Synergy of lytic phage pB23 and meropenem combination against carbapenem-resistant Acinetobacter baumannii

Abstract

Phage-antibiotic combination treatment is a novel noteworthy drug delivery method in anti-infection. In the current study, we have isolated a new phage, pB23, against carbapenem-resistant Acinetobacter baumannii 2023. Synergistic antibacterial effect between phage pB23 and meropenem combination could be more stable, using moderate doses of phage (multiplicity of infection ranging from 0.1 to 1,000) based on results of in vitro antibacterial activity. Phage pB23 and meropenem combination could effectively clear mature biofilms and prevent biofilm formation of carbapenem-resistant Acinetobacter baumannii in vitro. Phage pB23 and meropenem combination also has good synergistic antibacterial effects against carbapenem-resistant Acinetobacter baumannii in different growth phases under static culture conditions. The pig skin explant model shows that phage pB23 and meropenem combination has a synergistic effect to remove bacteria from wounds ex vivo. Phage pB23 and meropenem combination also exhibited a synergistic antibacterial effect in vivo using a zebrafish infection mode. The potential promotion of phage proliferation by meropenem and the sensitivity recovery of phage-resistant bacteria to meropenem might elucidate the mechanism of the synergistic antimicrobial activity. In summary, our study illustrates that phage pB23 and meropenem combination could produce synergistic antibacterial effects against carbapenem-resistant Acinetobacter baumannii under static growth conditions. This study also demonstrates that phage-antibiotic combination will become an effective strategy to enhance antibacterial activity of individual drug and provide a new idea of the drug development for the treatment of infections due to carbapenem-resistant Acinetobacter baumannii and other multidrug-resistant bacteria.

Keywords: carbapenem-resistant Acinetobacter baumannii; meropenem; phage; static growth conditions; synergistic antibacterial effect.

Fig 1

(A–E) Biological properties of phage pB23. (A) Thermal stability, (B) pH tolerance, (C) UV tolerance, (D) multiplicity of infection, (E) one-step growth kinetic curve, and (F) phylogenetic tree. Phage log concentration is determined based on the double-layer agar method. Data are presented as the mean plus standard deviation.

Fig 2

Kinetic curve of combined use of phage pB23 (2.5 × 10⁶ PFU/mL) and meropenem (4 µg/mL) (A) and antibacterial activity of the phage pB23 and meropenem (4 µg/mL) combination under different phage dose (B) in vitro. The y axis represents the active bacteria amount, which is calculated by log (CFU/mL) using the dilution plate-counting method. The x axis represents culture time.

Fig 3

Antibacterial activity of phage pB23 and meropenem (1× MIC) combination against both forming biofilms (A and B) and mature biofilms (C and D) under B.m#2023 static growth conditions in vitro. (A and C) Optical density at 600 nm was measured using the microplate reader. (B and D) Viable bacteria counting was detected using dilution plate-counting method. Error bars represent SD of three independent experiments (*, P value < 0.05).

Fig 4

Growth kinetics curve of B.m#2023 (A) and synergistic antibacterial effect of pB23 and meropenem combination against B.m#2023 (B) in different life stages under static growth conditions in vitro (*, P value < 0.05).

Fig 5

Effects of phage (MOI = 1) and meropenem (1× MIC) combination on bacterial growth revival (A) and number of phages (B). The experiment was repeated in triplicate, and the error bars represent the standard deviations (SD).

Fig 6

Antibacterial effect of pB23 alone or in combination with meropenem (1× MIC) in an ex vivo pig skin model of wound infection after 24-h posttreatment(*, P value < 0.05).

Fig 7

In vivo efficacy of pB23 and meropenem (1× MIC) alone or combination against B.m#2023 in a zebrafish model. Monitoring lasted for a period of 5 days.