Study On The Control Of Multidrug Resistant Acinetobacter Baumannii By Phages And Endolysin

Antibiotics have completely changed the treatment of infectious diseases and prolonged human life.However,antibiotic resistance has become a global public health threat.With a decrease in the discovery and development rate of novel antibiotics,phages have regained the favor of researchers and clinicians.Acinetobacter baumannii is a critical priority pathogen because of its high incidence of antibiotic resistance and significant role in nosocomial infections.In this study,the multidrug resistant A.baumannii phages and endolysin were used as the research object to explore and evaluate their application potential in the field of antiinfection.The antibacterial effects of phages and endolysin were deeply analyzed,which provided a research basis for the safe and effective application of them.13 clinical strains of A.baumannii were isolated from patient samples.The minimum inhibitory concentration test results for 12 kinds of antibiotics showed that all 13 strains were multidrug resistant.The A.baumannii clinical strains were used as hosts to isolate phages from hospital sewage.Lytic A.baumannii phages of D0,D2 and D22 were selected based on different plaque morphologies and clarity of the plaques and sizes.The three phages were all from the order of Caudovirales.D0,D2 and D22 were the member of the family Siphoviridae,Podoviridae and Myoviridae,respectively.Additionally,all phages were not sensitive to chloroform.The whole genome sequencing results showed that D2 and D22 were linear double stranded DNA,and D0 was circular double stranded DNA.The genome length of D0,D2 and D22 were 4,3051,3,9964 and 10,3539 bp,respectively.The GC contents of D0,D2 and D22 were 45.48%,39.23%and 37.45%,respectively.D0,D2 and D22 contained 55,47,and 159 open reading frames,and the number of open reading frames with known functions was 27,24 and 34,respectively.The life cycle of D0,D2 and D22 was lytic,and there were no genes for lysogen,drug resistance or virulence.Host range studied on A.baumannii clinical strains showed that phage D0,D2 and D22 was able to lyse 13,5 and 1 of the clinical strains,respectively.The phage D0 and D2 were combined into a phage cocktail,and the anti-infection effects of the phage cocktail and the single phage were further evaluated in vivo and in vitro.D0 has the latent period of 40 min,with the burst size of 39,while the latent of D2 lasted for 20 min and the burst size was 80.In vitro,the bactericidal ability of D0 was weak and dependent on the MOI.D2 was able to completely inhibit growth of the host cells up to 8 h at MOIs ranging from 0.01 to 100.The phage cocktail could completely suppress cell growth over 18 h.The results of colony count showed that the number of viable bacteria in the phage cocktail group was significantly lower than that in the single phage group(P<0.01).In vivo,both single phage and phage cocktail had protective effects on mice.Treatment with phage D0 yielded 50%survival.In contrast,90%or 100%of mice survived treatment with phage D2 or phage cocktail,respectively.To determine development of phage resistance among the treatment groups,we tested phage resistance from colonies isolated from samples of mice blood.The percentage of phage resistant mutants occurring in the phage D2 group was significantly higher than in the other two treatment groups(P<0.01).The endolysin of phage D2,namely Abtn-4,was found to exhibit broad antimicrobial activity.25 Gram-negative strains and 7 Gram-positive strains were susceptible to Abtn-4.Furthermore,Abtn-4 had the ability to reduce biofilm formation.Interestingly,Abtn-4 showed antimicrobial activity against phage resistant mutants.Laser scanning confocal microscopy revealed that Abtn-4 had a strong binding effect on bacteria surface.Bioinformatics analysis showed that Abtn-4 contained an amphipathic transmembrane helix and the positively charged amino acids were evenly distributed on the surface.Abtn-4 was bound to the surface of the bacteria through electrostatic adsorption,and then used the transmembrane helix to across the Gram-negative bacterial outer membrane.After reaching the peptidoglycan layer,Abtn-4 exerted enzymatic activity to hydrolyze the peptidoglycan,causing the bacteria to break and die.Based on these results,phage and endolysin may be a promising candidate therapeutic agent for multidrug resistant bacterial infections.