Isolation And Identification Of Bacteriophages To Multiple Drug Resistant Escherichia Coli And Therapeutic Study Of Cocktail

Diseases caused by Escherichia coli(E.coli)are very common in human beings and animals.High morbidity and mortality of E.coli cause serious economic loss,leading to an annual increase and abuse of antibiotics,but E.coli rapidly evolves resistance to antibiotics through various mechanisms,especially the emergence of multiple drug resistance(MDR),which makes epidemic diseases and intractable treatments much more serious.In recent years,bacteriophages have regained their popularity as antibiotic replacement therapy with their advantages of high lysis efficiency,safety,abundant sources and low cost.Firstly,we used 10 antibiotics for the determination of the minimum inhibiting concentration(MIC)to analyze the MDR of 100 E.coli strains isolated from duck cases.Results showed that the resistance rate reached 58.7%,the susceptible rate was only 37.5%,and the MDR rate up to 98%,indicating the severe situation of AMR.Screening by 2 MDR-E.coli isolates and ATCC 25922 strain,8 bacteriophages were obtained from numbers of water and feces samples,YZG5、TXC53、YZG5 53、JW5、ATD5、YZGJ2、JW2 and ATD25 were designated,respectively.The lysis spectrum of 100 E.coli was tested by all phages,in which ATD5,JW5 and YZGJ2 lysis rate up to 58%,58%and 52%,respectively.It should be noted that the host bacteria of ATD5,JW5 and YZGJ2 were all MDR-E.coli.Instead,the lysis spectrum of 2 bacteriophages targeted to ATCC 25922 was narrower in those phages.We mixed phages complementing on the lysis spectrum as cocktail EC1(YZGJ2+JW5)and EC2(YZGJ2+JW5+ATD5+YZG553+TXC553)before testing their lysis rate of 100 E.coli,which were reached to 75%and 95%.Three phages with the highest lysis rate were observed by transmission electron microscopy(TEM),and according to the guidelines proposed by the International Committee on Taxonomy of Viruses(ICTV),all three phages belonged to the order Caudovirales,the family Myoviridae,but ATD5 and JW5 belonged to the A1 morphology group whileYZGJ2 belonged to the A2.Next,we tested the biological characteristics of ATD5,JW5 and YZGJ2.The best optimal multiplicity of infection(MOI)was 1 for JW5,ATD5 was 10 and YZGJ2 was 0.001,respectively.One-step growth curve was determined with the best MOI,in which the incubation period of all phages was 10 min and the burst duration of 10-60 min.YZGJ2 was more acid tolerant than ATD5 and JW5 while all of them were intolerant to strong alkali,what’s more,they showed higher and more stable activity at pH4-9.Their activity remained stable at 40-60℃,ATD5 and YZGJ2 decreased at 60-70℃,and even JW5 lost its activity.The results of in vitro planktonic cell lysis assay of phages at different MOIs against host bacteria proved that phages have a better lysis effect on bacteria,while OD630 of the positive control group continued to rise within 12 h and OD630 of phages treated group generally showed a decreasing trend after 7 h.Besides,OD630 within 12 h was consistently lower than the positive control group.Observing the formation of E.coli biofilm after being treated with phages,we found that OD630 was lower than the positive control group,indicating that phages inhibited biofilm formation.ATD5 and YZGJ2 were sequenced by Illumina,results showed that they were all double-stranded DNA of 70,217 and 167,544 bp in length with GC contents of 41.28%and 35.53%,respectively.After searching with the National Center for Biotechnology Information ORF finder database,299 and 278 putative open reading frames were predicted by ATD5 and YZGJ2.71 and 264 coding sequences of ATD5 and YZGJ2 were predicted by Prokka,respectively.Finally,gene function annotations were predicted by UniProt,KEGG,go,RefSeq,and NR databases.The therapeutic ability of phage on ducklings and mice was tested after establishing the infection model with E coli 53,DJ2,3.Three strains of E.coli were mixed by median lethal dose and injection via different times and different numbers of administrations,results showed that the best therapeutic efficacy of phage by intraperitoneal injection around E.coli infection within an interval of 0-1 h,as well as improvement by continuous administration.Although results indicated that phagotherapy was not effective as expected,but also provided ideas for application to the clinical treatment of avian E.coli infection.