Characterization And Genomic Analysis Of Phage PAb-3, A Novel T4-like Bacteriophage Targets To Multidrug Resistant Acinetobacter Baumannii

Objective: The purpose of our study is to apply lytic bacteriophage to treat multi-resistant A. baumannii, and provide an alternative strategy for other multi-resistant bacteria. Meanwhile, the physico-chemical properties of the phage were analyzed and its whole genome was sequenced, which help us to evaluate the application safety of the phage in gene expression level.Methods: The pathogenic bacterium was isolated from a patient blood tissue using plate screening techniques. It was identified by morphological observation,physiological and biochemical indexes test, sequence similarity analysis of 16 S r RNA genes and drug sensitive test. The method of double-layer agar plate was used to isolate the phage targets to A. baumannii from swage in hospital. When the phage was purred, the transmission electron microscope was used to observe the morphology of this phage, and one-step growth curve, the sensitive to chloroform, the host range, p H and temperature stabilities were performed to characterize this phage. And then, After40 h density gradient ultracentrifugation, the DNA extraction was gained according to Phenol-Chloroform-Isoamyl-Alcohol Method. Subsequently, the Illumina High Throughput Sequencing Platform and the Heuristic Gene Mark S software were used forgenomic sequencing to analysis the component of phage genome.Results: A multidrug resistant A. baumannii was isolated and named as AB0815,and drug sensitive test suggest this bacterium was resistant to the fourth generation cephalosporins. A lytic bacteriophage named phage PAb-3 was isolated and it had a clear plaque(diameter 0.8mm) and was classified as Myoviridae by transmission electron microscopy. It owned host specificity and were insensitive to chloroform. It existed stable at a p H range of 4-10 and a temperature less than 50°C. One-step growth curve showed that it had a short latent period of 25 min and an estimated burst size of 243 PFU/cell. The result of SDS-PAGE indicated that this phage have eight kinds of structure protein. Genome sequencing and function gene analysis showed that phage PAb-3 had a genome of 44,781 bp with a G+C content of 42.14%(A=29.56%、T=28.30%、G=20.18%、C=21.96%). The phage genome has 44 CDSs,and 23 of them were clarified by Blastx against NR Database. In addition, there were no antibiotic resistant genes and virus genes in the genome.Conclusion: The bacterium named as AB0815 was confirmed this bacterium was resistant to the fourth generation cephalosporins, and was a multidrug resistant bacterium. A lytic bacteriophage named phage PAb-3 was isolated and classified as Myoviridae by transmission electron microscopy. One-step growth curve showed that it had a short latent period and estimated burst sizes, which suggest this phage has a strong lytic ability. In addition, this phage owned host specificity and were insensitive to chloroform. There were no antibiotic resistant genes and virus genes in the genome.This phage has a higher security and has potential application value. The phylogenetic analysis indicated this phage belonged to T4-like phage.