In Vitro/ Vivo Activity of Potential MCR-1 Inhibitor in Combination With Colistin Againsts mcr-1-Positive Klebsiella pneumonia

Abstract

Carbapenem resistance among strains of the nosocomial pathogen Klebsiella pneumoniae is increasing worldwide, causing serious clinical infections and higher mortality rates. Polymyxins are some of the few "last resort" options for treatment of carbapenem-resistant Enterobacteriaceae, including K. pneumoniae, however, the emergence of plasmid-mediated colistin resistance gene mcr-1 has largely rendered polymyxin-class antibiotics ineffective in a clinical setting. We previously identified a natural compound, pterostilbene, which has a synergistic effect in combination with polymyxins. Here, we aimed to determine whether pterostilbene application can restore the bactericidal activity of polymyxins against mcr-1-positive K. pneumoniae. Checkerboard MIC studies confirmed that pterostilbene reduces the MIC of colistin against mcr-1-positive clinical K. pneumoniae isolates, with the bacteria going from resistant to sensitive, and also demonstrated a synergistic effect with colistin (FIC index = 0.11 ± 0.04 or 0.28 ± 0.00). Time-killing assays showed that individually, both pterostilbene and colistin failed to eradicate K. pneumoniae strains, while in combination, the two drugs effectively eliminated K. pneumoniae ZJ02 and K. pneumoniae ZJ05 by 1-3 h post-inoculation. The combined disk test also showed increases in the zones of inhibition only for mcr-1-positive Escherichia coli and K. pneumoniae isolates. A mouse infection model demonstrated that the survival rate of mice at 7 days post-intraperitoneal injection with a lethal dose of K. pneumoniae ZJ05 was significantly promoted from 0 to 67% following combination therapy. This is the first time a MCR-1 inhibitor has successfully been used in combination with colistin against human clinical MCR-1 producing K. pneumoniae ZJ05 isolate.

Keywords: K. pneumonia; MCR-1 inhibitor; colistin; combination therapy; pterostilbene.

Figure 1

Pterostilbene in combination with colistin restores the in vitro sensitivity of K. pneumoniae to polymyxins. (A,B) Growth curves for K. pneumoniae ZJ02 (A) and K. pneumoniae ZJ05 (B) cultured in the presence of various concentrations (0–128 μg/mL) of pterostilbene. Values represent the averages of three independent experiments. (C,D) Time-killing curves for colistin, pterostilbene, colistin + pterostilbene, and control treatment (medium only) against K. pneumoniae ZJ02 (C) and K. pneumoniae ZJ05 (D). Values represent the averages of three independent experiments.

Figure 2

Zones of inhibition surrounding colistin disks supplemented with 0, 8, or 32 μg/mL of pterostilbene on lawns of K. pneumoniae ZJ02, K. pneumoniae ZJ05, and E. coli DH5α (pUC19-mcr-1) on MHB agar plates.

Figure 3

Effects of pterostilbene and colistin combination therapy in vivo. Mice were infected with K. pneumoniae ZJ05 and then treated with pterostilbene, colistin, pterostilbene combined with colistin (combination), or control solvent treatment (model). Uninfected mice were used as a healthy control (normal control). At 48 h post-inoculation, mice were euthanized and the bacterial burden (A) and wet/dry weight ratio (B) of lungs were calculated. **P < 0.01. (C) Survival curve of mice infected with K. pneumoniae ZJ05 and monitored for 7 days post-infection. The data represent the means and standard deviations from three separate experiments (18 mice per group). Gross pathological changes (D) and histopathology (E) of the lung tissue of mice from the first experiment were also assessed.