Study On The Effect Of Bacteriophage On The Horizontal Transfer Of Escherichia Coli Resistance Genes

World Health Organization had characterised antimicrobial resistance as a global public health crisis.Bacteria can acquire antibiotic resistance through chromosomal mutations or horizontal gene transfer,and the latter is the main cause of antibiotic resistance transmission.This process is generally driven by means of transformation,conjugation and transduction.The role of bacteriophages as vehicles for antibiotic resistance genes(ARGs)is one of the vital approaches of horizontal transfer of bacterial genes,but it is always ignored.The horizontal transfer of ARGs was evaluated through both virulent phage and lysogenic phage in this paper.On the genomes of virulent phages and lysogenic phage,we all have found a nonnegligible ARG fragments.Bacteriophage play a vital role in the horizontal transfer of ARGs,the transduction cannot be ignored.In this paper,135 strains E.coli were isolated from pig feces in a pig farm in Guangdong.Using these 135 strains E.coli as host bacteria,two virulent phages were successfully isolated from the wastewater pool of the same farm,they were named HZP2 and HZ2R8.Their hosts are HZA135 and HZA50 respectively.Both HZP2 and HZ2R8 belong to the Podoviridae family,with stable biological characteristics and the ability of rapid propagation.The average burst size of HZP2 and HZ2R8 are 120 pfu/cell and 93 pfu/cell,respectively.HZP2 genome was 32448 bp in length with a GC content of 48.30%and the accession number is MK542821.HZ2R8 genome was 39482 bp in length with a GC content of 48.61%and the accession number is MG832642.The genome sequence structure and composition of HZP2 and HZ2R8are similar.HZP2 has 43 annotated open reading frames,and HZ2R8 has 38 annotated open reading frames.The open reading frames are mainly divided into lysis module,DNA replication and regulation module,packaging and structure module,and host suppression module.The phylogenetic tree based on the large subunit of the phage terminase shows the close evolutionary relationship between HZP2 and HZ2R8,which was just consistent with the fact that HZP2 and HZ2R8 were isolated from the same wastewater pool.The host bacteria were carried out for antimicrobial susceptibility test.According to the results of the antimicrobial susceptibility test,the related ARGs were quantified by q PCR.The virulent phages were enriched at the MOI of 10:1,and transduction frequency(TF)and transduction ability(TA)were calculated to further evaluate the level of horizontal transfer of ARGs by the virulent phages.The results show that the TF of HZP2 for ARGs is between4.09E-6 and 4.36E-3 and the TF of HZ2R8 for ARGs is between 2.49E-6 and 6.22E-5.The TA of HZP2 for ARGs is sul3(1.80E-2)>tetM(3.21E-3)>mcr-1(2.23E-3)>bla_TEM(1.28E-3)>cmlA(9.52E-4)>qnrS(2.28E-4)and the TA of HZ2R8 for ARGs is mcr-1(8.21E-4)>bla_TEM(2.51E-4)>cmlA(2.40E-4)>qnrS(7.96E-5).The lysogenic phage was induced by Mitomycin C(MMC),and the lysogenic phage genomes were extracted.PCR was used to evaluate the level of horizontal transfer of ARGs by the lysogenic phage.First,12 specific ARGs were detected by PCR,and 112 strains E.coli carrying three or more ARGs were screened.Furthermore,the induction test was carried out under the condition that the final concentration of MMC was 1?g/m L and the induction time was 12 h.A total of 97 strains of E.coli were successfully induced to obtain lysogenic phage.The PCR results showed that 8 ARGs(mcr-1,bla_TEM,erm B,qnrS,flo R,aac-(6?)-lb-cr,tetM and sul1)were detected in the lysogenic phage genomes,indicating that these eight ARGs may have random horizontal transfer between E.coli and lysogenic phage.Among them,qnrS was detected both in the genomes of 63 strains E.coli and 46 lysogenic phage,the resistance transduction ratio(RTR)was as high as 73.02%,which indicated that qnrS is easy to be transduced by lysogenic phage.mcr-1 was detected both in the genomes of 53 strains E.coli and 19 lysogenic phage,the RTR was as high as 35.85%,which indicated that the transduction effect of lysogenic phage on mcr-1 cannot be ignored.In general,this paper evaluated the influence of E.coli phage on horizontal transfer of ARGs to some extent through both virulent phage and lysogenic phage,and also made a theoretical basis for the follow-up study on phage transduction mechanism and even the retransfer of antimicrobial resistance through phage transduction.