Characterization,Genomic Analysis And Application Of Bacteriophages That Infects Antibiotics-resistant Acinetobacter Baumannii Clinical Isolates

Objective:Acinetobacter baumannii is an emerging nosocomial pathogen with increasing prevalence of antibiotics resistance. The hospital cross-infection could be a significant factor in contributing to its outbreaks in the community and seriously threaten the lives of patients. It is imperative to find the effective methods of prevention and treatment. The recent renaissance of bacteriophage therapy may provide new treatment strategies for combatting drug-resistant bacterial infections. In this study, we described preliminary application of bacteriophages against antibiotics-resistant bacteria which may be potential targets for the development of novel regimens to treat multidrug-resistant Acinetobacter baumannii in clinical medicine.Methods:The alarming rise in antibacterial resistance among bacteria has led to interests in research of bacteriophage. A total of40Acinetobacter baumannii isolates derived from hospitalized patients were genotyped by using Pulsed-field Gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Bacteriophages were isolated from raw sewage using these Acinetobacter baumannii strains as host cells. Characterization, sequencing and comparative genomic analysis of a novel lytic bacteriophage IME-AB2was carried out. Surface spraying with aerosolized IME-AB2reduced bacterial counts on an A. baumannii-treated surface in vitro. Using a mouse lung-infection model prepared with MDR-AB, we evaluated the efficacy of bacteriophage treatments. Survival was monitored and bronchoalveolar lavage fluids (BALF) were analyzed. Quantification of bacteria, bacteriophages, as well as histology analyses were performed.Results:The40Acinetobacter baumannii strains were resistant to multiple antibiotics including carbapenems, β-lactamase inhibitors. The PFGE genotypes were grouped into three clones A,B,C and the clone C was the major one which contains26strains of the40(65%) strains. MLST analysis showed that the genotypes can be grouped into thirteen clones and the largest clone ST208contained24isolates of the40(60%). Sixteen newly isolated Acinetobacter baumannii bacteriophages were found to lyse31of the above mentioned40strains (78%). We isolated a lytic bacteriophage IME-AB2which produced a titer of1×1011pfu/ml in LB culture and had a short latent period and a short burst period. The results showed that phage IME-AB2also had a short adsorption rate.4℃preservation was proved to be a good and convenient method for the storage of the virus. The bacteriophage IME-AB2had an icosahedral head and displays morphology resembling Myoviridae family phages. Whole genome of IME-AB2was sequenced and annotated and the results showed that its genome is a double-stranded DNA molecule consisting of43,665nucleotides. The genome has a G+C content of37.5%and82putative coding sequences (CDSs) and was selected for characterization and a genomic comparative study. The whole-genome sequence of phage IME-AB2has been deposited in the NCBI nucleotide sequence database under GenBank accession number:JX976549. It could clear its host bacteria suspension quickly and reduce bacterial counts on an A. baumannii-treated surface. Intranasal treatment by bacteriophage in mice pneumonia derived by A.baumannii infection at30min,4h and24h after bacterial inoculation could rescue100%,50%,20%of the mice respectively. About100%of mice survived with treatment at the multiplicity of infection (MOI) of1. About80%of mice survived with treatment at the MOI of0.1. About60%of mice survived with treatment at the MOI of0.01. About50%of mice survived with treatment at the MOI of0.001. About10%of mice survived with treatment of phosphate-buffered saline (PBS). Bacterial counts were significantly lower in the BALF from mice that had received curative bacteriophage treatment than that in the control treatment. Bacteriophage counts did not significantly change in the BALF from mice that had received bacteriophage treatment compared with the non-infected animals. This histology of mice given curative bacteriophage treatments was different from the untreated mice. Conclusions:This encouraging data showed that bacteriophage had good antibacterial activity against Acinetobacter baumannii in vitro and in vivo. It may benefit future phage therapy studies of multidrug-resistant A.baumannii infection.16figures,13tables,41references.