Activity of novispirin G10 against Pseudomonas aeruginosa in vitro and in infected burns

Abstract

The emergence of multidrug-resistant microbes has serious implications for managing infection and sepsis and has stimulated efforts to develop alternative treatments, such as antimicrobial peptides. The objective of this study was to test a designer peptide, novispirin G10, against multidrug-resistant microorganisms. By two-stage radial diffusion assays, its activity against such organisms compared favorably with that of standard antibiotics and other antimicrobial peptides. It killed bacteria very rapidly, was nonhemolytic, and was relatively noncytotoxic. The peptide induced an immediate, massive efflux of potassium from Pseudomonas aeruginosa, suggesting that it altered the permeability of its inner membrane. The presence of human serum reduced but did not eliminate its activity. We tested the in vivo activity of novispirin G10 in rats with an infected, partial-thickness burn that covered 20% of their total body surface area. The burned area was seeded with 10(6) CFU of a Silvadene-resistant P. aeruginosa strain, and 24 h later a single treatment with 0, 1, 3, or 6 mg of synthetic novispirin G10 (n = 16 at each concentration) per kg was given intradermally. Significant bacterial killing (P < 0.0001) was evident within 4 h in each peptide group compared to controls receiving vehicle. Antimicrobial peptides such as novispirin G10 may provide a useful alternative or adjunct to standard antibiotic agents in treating burns or other wound infections.

FIG. 1.

Microbicidal kinetics. P. aeruginosa strain 2 was exposed to the indicated concentrations of novispirin G10 in a medium composed of 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7.4), and 0.3 mg of Trypticase soy broth powder/ml. Aliquots were removed after 5 and 10 min, diluted, spread on Trypticase soy agar plates, and incubated overnight to allow colony development.

FIG. 2.

Induction of potassium efflux. (Left panel) Potassium efflux from stationary-phase P. aeruginosa strain 2 exposed to 5 or 15 μg of novispirin G10 per ml was monitored with an ion-selective potassium electrode. (Right panel) Colony counts were done under the same conditions after exposing two P. aeruginosa strains (strains 2 and MR 3007) to 15 μg of novispirin G10 per ml.

FIG. 3.

Cytotoxic properties. The lowest cytotoxic effect was seen with novispirin G10 compared to the other antimicrobial peptides tested in the ME-180 cervical epithelial or A549 pulmonary epithelial cell line.

FIG. 4.

Hemolytic properties. A suspension of washed human cells (2.8%, vol/vol) was incubated with various concentrations of PG-1, PGG, ovispirin, or novispirin (G7, G10, T7, and T10) for 30 min at 37°C and then centrifuged. The percentage of total hemoglobin released to the supernatant was measured.

FIG. 5.

Bacterial clearance in an infected burn wound model. This graph summarizes the bacterial counts for P. aeruginosa in the burn wound 4 h after intradermal injection (500 μl/animal) into the burn wound of either 1, 3, or 6 mg of novispirin G10 (n = 16 for each concentration) or vehicle control (n = 16) per kg. ∗, P < 0.05, novispirin G10 versus vehicle control; #, P < 0.05, novispirin G10 at 1 mg/kg versus 6 mg/kg.