Synergistic Antibacterial Effect of Phage pB3074 in Combination with Antibiotics Targeting Cell Wall against Multidrug-Resistant Acinetobacter baumannii In Vitro and Ex Vivo

Abstract

Synergistic effects of phages in combination with antibiotics have received increasing attention. In this present study, we isolated a new phage pB3074 against clinically isolated multidrug-resistant Acinetobacter baumannii. Phage pB3074 combined with cell wall-targeting antibiotics could produce synergistic antibacterial effect in vitro bactericidal activities. Further research indicates that the bacteriophage dose is critical to synergistic antimicrobial effect of phage and antibiotic combination. Cefotaxime and meropenem were selected as the representative cell wall-targeting antibiotics for further synergistic antibacterial study. Results illustrated that phage pB3074 and cefotaxime or meropenem combination was very effective for the removal of mature biofilm and inhibition of biofilm formation. In a pig skin explant model, results also showed that phage pB3074 and cefotaxime or meropenem combination was very effective for the treatment of wound infection ex vivo. Subsequent studies showed that some extent recovery of drug sensitivity to cell wall-targeting antibiotics might be vital mechanism of synergistic antibacterial effect between bacteriophage pB3074 and these antibiotics. The existence of antibiotics could promote phage adsorption and proliferation, which might also be potential mechanism for synergistic antibacterial activities and have been observed in cefotaxime and meropenem application. In summary, results in the current study demonstrated that phage pB3074 has the potential to be developed as an antibacterial agent and combined application of phages and antibiotics might be a new choice for the treatment of current multidrug-resistant bacterial infections. IMPORTANCE Combined application of phages and antibiotics cannot only effectively inhibit the appearance of phage-resistant bacteria, but also reduce the effective use concentration of antibiotics, and even make some bacteria regain sensitivity to some resistant antibiotics. Therefore, phage-antibiotic combination (PAC) could improve the antibacterial activity of individual drug, providing a new choice for clinical treatment of multidrug-resistant bacterial infections.

Keywords: ex vivo; in vitro; mechanism; multidrug-resistant A. baumannii; phage-antibiotic synergy.

FIG 1

(A to E) Biological properties of phage pB3074. (A) pH sensitivity; (B) temperature tolerance; (C) UV tolerance; (D) determination of the optimal MOI; and (E) one-step growth curve. (F) Phylogenetic tree. Phage log concentration are calculated based on the double-layer agar method. Data are presented as the mean plus standard deviation (SD).

FIG 2

The effect of phage dose on the combination of phage pB3074 and cell wall-targeting antibiotics, including (A) piperacillin; (B) ceftazidime; (C) cefotaxime; (D) imipenem; (E) meropenem; and (F) aztreonam. The y axis represents the active bacteria amount, which is calculated by log (CFU/mL). The x axis represents culture time. Piperacillin, 128 μg/mL; ceftazidime, 16 μg/mL; cefotaxime,16 μg/mL; imipenem, 8 μg/mL; meropenem, 2 μg/mL and aztreonam, 8 μg/mL. L, low dose (MOI = 0.01); M, middle dose (MOI = 1); H, high dose (MOI = 100).

FIG 3

Evaluation of the combination of pB3074 and antibiotic in vitro activity against both (A, B) forming; and (C, D) mature biofilms of Bm3074. Antibiotics include cefotaxime (2×MIC) or meropenem (0.5×MIC). (A and C) Optical density at 600 nm (OD₆₀₀) was measured using the microplate reader. (B and D) Viable bacteria counting was detected using dilution plate counting method. For the biofilm formation inhibition experiment in panels A and B, the phage and B.m3074, antibiotic and B.m3074, antibiotic, phage and B.m3074 were cocultured. For the mature biofilm-reducing experiment in panels C and D, phage, antibiotic, and antibiotic plus phage were added when mature biofilm was observed. Error bars represent SD of three independent experiments (*, P value < 0.05).

FIG 4

Antibacterial effect of pB3074 alone or in combination with antibiotic (cefotaxime [2×MIC] or meropenem [0.5×MIC]) in an ex vivo pig skin model of wound infection and treatment.

FIG 5

Bacteriophage proliferation with and without antibiotic (A to C) and diameter of bacteriophage plaque measured and statistically compared (D). Cefotaxime (2×MIC) or meropenem (0.5×MIC) used in this study.