Ca-EDTA restores the activity of ceftazidime-avibactam or aztreonam against carbapenemase-producing Klebsiellapneumoniae infections

Abstract

Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

Keywords: Biological sciences; Health sciences; Immunology; Medical microbiology; Medical specialty; Medicine; Microbiology.

Graphical abstract

Figure 1

Synergistic and Time-kill effect on carbapenemase-producing K. pneumoniae clinical isolates (n = 29) (A) Synergistic, and Time-kill data for (B) Ca-EDTA + ceftazidime/avibactam, (C) Ca-EDTA +aztreonam, (D) aztreonam + ceftazidime/avibactam. All experiments in (A) and (B) was performed as three biologically independent experiments, and the mean ± s.d. is shown. p values were determined using an unpaired, two-tailed Student’s t test.

Figure 2

Effects of Ca-EDTA, aztreonam and ceftazidime/avibactam and their combination in vitro (A) biofilm biovolume and (B) biofilm cell viability (%) of carbapenemase-producing K. pneumoniae clinical isolates (n = 29) at different time intervals. All experiments in a–b was performed as three biologically independent experiments, and the mean ± s.d. is shown. p values were determined using an unpaired, two-tailed Student’s t test.

Figure 3

Effects of Ca-EDTA, aztreonam and ceftazidime/avibactam and their combination on catheter biofilm infections Effects of PBS (control), aztreonam (16 μg/mL), ceftazidime/avibactam (1 μg/mL) (16 μg/mL), ceftazidime-avibactam + aztreonem (16/16 μg/mL), Ca-EDTA (24 mg/mL), aztreonam -Ca-EDTA (4/12 mg/mL) and ceftazidime/avibactam- Ca-EDTA (4 μg/mL + 12 mg/mL) on carbapenemase-producing K. pneumoniae clinical isolates (n = 7) catheter biofilm infection model in vivo (A) biovolume (B) Live/Dead cell ratio (C) bio-volume inhibition, and (D) survival of mice. Values were measured each of the 10 catheters and represented the as averages. All experiments in A–E were performed as three biologically independent experiments, and the mean ± s.d. is shown. p values were determined using an unpaired, two-tailed Student’s t test.

Figure 4

Confocal images of in vivo catheter biofilm Confocal imaging analysis (3D) z axis image stack visualizing (40x magnification) of (A) aztreonam (16 μg/mL), (B) ceftazidime/avibactam (1 μg/mL) (16 μg/mL), (C) ceftazidime-avibactam + aztreonem (16/16 μg/mL), (D) Ca-EDTA (24 mg/mL), (E) aztreonam -Ca-EDTA (4/12 mg/mL) and (F) ceftazidime/avibactam- Ca-EDTA (4 μg/mL + 12 mg/mL) on carbapenemase-producing K. pneumoniae clinical isolates (n = 7) catheter biofilm infection model in vivo. One catheter was selected from the 10 catheters after confocal scanning in each treatment group for visualization purposes and live bacteria were stained green and dead bacteria were stained red.

Figure 5

Effects of Ca-EDTA, aztreonam and ceftazidime/avibactam and their combination on bacteremia Effects of PBS (control), aztreonam (700 mg/kg), ceftazidime/avibactam (375 mg/kg), ceftazidime-avibactam + aztreonam (375 mg/kg + 700 mg/kg), Ca-EDTA (80 mg/kg/day), aztreonam -Ca-EDTA (700 mg/kg + 80 mg/kg/day) and ceftazidime/avibactam- Ca-EDTA (375 mg/kg + 80 mg/kg/day) on carbapenemase-producing K. pneumoniae clinical isolates (n = 7) mouse bacteremia infection model (A) illustration of a mouse model, (B) bacterial load in internal organs, (C) survival of mice. p values were determined using a two-sided Mann–Whitney U-test. All data were presented as means ± s.d.

Figure 6

Effects of Ca-EDTA, aztreonam and ceftazidime/avibactam and their combination on pneumonia Effects of PBS (control), aztreonam (700 mg/kg), ceftazidime/avibactam (375 mg/kg), ceftazidime-avibactam + aztreonam (375 mg/kg + 700 mg/kg), Ca-EDTA (80 mg/kg/day), aztreonam -Ca-EDTA (700 mg/kg + 80 mg/kg/day) and ceftazidime/avibactam- Ca-EDTA (375 mg/kg + 80 mg/kg/day) on carbapenemase-producing K. pneumoniae clinical isolates (n = 7) mouse lung infection model (A) illustration of a mouse model, (B) bacterial load in internal organs, (C) survival of mice. p values were determined using a two-sided Mann–Whitney U-test. All data were presented as means ± s.d.

Figure 7

Effects of Ca-EDTA, aztreonam and ceftazidime/avibactam and their combination on thigh infection Effects of PBS (control), aztreonam (700 mg/kg), ceftazidime/avibactam (375 mg/kg), ceftazidime-avibactam + Aztreonam (375 mg/kg + 700 mg/kg), Ca-EDTA (80 mg/kg/day), aztreonam -Ca-EDTA (700 mg/kg + 80 mg/kg/day) and ceftazidime/avibactam- Ca-EDTA (375 mg/kg + 80 mg/kg/day) on carbapenemase-producing K. pneumoniae clinical isolates (n = 7) mouse thigh infection model (A) illustration of a mouse model, (B) bacterial load in thigh, (C) serum creatinine of mice (D) relative expression of virulence genes. p values were determined using a two-sided Mann–Whitney U-test. All data were presented as means ± s.d.