Isolation And Characterization Of Phages Against Multidrug-resistant Escherichia Coli And Pseudomonas Aeruginosa

Objectives:Escherichia coli(E.coli)is the most common opportunistic pathogenic bacteria in clinical.It has a wide range of infection sites,and some strains have virulence factors,which can cause intestinal infection.E.coli can also invade intestinal tissues or organs and cause extra intestinal infections.Pseudomonas aeruginosa is an important opportunistic pathogen causing nosocomial infection,which can cause many diseases in clinical practice.In recent years,the widespread use and irrational application of antibiotics have made the resistant strains increasing and the bacterial resistance is becoming more and more serious,especially the clinical monitoring separation rate of multidrug resistant Escherichia coli and Pseudomonas aeruginosa strains has also increased year by year,which has brought great difficulties to the prevention and treatment of infectious diseases.Bacteriophages are being reconsidered for their use in killing bacterial.Bacteriophage is a kind of virus that can specifically infect bacteria.It is widely existed in environment and organism.Some bacteriophages have the ability to kill bacteria,and the application of phage therapy has attracted more and more attention.A lots of new phages were discovered and studied well recently.Therefore,we identified and characterized the bacteriophages of B2 and YP3..Escherichia coli phage B2 and Pseudomonas aeruginosa phage YP3 are virulent phages.Genomic and proteome analysis show that B2 and YP3 do not encode genes associated with toxins or other virulence factors.B2 and YP3 significantly reduced the growth of their host multi-drug resistant clinical stains.And YP3 can significantly reduce the mouse infection in vivo.Further well study the interaction between these phages and their host,this phages scould be used to interrupt or reduce the spread of mutidrug resistant E.coli and Pseudomonas aeruginosa.Methods:1.Escherichia coli and Pseudomonas aeruginosa bacteriophages with good lysate bacteria were isolated and screened from waste water of Nanjing by using the strain MG1655,PAO1,PA14 and clinical strains of Escherichia coli and Pseudomonas aeruginosa isolated from patients.The bacteriophages of Escherichia coli and Pseudomonas aeruginosa were named as: B2 and YP3 respectively.2.By continuously picking up plaque and infecting host bacterium suspension,we first purified phage and observed its plaque morphology,growth pattern and host range identification.3.Molecular cloning technology was used for preliminary phage genomics analysis.4.Ultra high speed cesium chloride density gradient centrifugation was used to purify phage B2 and YP3.The morphology were observed by electron microscopy.Growth curve,temperature stability and bactericidal curve were determined.5.Phage proteins were detected and analyzed by SDS-PAGE and mass spectrometry.6.Sequencing and analysis of the whole genome of phage using next-generation sequencing.7.Amouse model of Pseudomonas aeruginosa lung infection was established,YP3 was used to treat the lung infection in mice caused by Pseudomonas aeruginosa.Results:New bacteriophages were isolated and screened by using the strain MG1655 and the P2321 isolated from clinical patient,named as B2 and YP3 respectively.First,the biological characteristics of phage are studied: B2 is a specific lysis Escherichia coli and YP3 specific lysis Pseudomonas aeruginosa,both of which form a clear and transparent phage plaque;is found to Phages morphology illustrated that they belongs to the family Siphoviridaey and Myoviridae family respectively.The bacteriophage B2 and YP3 can maintain stable activity in a wide range of temperature,that the phage has good temperature stability and easy to preserve and transport.The one step growth curve indicates that the incubation period of phage B2 is short,the explosive amount is large and the replication ability is strong;the latent period of phage YP3 is short,and the bacteriophage can be killed in a short time.Secondly,wedetected the bacteriophages' host spectrum and bactericidal efficacy: B2 can cleave 7 clinically randomly separated multidrug resistant Escherichia coli strains(cracking rate: 19%),and YP3 can cleave 15 strains of Pseudomonas aeruginosa(cracking rate: 75%);bacterial challenge experimentactually verified that B2 and YP3 can effectively inhibit their host bacteria and kill bacteria in a short time.Therefore,bacteriophages B2 and YP3 become good candidates for biological control of the corresponding pathogens.In addition,we detected and analyzed the genome and the structural protein of virus B2 and YP3.The full length of the genome of phage B2 is 44283 bp,consisting of cyclic double stranded DNA,with a GC content of 54.77% and 65 open reading frames(ORF).The 25% functional proteins are divided into five functional groups: DNA replication or modification,host cleavage,assembly,structural protein and additional functions.Phage B2 is a kind of lytic phage,and genomic analysis shows that B2 does not encode genes associated with toxins or other virulence factors,which have potential for application in phage therapy.21 proteins of B2 were identified by mass spectrometry,of which 12 structural proteins of 13 known structural proteins were identified,and some hypothetical proteins were identified.The evolution of B2 and its related homologous phage genome shows that horizontal and vertical sequence rearrangement may participate in the evolution of the phage genome,and then participate in the evolution process of phage adapted to the host bacteria.The total length of the genome of phage YP3 is 92709 bp,consisting of linear double stranded DNA,GC content of 49.4%,190 open reading frames(ORF),and 63 proteins identified by mass spectrometry,including known structural and hypothetical proteins.The study found that the genome of YP3 encodes many hypothetical proteins,which may contain antiseptic enzymes,and the full sequence analysis of phage genome is also beneficial to further study the interaction between phage and host,which lays the foundation for the real application of phage against multi resistant Pseudomonas aeruginosa infection.Finally,in the mouse lung infection model caused by multidrug-resistant Pseudomonas aeruginosa strain,the survival rate of infected mice was obviously enhanced after the addition of phage YP3.It was proved that YP3 could inhibit the infection caused by Pseudomonas aeruginosa in vivo.