Fig 1.
In vivo tracking of bioluminescently labeled (live) bacterial infections.
CD-1 female mice were injected with individual bacterial strains carrying plasmids constitutively expressing lux reporter genes. Bacterial strains were injected subcutaneously at a dose of 1 × 109 CFU E. faecium, 5 × 107 CFU S. aureus, 1 × 109 CFU K. pneumoniae, 1 × 109 CFU A. baumannii, 5 × 107 CFU P. aeruginosa, 2.5 × 108 CFU E. cloacae, and 1 × 108 CFU E. coli. The infection was monitored 1 h post infection and then every 24 h until day 3. Representative images for day 0 and day 3 are shown. Mice were imaged using a Perkin Elmer in vivo imaging system (IVIS) and the experiment was repeated twice with three mice/group. The scale at the bottom indicates radiance x 106.
Table 1.
In vitro MICs of antibiotics and peptides against clinical isolates.
Fig 2.
Antibiotic and synthetic peptide mono- and combinatorial therapy in a murine cutaneous abscess model using female CD-1 mice and clinical drug-resistant bacterial isolates.
Bacterial strains were injected subcutaneously and treated one hour post infection with either saline (control), synthetic peptides, antibiotics, or antibiotic-peptide combinations. Synthetic peptide concentrations for all conditions were as follows: 1002, 10 mg/kg (3 mg/kg for E. faecium); 1018, 10 mg/kg; HHC-10 10 mg/kg, and DJK-5, 3 mg/kg (0.25 mg/kg for S. aureus). Infected and inflamed tissue was measured three days post infection and pus-containing abscess lumps excised to determine CFU. Abscess sizes are in box and whiskers plots (left panel) and counted CFU/abscess data expressed with geometric mean (right panel). A. P. aeruginosa LESB58, ciprofloxacin 0.4 mg/kg. B. A. baumannii Ab5075, erythromycin 6 mg/kg, meropenem 6 mg/kg. C. K. pneumoniae KPLN649, meropenem 10 mg/kg, ciprofloxacin 30 mg/kg. D. E. cloacae 218 R1, ciprofloxacin 0.006 mg/kg. E. E. coli E38, ciprofloxacin 4 mg/kg, F. E. faecium #1–1, gentamicin 16 mg/kg. G. S. aureus LAC, clindamycin 0.01 mg/kg; vancomycin 0.15 mg/kg. (A-G) n = 368 biologically independent animals. All experiments were done at least three times with 2–4 mice/group. Statistical analysis was performed using One-way ANOVA, Kruskal-Wallis test with Dunn’s correction (two-sided). The asterisk indicates significant differences to the wild-type (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The hash indicates significant differences of the combination therapy over the sum of the effects of each agent alone (#, p < 0.05; ##, p < 0.01; ###, p < 0.001).
Table 2.
Outer membrane permeabilization by peptides cf. antibiotics at their corresponding MICs.
The uptake of the fluorophore NPN in the presence of different antibiotics and synthetic peptides was determined by assessing increased fluorescence at an excitation wavelength of 350 nm and an emission wavelength of 420 nm due to partition of the normally impermeable hydrophobic NPN into bacterial membranes. Relative fluorescence values of at least three biological replicates were determined by subtracting the fluorescence value without test substance.