Prevalence And Characteristics Of Multi-drug Resistant Plasmids IncHⅠ2 And IncFⅡ33 In Escherichia Coli From Chicken

The development of bacterial resistance has become increasingly severe in recent years,especially the emergence and prevalence of multidrug-resistant,extensively drug-resistance and pandrug-resistant bacteria,leading a global public health problem.As a vectors of antimicrobial resistance genes,plasmids can spread drug resistance among bacteria of different regions,origins and species,such as IncHⅠ2 and IncFⅡ plasmids,the main popular plasmids in Enterobacteriaceae bacteria,which can spread drug resistance through horizontal transmission.IncFⅡ33,belonging to IncFⅡ plasmid subtype,is one of the popular plasmids in Escherichia coli from domestic animals.Evidence has showed that some important resistance genes are located on IncHⅠ2 and IncFⅡ plasmids,such as blaCTX-M,fosA3,floR,tetA,oqxAB and mcr-1.Therefore,it is necessary to explore the prevalence and characteristics of IncHⅠ2 and IncFⅡ in Escherichia coli from animals to provide a theoretical basis to slow down the development of multidrug-resistant bacteria.The prevalence of IncHⅠ2 and IncFⅡ33 plasmids in 6173 animal-derived E.coli of different years,regions and origins was investigated.It was found that the detection rate of IncHⅠ2 among all years was relatively stable,and the detection rate of IncFⅡ showed a downward trend,while the positive rate of IncFⅡ33 showed a slow upward trend after the year of 2007.Specifically,the detection rates of IncHⅠ2 and IncFⅡ in 174 E.coli from a chicken farm in Anhui from 2015 to 2018 were 31.61%(55/174)and 52.30%(91/174),respectively.34.07%(31/91)IncFⅡ plasmid belonged to IncFⅡ33 subtype,and its detection rate was on the rise.Additionally,The detection rates of IncHⅠ2 and IncFⅡ33 in E.coli of different regions were significantly higher than those from other sources.The antibiotic sensitivity test revealed that IncHⅠ2 and IncFⅡ33-positive strains had a significantly higher resistance to cefotaxime,cefotaxime,florfenicol,colistin,fosfomycin,and other drugs than those strains that did not carry these two plasmids.The result of PFGE typing showed that except some strains that might be clonal transmission,other strains carrying IncHⅠ2 and/or IncF33 plasmids were non-clonal strains,suggesting that there may be a long-term horizontal transmission of IncHⅠ2 and IncFⅡ33 plasmids in this farm.Conjugation and transformation experiments were performed on IncHⅠ2 and IncFⅡ33-positive strains,and 17 transconjugants and 18 transformants were obtained.Among them,the size of IncHⅠ2 plasmid was 180 kb to 336.5 kb,and harbored blaCTX-M-14(n=8),blaCTX-M-55(n=2),blaCTX-M-65(n=2),floR(n=14),rmtB(n=2)and mcr-1(n=3)and others.The size of IncFⅡ33 plasmid ranged from 54.7 kb to 160 kb and mainly harbored blaCTX-M-55(n=15),fosA3(n=16)and rmtB(n=8).The factors influencing the prevalence of these two plasmids were studied by performing conjugation frequency assay and stability assay.The conjugation frequency of IncHⅠ2 and IncFⅡ33 plasmids were 10-6-10-4 and 10-3-10-2,respectively.Without drug selection pressure,the stability experiments of IncHⅠ2 and IncFⅡ33-positive strains were carried out for 32 days,and it was found that the IncFⅡ33 plasmid was still stable while the loss of IncHⅠ2 plasmid was obvious.WGS showed that the backbone of two IncHⅠ2 plasmids pHNA8M074 and pHNA8M068-1,and one F33:A-:B-plasmid pHNA8M068-2,were relatively conserved.However,the variable regions were significantly different due to various insertion elements,resulting from rearrangement,insertion,and deletion of DNA molecules.In summary,IncHⅠ2 and IncFⅡ33 in E.coli were the main popular plasmids in the chicken farm,which mediated the spread of resistance genes,such as blaCTX-M,fosA3,floR,tetA,oqxAB and so on.Compared with IncHⅠ2 plasmid,IncFⅡ33 had a good stability and high conjugation frequency,which could promote a wide dissemination of drug resistance between different bacteria.In addition,these plasmids could harbor mutiple drug resistance genes by mobile elements,thus under the selective pressure of antibiotics,these multiple drug-resistant plasmids could be prevalent in this farm.