS-3PR, respectively. DRS-3PR had lower MICs against three of the four resistant clinical isolates and showed greater cytotoxicity and hemolytic activity than DRS-2PR. For both peptides, the IC₅₀ was lower in HaCaT than in HeLa cells, indicating a lack of tumor selectivity under the conditions tested.