Bacteriophage Rescues Mice From Acinetobacter Baumannii-mediated Pneumonia

Objective: Non-fermentative bacteria, represented by A. baumanii, has become a major causative pathogen of nosocomial infection in the 21 st century. With the rapid transmission of drug-resistant bacteria,we are facing a dangerous situation in which no more drugs can be used. Under this circumstances, in hope of seeking alternative options of antibiotics, bacteriophage therapy, the history of which dates back a hundred years ago, is gaining more and more attention. In this research, we evaluated the potential efficacy and safety of a lytic bacteriophage(IME-AB2) for controlling A. baumannii mediated mouse pneumonia.The bacteriophage(IME-AB2), which was previously isolated, has already been whole genome sequenced and registered on Genebank. Therefore, its biological information is clear. The result of this research will provide us with new vision and experimental basis for the treatment of drug-resistant A. baumanii. The research methods will offer technical support for individualized treatment of phage therapy.Methods: According to previous assays, A mouse pneumonia model was developed by combining cyclophosphamide pretreatment and the culture of Acinetobacter baumannii which was administered intranasally to the BALB/c mice, and a lytic bacteriophage was evaluated for its therapeutic efficacy in this model by examining the survival rate, clinical evaluation,histopathological examination,microcomputed tomography and some other indexes. The dose-efficacy analysis and time-efficacy of phage therapy were also carried out. The variation of bacteria count and phage count were analyzed to investigate the therapeutic mechanism. Safety evaluation was carried out by observing the clinical score of mice, by examining the pathological change in mice spleen and liver, and by titrating the phage antibodies in mice blood.Results: According to clincal symptom scoring and Micro-CT examination, a mouse model with A.baumanii pneumonia was successfully developed. By injection intraperitoneally with cyclophosphamide at3 days prior to bacterial inoculation, a dose of 2×108 cfu of A.baumannii was administered intranasally to the BALB/c mice; a pneumonia model induced by A.baumannii was developed. Results of the survival curve showed that the therapeutic efficacy of 10 MOI doses of bacteriophage administered at 1 h post-bacterial inoculation was best, which led to a 100% survival, while the entire control group(PBS treatment) died between 60~144 h post-challenge. Half of the control animals died in the first 83 h post-challenge(P<0.001). 1h after challenged with A. baumanii, mice were given phage therapy with different MOI, 10, 1, 0.1, respectively. The survival rate was 100%, 70%, 20%, respectively. Fixating MOI at 10, phage was administered to 3 groups of mice at 1h, 4h, 24 h. The survival rate was 100%, 60%, 20%,respectively. The results well established dose-dependence and time-dependence of phage therapy.Micro-CT of mice lungs showed that the infiltration range of lung was smaller in the therapy group, and not as severe as the control group. Histopathological examination showed that hemorrhage, edema and leukocyte infiltration were much more severe in the control group, and massive lung consolidation was observed. At each time points between 2-144 h observation period, the bacteria counts of lung in the treatment group are significantly lower than that of the control group. The bacteria counts declines as the time proceeds. As the bacteria counts declines, the phage counts also declines. Clinical evaluation indicated that all challenged mice became sick, but that bacteriophage treatment significantly alleviated clinical symptoms from 36 h post-challenge until complete recovery. However, the health status of the control group worsened until death(P < 0.001). Mice spleens shrinked significantly after cyclophosphamide injection. Somehow, they recover to its original size or even larger after phage treatment. Neutralizing antibodies of phage are detectable 216 h after phage administration.Conclusion: The results of this research indicated that IME-AB2 are effective in the treatment of Drug-resistant A. baumanii mediated pneumonia in mice. Safety of phage therapy was also promising.Further studies in phage therapy are anticipated. The immunogenecity of bacteriophage should be taken into account. Therefore, the tactic of intravenous administration and repeated administration needs further research.