Potential Of Lytic Bacteriophages To Disrupt Antibiotic Resistant Acinetobacter Baumannii Biofilms In Vitro

Acinetobacter baumannii is a commonly opportunistic pathogen that can cause major health problems in respiratory system.With the emergence of multidrug-resistant A.baumannii(MDRAB)and even pandrug-resistant A.baumannii(PDRAB),it is difficult to control and treat clinical infections caused by these bacteria.In addition,most clinical A.baumannii isolates can develop biofilms,which benefits A.baumannii survival and increases their antibiotic resistance.Therefore,one of important strategies to fight against antibiotic resistance is inhibiting or eliminating biofilms.Bacteriophage can specifically infect and lyse A.baumannii with a different antibacterial mechanism to antibiotics,and phage therapy has received much attention again by researchers when in face to infections caused by seriously drug-resistant bacteria.In addition,therapies based on bacteriophage or its derivatives were considered as potential effective alternatives to conventional antibiotics for controlling seriously drug-resistant A.baumannii infections.In this study,a novel lytic bacteriophage was isolated from PLA Hospital 307 sewage through using an indicated pandrug-resistant A.baumannii from patients’ sputum samples in the intensive care unit at hospital.After its biological characteristics and complete genome sequence were studied,we cultivated biofilms using A.baumannii isolates in vitro and explored the potential of lytic bacteriophages to disrupt biofilms.In order to study its biological characteristics,the phage’s lytic potential and ability inducing development of halos in bacterial lawnwere evaluated on double-layer agar plate,and morphology of the phage was observed with transmission electron microscope.The lytic spectrum and one-step growth curve of the bacteriophage were also determined.To disclose its genetic components and information,complete genome sequence of the phage was obtained and assymbled by high throughput sequencing,and annotated with RAST programme.The results showed that the lytic phage develops round and clear plaques surrounded by obvious halos on its host bacterium lawn.The phage had a hexagonal head with a diameter of 48~50 nm,and a contractile tail of 140~150 nm in length,which was assigned to the Myoviridae family.The phage can lyse nine strains of A.baumannii including host bacteria among 31 strains in vitro,while it has a relatively short latent period of 10 min and a relatively large burst size of about 229 plaque forming units per cell.Genomic analysis indicated that the phage has a linear double-stranded genome of 41 243 bp,with a G+C content of 39.3 %,and 16 putative open reading frames were annotated with known functions among total52 open reading frames.No integrase,virulence factors or transposon-like components were annotated in the phage genome.The phage has a genome with high similarity to Acinetobacter phages AB3,Fri1,Abp1,phiAB1,and vB_AbaP_PD-6A3.In order to explore the potential of lytic bacteriophages to disrupt biofilms,we infected and evaluated A.baumannii biofilms obtained with methods of scanning electron microscopy,confocal laser scanning microscopy,semi-quantitation through staining with crystal violet,bacterial enumberation of A.baumannii biofilms and ATP release experiment.Results of microscopy observation showed that the lytic bacteriophage can obviously disrupt antibiotic-resistant A.baumannii biofilms,while results of bacterial counts indicated that the lytic phage inhibited the formation of A.baumannii biofilms with a proportion of88.5 % and decreased the proportion of A.baumannii biofilm cells inpreformed biofilms by 52 %~90 %.Moreover,results of ATP release experiment further proved that the lytic phage can infect and disrupt A.baumannii biofilms,and suggested that the phage can enter and infect bacteria in inner biofilms.Additionally,we also observed that both exponential A.baumannii culture and A.baumannii biofilms can develop resistant to the lytic bacteriophage,and the frequency of bacteriophage insensitive mutation formed by exponential A.baumannii culture when infected with the lytic phage was estimated to be 3 × 10-6 cfu.In conclusion,we successfully isolated a lytic phage,and explored its biological characteristics and genetic components,while we found that the phage not only can effectively infect and lytic its host bacteria,but also can inhibit and disrupt its biofilms.Therefore,the lytic bacteriophage has potential to be developed as an alternative strategy for controlling drug-resistant A.baumannii infections.However,we also found some problems in this research,for example,the isolation and purification of phage depolymerase,the combined effect of phage depolymerase and other anti-bacteria materials,and the resolution of bacteria resist to phages,while the related research is under way.