Characterization And Genome Analysis Of Phage VB_KpnS_IME279 And Phage A18P4

Objectives: The biological and genomic characteristics of phages of Klebsiella pneumoniae that are pathogenic to humans and and Aeromonas hydrophila that are pathogenic to humananimal-fish were studied.It will provide theoretical basis and data support for the future application of phage therapy and the further development of phage genomics.Methods:Phage vB_KpnS_IME279 was isolated from Beijing 307 hospital sewage by using Klebsiella pneumoniae(530 strain)as indicator bacteria,meanwhile using Aeromonas hydrophila(A18 strain)as indicator bacteria,phage A18P4 was isolated from the sewage of Nanjing Zhongcai logistics Aquatic Market phage.By observing the plaques produced by the two phages after lysing the indicator bacteria on a double-layer plate,the morphology of the two phages was observed under a transmission electron microscope.In addition,the biological characteristics of these two phages were analyzed by determining the optimal multiplicity of infection,the one-step growth curve,the sensitivity of the phages to temperature and pH,and the phages lysis spectrum.The genomic characteristics of the two phages were analyzed by the second generation high-throughput sequencing technology,the whole genome sequence of the sequenced phage was annotated and the evolutionary tree was constructed.The end of phage genome was analyzed by bioinformatics method.Results:(1)In this study,Klebsiella pneumoniae(strain 530)isolated from the urine of hospital patients was used as an indicator to isolate a strain of Klebsiella pneumoniae phage from the hospital’s sewage,named vB_KpnS_IME279.The experimental results showed that the phage lysed the host bacteria to form a transparent and uniform plaque.Tansmission electron microscope images showed that the phage vB_KpnS_IME279 belonged to the family Podoviridae.The optimal multiplicity of infection for phage vB_KpnS_IME279 was 0.1,and the one-step growth curve showed that the incubation period of the phage was about 20 min,and the lysis amount of the phage during the lysis period of 80 min was about 138PFU/Cell.The phage was more sensitive to high temperature and strong acid and alkali.The phage has a narrow cleavage spectrum,and the ST and wzi genotypes of Klebsiella pneumoniae that can be lysed are ST11,ST37,ST375 wzi209,wzi52,wzi12 and wzi72,respectively.The whole genome sequence of the phage was obtained by high-throughput second-generation sequencing technology.The phage’s genome sequence was double-stranded linear DNA with a full-length genome of 42621 bp and a GC content of 59.3%(GenBank accession number: MF614100.1).After RAST online annotation,61 open reading frames(ORF)were obtained,of which 41 ORFs encoded hypothetical proteins,and the remaining 20 ORFs encoded functional proteins.BlastN nucleic acid alignment results showed that the phage vB_KpnS_IME279 had low homology with other phages in the NCBI library.(2)Using Aeromonas hydrophila(Strain A18)as an indicator,an Aeromonas hydrophila phage A18P4 was isolated from the sewage of Nanjing Zhongcai logistics Aquatic Market.Transmission electron microscopy showed that the phage belonged to the family Siphoviridae.The optimal multiplicity of infection of phage A18P4 was 0.1.The one-step growth curve showed that the incubation period of the phage was 1 h,and the amount of lysis during the 8 h lysis period was 202 PFU / Cell.The survival rate of phage was higher at 40-60℃,and the phage was more stable at pH of 4-11.In addition,the phage has a wide lysis spectrum.The total length genome of phage A18P4 obtained by second-generation high-throughput sequencing was 60795 bp,which is a linear double-stranded DNA structure with a GC content of 62%(GenBank accession number MN317029.1).After online annotation by RAST,73 ORFs were obtained,of which 48 ORFs were predicted to encode hypothetical proteins,and the other 25 ORFs encoded functional proteins.Using the end analysis technology established in our laboratory for analysis,the results show that there is no fixed end in the phage,which indicated that the ends gene of the phage was random.Conclusion:(1)In this study,the Klebsiella pneumoniae phage vB_KpnS_IME279 belongs to the family Podoviridae.The phage has a short latent period,strong lysis,and a narrow lysis spectrum.The whole genome of the phage was 42621 bp,contains 61 ORFs,and no toxins gene.(2)Aeromonas hydrophila phage A18P4 belongs to the Chivirus of the Siphoviridae family.The phage has a long latent period,strong lysis and a wide lysis spectrum.The bacteriophage has a genome length of 60795 bp and contains 73 ORFs.