Isolation And Identification Of Lytic Bacteriophages Of A Strain Of Pseudomonas Aeruginosa And Genomics Study

Objective: Take Pseudomonas aeruginosa as the host bacteria,screen its lytic phage,investigate the biological characteristics of the phage,sequence the whole genome,and analyze the genome sequence information of the phage,in order to provide a new choice for the clinical treatment of drug-resistant Pseudomonas aeruginosa infection,reduce the abuse of antibiotics,and provide scientific basis for the treatment of other drug-resistant bacterial infections.Method:1.Drug sensitivity test was used to determine the drug resistance of the strain,and 16 S r DNA technology was used for molecular biological identification.2.Take drug-resistant Pseudomonas aeruginosa as the host,screen its lytic bacteriophage,and investigate the basic biological characteristics of the bacteriophage,including the observation of specific morphology by transmission electron microscope,the detection of its stability in different acid and alkali environments by setting different temperatures and environments,and the exploration of the optimal infection complex and one-step growth curve.3.DNA samples were extracted for whole-genome sequencing,ORF Finder was used to predict the open reading frame of bacteriophages,NCBI Protein BLAST was used to search for homologous protein genes,CG View was used to draw the whole genome circle of bacteriophages,Mauve software was used for collinearity analysis,MEGA X software was used to construct the phylogenetic tree,and TMHMM-2.0 and Signa IP4.1 were used to predict the transmembrane region and signal peptide of the functional gene lyase and perforin of bacteriophages,Ex PAsy website predicts the molecular formula,hydrophobicity and other physical and chemical properties,Di NNA1.1 predicts whether there is disulfide bond in the protein structure,and SWISS-MODEL and Rose TTAFold construct their tertiary structure and analyze their reliability.Result: 1.16 S r DNA test confirmed that the host strain was Pseudomonas aeruginosa,and drug sensitivity test confirmed that it was a multi-drug resistant strain.2.A Pseudomonas aeruginosa bacteriophage was successfully isolated from sewage v B_Pae S_HP1,potency 8 × 1010(PFU/m L);According to transmission electron microscope,it belongs to the family of short tailed bacteriophages;Wide temperature tolerance range,good activity at-20-60℃,good acid-base stability,p H range suitable for growth of 4-9,insensitive to chloroform;The incubation period is 0~30min,the cracking period is 30~80min,and the cracking amount is 120 PFU/infected cell;The Muhiplieity of Infection is 1.3.After sequencing,HP1 was confirmed to be double-stranded linear DNA with a total length of 40728 bp and a GC content of 62.4%;It is predicted that 520 ORFs,270 on the justice chain and 250 on the negative chain;It is predicted that 67 CDS,including 43 functional proteins and 24 hypothetical proteins,including 17 proteins related to DNA structure,15 proteins related to DNA transcription and translation,and 6 proteins related to DNA assembly,with a gene density of 1.65,are Phikmvvirus phages;No virulence factor and antibiotic resistance gene were found;The results of genome circle map showed that it had relatively complete gene structure;The four phage strains with high consistency between HP1 and its genome have good co-linearity and no gene mutation,but there is translocation;Lyase and perforin protein do not contain transmembrane domain structure;Lyase has no signal peptide structure,and perforin has signal peptide structure;The average hydrophilicity coefficient of endolysin is-0.090,which is hydrophilic protein,and the average hydrophilicity coefficient of perforin is 0.609,which is hydrophobic protein;There are three disulfide bonds in the protein structure of endolysin,no protein curl helix structure,and no disulfide bond in the protein structure of perforin,but there is one protein curl helix structure;In this experiment,the tertiary structures of endolysin and perforin were successfully constructed and their conformational rationality was confirmed.Conclusion: This experiment successfully screened a phage strain of Pseudomonas aeruginosa,and studied its biological characteristics and genomics related content to preliminarily prove its safety and effectiveness.It was determined that it can provide a new choice for the clinical treatment of drug-resistant Pseudomonas aeruginosa infections.