Comparison of antimicrobial and cytotoxicity profiles of skin secretions from endemic polypedates cruciger and cosmopolitan Duttaphrynus Melanostictus

Abstract

Antimicrobial peptides (AMPs) in amphibian skin secretions are being explored as potential new antibiotics. Cytotoxicity to mammalian cells is a major challenge in their clinical usage. This study explored antimicrobial properties of skin secretions from endemic Polypedates cruciger (common hourglass treefrog) compared to cosmopolitan Duttaphrynus melanostictus (Asian common toad) with known AMPs. Secretions were obtained from specimens collected at University of Peradeniya premises by electrical stimulation (5V, DC) and washing skin with deionized water. Secretions were stabilized with glacial acetic acid (50:1 v/v) and sterile filtered. Activity against Staphylococcus aureus, multidrug resistant (MDR) Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were determined using microdilution in 96-well plates with two-fold serial dilutions at 37⁰ C for 18-24 hours. Absorbance was measured at 620 nm, and viability was verified by growth on blood agar. Cytotoxicity was determined by incubating secretions with canine red blood cells (RBCs) at 37⁰ C with 0.1% Tween 20 as positive control. Kill curve assays were conducted against S. aureus and E. coli with undiluted skin secretions at 37⁰ C. All experiments were duplicated. S. aureus and E. coli were inhibited by P. cruciger and D. melanostictus secretions in 1:4 and 1:8 dilutions respectively. Both secretions also inhibited MDR A. baumannii, and P. aeruginosa at 1:4 dilution, indicating broad-spectrum activity. However, both secretions were inefficient against K. pneumoniae. E. coli was rapidly killed by P. cruciger secretions (10 min post exposure), while D. melanostictus secretions needed 1 hr exposure. Both secretions needed at least 2 hr exposure to kill S. aureus. Canine RBCs were not affected by P. cruciger secretions after 1 hr exposure, while ~20% haemolysis was caused by D. melanostictus secretions suggesting significantly lower cytotoxicity in P. cruciger secretion, indicating potential utility of AMPs from P. cruciger with reduced cytotoxicity as an antibiotic candidate.