| Objective: A phage that can specifically lyse multidrug-resistant Acinetobacter baumannii(MDRAB)was isolated and identified,and its biological characteristics were analyzed.The genome of the bacteriophage and the functional genes of its lysing bacteria were analyzed.The carrying status of prophages in 82 genome-wide sequencing A.baumannii strains was predicted,and the antibiotic resistance genes and virulence factors encoded by prophages were analyzed.Methods: The phages that lysed A.baumannii were isolated and purified from pond water using the mixture of 10 A.baumannii strains as host bacteria.Phages were purified from A.baumannii Ab1,and their morphological characteristics,host spectrum,optimal multiplicity of infection,adsorption curve and one-step growth curve were analyzed.The high concentration of lysis buffer was obtained by double agar plate method with the host bacteria Ab1 proliferative phage 5W.The filtrate was collected and purified by centrifugation and filtration.Mi Seq platform was used for sequencing,and Newbler 3.0 was used to splice the sequence.Bioinformatics analysis of phage 5W genome was performed using BASys,t RNAScan-SE v2.0,CGView Serve,TMHMM Server v.2.0,MEGA 5.0,Mauve 2.4.0 and ACT software.82 strains of A.baumannii were screened and downloaded from NCBI.PHASTER software was used to predict the prophage carried by A.baumannii,and CARD and VFDB online analysis software were used to predict the antibiotic resistance genes and virulence factors encoded by the prophage.Results: A phage 5W that can lyse multi-drug resistant A.baumannii was isolated.This phage is a member of the Siphoviridae family of the order Caudoviruses.The host spectrum of phage 5W is relatively narrow.Among the 19 clinical strains,4 strains could be lysed,and only Ab1 has a strong lysis effect.5W can quickly adsorb the host bacteria Ab1,and the adsorption rate of the phage after mixing for 2 min was over 80%.The one-step growth curve shows that the incubation period was 20 min,the burst period was about 240 min,and the burst volume was about 180PFU/cell.The phage 5W genome is ds DNA with a total length of 43,032 bp,a GC content of 39.85%,and 61 open reading frames,of which 19 coding sequences can predict its function.5W does not contain known virulence genes and drug resistance genes.It encodes a ? type holin and a C-terminal N-acetyl-?-D-Muramidase belonging to the GH?108 family,which is rich in positively charged amino acids.The lytic gene cluster contains the M/S subunit of restriction endonuclease.Both holin and lysin do not contain molecular signal peptides,and the secondary structure components of the latter are mainly ?-helix and random coil.The 5W lysin has high homology with the lysin of phage Ab105-2phi.The front and rear half of the 5W genome were highly similar to the two prophages phi ABCR01-03 and phi ABCR01-02 in the A.baumannii ABCR01 genome,respectively,and they were closely related to the phage ctbh1,suggesting that it might be composed of two different prophages.A total of 472 prophages were predicted from 82 A.baumannii strains,including 201 intact prophages,91 suspected prophages and 180 defective prophages.On average,about 2.45 intact prophages can be carried in each genome of A.baumannii.The proportion of all prophages carried by each A.baumannii was about 4%-6%.Twenty-nine prophages carried resistance genes,and there were 14 types of resistance from 15 different families,covering the mechanism of 6 kinds of antibiotic resistance.A total of 132 prophages carried 38 virulence genes and 34 virulence factors.Different types of prophages generally carried 1-2virulence factors,and a few prophages carried 3 or more virulence factors.By analyzing the possible host source composition of virulence factors,it was found that in addition to A.baumannii,Neisseria meningitidis,Shigella dysenteriae,Legionella pneumophila and their subspecies also had a high structural proportion,which possible host sources.Conclusions: In this study,a new mild phage 5W of A.baumannii was successfully isolated and identified.The phage has biological characteristics such as narrow host spectrum,short incubation period,large amount of cleavage and fast adsorption.The genome of phage 5W contains no known virulence genes and drug resistance genes.The C-terminal of the lysin is rich in positively charged amino acids,belonging to the N-acetyl-?-D-cytoplasmic enzyme of GH?108 family.Restriction endonuclease M/S subunit was inserted into the soluble gene cluster.The results of comparative genomic analysis suggested that 5W might be recombined by two different prophages.A.baumannii generally carries prophages,but the proportion of prophage genes in A.baumannii genome is not high,about 4?6%.Some prophages encode antibiotic resistance genes,mainly aminoglycosides,sulfonamides and ?-lactams.Prophages carry a high proportion of virulence genes.Prophages may play an important role in the development and pathogenesis of antibiotic resistance in A.baumannii. |
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