Synergistic Killing and Re-Sensitization of Pseudomonas aeruginosa to Antibiotics by Phage-Antibiotic Combination Treatment

Abstract

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections pose a serious health threat. Bacteriophage-antibiotic combination therapy is a promising candidate for combating these infections. A 5-phage P. aeruginosa cocktail, PAM2H, was tested in combination with antibiotics (ceftazidime, ciprofloxacin, gentamicin, meropenem) to determine if PAM2H enhances antibiotic activity. Combination treatment in vitro resulted in a significant increase in susceptibility of MDR strains to antibiotics. Treatment with ceftazidime (CAZ), meropenem, gentamicin, or ciprofloxacin in the presence of the phage increased the number of P. aeruginosa strains susceptible to these antibiotics by 63%, 56%, 31%, and 81%, respectively. Additionally, in a mouse dorsal wound model, seven of eight mice treated with a combination of CAZ and PAM2H for three days had no detectable bacteria remaining in their wounds on day 4, while all mice treated with CAZ or PAM2H alone had ~10⁷ colony forming units (CFU) remaining in their wounds. P. aeruginosa recovered from mouse wounds post-treatment showed decreased virulence in a wax worm model, and DNA sequencing indicated that the combination treatment prevented mutations in genes encoding known phage receptors. Treatment with PAM2H in combination with antibiotics resulted in the re-sensitization of P. aeruginosa to antibiotics in vitro and a synergistic reduction in bacterial burden in vivo.

Keywords: Pseudomonas aeruginosa; antimicrobial resistance; phage therapy; phage–antibiotic synergy; re-sensitization.

Figure 1

IVIS images showing the radiance (p/s/cm²/sr) of PAO1::lux bacteria in dorsal wounds of mice on day 4 post-surgery after completion of treatments. (A). IVIS of a mouse from the control-treated group receiving only PBS, (B). PAM2H (phage cocktail) only treated group, (C). CAZ only treated group, (D). combination treated group receiving both PAM2H and CAZ.

Figure 2

Quantification of the bacterial luminescence for mice from each treatment group as determined by IVIS on day 4 at the completion of treatment. * p < 0.05.

Figure 3

Colony forming units obtained from excised wound tissue on day 4 post treatment. * p < 0.05, **** p < 0.0001.

Figure 4

Mouse dorsal wound sizes as measured by an Aranz wound measurement device over 21 days. On day 10, the wound size for the combo-treated group was significantly smaller than that in the control or in the other treatment groups (p < 0.005), and the median wound size on day 10 for the combo group was less than the median wound size on day 0, meaning the wounds had begun to contract and heal.

Figure 5

Kaplan–Meier survival curve for Galleria wax worms inoculated with PAO1::lux mutants recovered from mouse dorsal wounds post-treatment. Original PAO1::lux was used as a positive control, and an untouched group and PBS injected group served as negative controls. PH1 and PH2 were isolates obtained from mice in the phage-only treatment group. CA1 was collected from the CAZ-only treatment group, and CAPH1 was obtained from the combination treatment group. PH2, PH1, and CAPH1 showed a significant increase in survival compared to the PAO1::lux (p < 0.0001). Note: the survival curves for PH1 and CAPH1 were identical; thus, the survival curve for CAPH1 was moved below PH1 so that the symbols for each curve are discernible on the graph.