Efficacy of OH-CATH30 and its analogs against drug-resistant bacteria in vitro and in mouse models

Abstract

Antimicrobial peptides (AMPs) have been considered alternatives to conventional antibiotics for drug-resistant bacterial infections. However, their comparatively high toxicity toward eukaryotic cells and poor efficacy in vivo hamper their clinical application. OH-CATH30, a novel cathelicidin peptide deduced from the king cobra, possesses potent antibacterial activity in vitro. The objective of this study is to evaluate the efficacy of OH-CATH30 and its analog OH-CM6 against drug-resistant bacteria in vitro and in vivo. The MICs of OH-CATH30 and OH-CM6 ranged from 1.56 to 12.5 μg/ml against drug-resistant clinical isolates of several pathogenic species, including Escherichia coli, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. The MICs of OH-CATH30 and OH-CM6 were slightly altered in the presence of 25% human serum. OH-CATH30 and OH-CM6 killed E. coli quickly (within 60 min) by disrupting the bacterial cytoplasmic membrane. Importantly, the 50% lethal doses (LD(50)) of OH-CATH30 and OH-CM6 in mice following intraperitoneal (i.p.) injection were 120 mg/kg of body weight and 100 mg/kg, respectively, and no death was observed at any dose up to 160 mg/kg following subcutaneous (s.c.) injection. Moreover, 10 mg/kg OH-CATH30 or OH-CM6 significantly decreased the bacterial counts as well as the inflammatory response in a mouse thigh infection model and rescued infected mice in a bacteremia model induced by drug-resistant E. coli. Taken together, our findings demonstrate that the natural cathelicidin peptide OH-CATH30 and its analogs exhibit relatively low toxicity and potent efficacy in mouse models, indicating that they may have therapeutic potential against the systemic infections caused by drug-resistant bacteria.

Fig 1

Toxicity of OH-CATH30 and its analogs in vitro and in vivo. (A) Hemolytic activity of peptides. Peptides at various concentrations were incubated with human red blood cells for 4 h at 37°C. (B) Survival of the HaCaT cells after being treated with peptides for 48 h. (C) Acute toxicity of peptides in Kunming mice via a single i.p or s.c injection.

Fig 2

Killing kinetics of OH-CATH30 and OH-CM6. E. coli ATCC 25922 (A) and E. coli clinical strain 1 (B) (5 × 10⁵ CFU/ml) were exposed to OH-CATH30 and OH-CM6 at 2× the MIC for 0, 5, 10, 30, 45, 50, and 60 min at 37°C.

Fig 3

Effect of OH-CATH on the fluorescence intensity changes of E. coli 25922 incubated with diSC3-5. (A) OH-CATH30 and OH-CM6 result in the leakage of dye in a dose-dependent manner. (B) The kinetics of fluorescence intensity changes in the presence of OH-CATH30 and OH-CM6 at 50 μg/ml.

Fig 4

Effect of serum on the antibacterial activity of OH-CATH30 and its analogs. (A) Peptides were preincubated with human serum for 0 to 12 h. Aliquots were taken at different time intervals, and the antibacterial activities of the aliquots against E. coli 25922 were determined using an agar radial diffusion method. (B) The antibacterial activity of the peptides was tested in the presence of 25% human serum using a microdilution assay. Each value shown is the mean ± standard error of results from three experiments.

Fig 5

Efficacy of OH-CATH30 and OH-CM6 in the E. coli 25922-induced murine thigh infection model. (A) Thigh viable bacterial counts. (B) Thigh weight. (C) Level of TNF-α in thigh tissue. ICR mice (n = 6) were made neutropenic by the administration of cyclophosphamide and treated with OH-CATH peptides (1, 10, or 20 mg/kg) or CFP (40 mg/kg) immediately via a single s.c. injection after the bacterial challenge. *, P < 0.05 versus the untreated group.

Fig 6

Survival of mice in a bacteremia model induced by the MDR E. coli clinical strain. Mice received treatment with OH-CATH peptides (1, 10, or 20 mg/kg) or CFP (40 mg/kg) via i.p. injection. (A and B) Treatment initiated at 1 h postinoculation (n = 10). (C and D) Treatment initiated at 4 h postinoculation (n = 7).

Fig 7

Blood bacterial counts in a bacteremia model at 2 h after the last treatment. Mice received treatment with OH-CATH peptides (10 mg/kg) or CFP (40 mg/kg) via i.p. injection. (A) Treatment initiated at 1 h postinoculation (n = 10). (B) Treatment initiated at 4 h postinoculation (n = 7). Each point represents the determination from a single animal, and the line shows the mean value. *, P < 0.05 versus the untreated and CFP-treated groups.