Isolation And Characterization Of Bacteriophages Against Three Common Pathogens Of Hospital-acquired Pneumonia

ObjectiveTo isolate and purify bacteriophages of common pathogens associated with hospital-acquired pneumonia from hospital sewage,observe its morphology,study its host range,biological and genetic characteristics,to lay an experimental foundation for the clinical application of bacteriophage.MethodsUsing six standard strains as the host bacteria to isolate and purify bacteriophage from hospital sewage by the double-layer agar plate assay,which was Acinetobacter baumannii ATCC17978,Pseudomonas aeruginosa ATCC27853,Staphylococcus aureus ATCC25923,and ATCC29213,Klebsiella pneumonia ATCC700603 and Stenotrophomonas maltophilia ATCC17666.Observing bacteriophage morphology by transmission electron microscope,determining its optimal MOI,one-step growth curve and host range.Sequencing the whole genome,and annotating gene function,studying gene structure,and analyzing the relationship of genetic evolution.Results1.Six bacteriophages were isolated and purified,including four strains of Stenotrophomonas maltophilia bacteriophages,named:vB_SmaM_SMP(1-4);one strain of Staphylococcus aureus bacteriophage,named:vB_SauH_SAP1;one strain of Klebsiella pneumonia bacteriophage,named:vB_KpnM_SYP2.Transmission electron microscopy observation shows they all had icosahedron head and a contractile tail,vB_SmaM_SMP(1-4)and vB_SauH_SAP1 belong to Al-shaped Myoviridae family,vB_KpnM_SYP2 belongs to A3-shaped Myoviridae family.2.The isolated Stenotrophomonas maltophilia and Staphylococcus aureus bacteriophages have strict species specificity.vB_SmaM_SMP(1-4)can only lysis Stenotrophomonas maltophilia,and the lysis rate is that vB_SmaM_SMP1:67%,vB_SmaM_SMP2:58%,vB_SmaM_SMP3:42%,vB_SmaM_SMP4:71%;vB_SauH_SAP1 can lysis Staphylococcus strains,such as Staphylococcus epidermidis,especially MRSA,with a lysis rate of 27%.3.The biological characteristics of vB_SmaM_SMP1,vB_SmaM_SMP3,and vB_SauH_SAP1 were studied.Their optimal MOI ware all 0.1.One-step growth curve showed that the latent phase was of vB_SmaM_SMP1 and vB_SmaM_SMP3 was 60-70 min,one whole growth cycle was about 140min,the burst size of vB_SmaM_SMP1 was 195PFU/Cell and vB_SmaM_SMP3 was 163PFU/Cell;vB_SauH_SAP1 has a high lysis efficiency,and the latent phase was only 10 min,and one whole growth cycle was about 30 min.The three bacteriophages were insensitive to chloroform,indicating that there were no liposomes in the capsid.The thermal stability test showed that the temperature higher than 60 ℃ had a great effect on the activity of the three bacteriophages.Both vB_SmaM_SMP1 and vB_SmaM_SMP3 have a wide stability range of pH and still have high activity at pH 2-10.vB_SauH_SAP1 can maintain a good activity only when pH is 5-8,but shows strong tolerance to an alkaline environment.4.The whole-genome sequencing showed that the genome of vB_SauH_SAPl was 143375 bp in length,with a G+C content of 30.2%,encoding 226 ORFs.No lysogen genes and known virulence-related genes or antibiotic resistance genes have been predicted.The function of the gene was deduced and divided into five modules:nucleotide metabolism and replication,morphogenesis,lysis,DNA packaging,and metabolism.The results were submitted to the GenBank database with the accession number:MT786458.Comparative genomic and phylogenetic analysis showed that it has high homology with Kayvirus Staphylococcus bacteriophages.ConclusionSix bacteriophages against Staphylococcus aureus,Klebsiella pneumoniae and Stenotrophomonas maltophilia were isolated,among which Staphylococcus aureus phage vB_SauH_SAP1 had a potential application value,four bacteriophages vB_SmaM_SMP(1-4)were morphologically specific to the isolated Stenotrophomonas maltophilia bacteriophages with a high burst size,which had the value of further research in the genome.