The Co-dissemination Mechanism Of Bla_CTX-M And Mcr-1 In Escherichia Coli From Chicken

In recent years,Escherichia coli(E.coli)that simultaneously detected bla_CTX-M and mcr-1 has been reported,but the genetic background and transmission mechanism of these in E.coli from chicken in Henan province remained unclear.Therefore,in this study,E.coli from chicken carrying both bla_CTX-M and mcr-1 in Henan province as the purpose to explore the common dissemination mechanism of these,and to provide theoretical basis for clinical prevention,control and treatment.In this experiment,among 14 clinical isolates of E.coli from chicken,12 bacteria strain were detected to carry both bla_CTX-M and mcr-1.and 9 of them were screened to obtain conjugations containing both bla_CTX-M and mcr-1.The susceptibility results showed that 9 clinical isolates and transconjugants appear different degrees of resistance to cefotaxime,colistin,aminoglycosides,tetracyclines,amide alcohols and quinolones.The blacCTX-M typing result showed that all the 9 clinical isolates carry 1-2 blaCYX-M subtypes,which belong to bla_CTX-M-1 subgroups(bla_CTX-M-55 subtype)and bla_CTX-M-9 subgroups(bla_CTX-M-14 and bla_CTX-M-27 subtypes),and only a single bIaCTX-M subtype was detected by the corresponding transconjugant.At least two replicon types were detected in clinical isolates and transconjugant(except for transconjugant LWY6J),and the main replicon types were IncHI2 and IncI1.Through S1-PFGE and WGS,the genetic maps of isolates LWY24 and LWY6 and their plasmids were analyzed.The results showed that there are 4 plasmids in LWY24,including three conjugative plasmids which IncFII plasmid pLWY24J-3 carrying bla_CTX-M-55,blaTEM-1b and rmtB,IncI2 plasmid pLWY24J-mcr-1 carrying mcr-1,and Incll plasmid pLWY24J-4 without resistance.And a non-conjugative IncHI2/IncHI2A plasmid pLWY24 which carrying 15 resistance gene of bla_CTX-M-14,fosA3,floR and so on.There were two conjugative plasmids simultaneously in LWY6,one was IncHI2 plasmid pLWY6J-MC,which carried 11 resistance genes such as fosA3 and sul2 in addition to mcr-1 and bla_CTX-M-14,the other was IncF? type non-resistance plasmid pLWY6-2.At the same time,LWY6 also carried bla_CTX-M-55 on the chromosome.In addition,the above resistant plasmids all carried a large number of insertion sequences and transposons.indicating that horizontal transmission of bla_CTX-M and mcr-1 was mainly through plasmid conjugation,rearrangement and transposition.By re-conjugative assay,determination of growth curve,plasmid stability and competitive assay to evaluate the adaptability of the two plasmids and influence each other in the process of transfer.The results showed that under different screening conditions,received six carry different plasmids combination of transconjugants,which were C600F?(only carrying IncF? plasmid containing bla_CTX-M-55),C600F?+I1(carrying IncFII plasmid and IncI1 plasmid non-resistance),C600I2(only carrying IncI2 plasmid containing mcr-1),C600I2+I1(carrying IncI2 plasmid and IncI1 plasmid),C600F?+I2(carrying IncF? plasmid and IncI2 plasmid),C600F?+I2+I1(carrying the three plasmids).The results of growth curve showed that the addition of plasmids had no significant effect on the growth rate of the recipient C600.Re-conjugation experiments showed that compare with single plasmid(C600F?+I2),the conjugation frequency of pLWY24J3 was significantly increased(p<0.05).At the same time,in the presence of pLWY24J3(C600F?+I1),conjugation frequency of pLWY24J-3 was significantly decreased(p<0.0001),however,when the third plasmid pLWY24J-mcr-l coexisted(C600F?+I2+I1).conjugation frequency of pLWY24J-3 was significantly increased(p<0.0001).Plasmid stability results showed that compared with pLWY24J-3 alone(C600F?),the stability of pLWY24J-3 and pLWY24J-mcr-1 coexisting(C600F?+12)was significantly improved.In vitro competition experiments showed that the acquisition of plasmid pLWY24Jmcr-1 did not cause considerable adaptability cost to pLWY24J-3.In summary,in the coexistence of bla_CTX-M and mcr-1,the horizontal transfer modes were diverse,which can be transferred simultaneously or separately,and plasmid conjugation,transposition of insertion sequences and rearrangement were the main reasons for the rapid spread of bla_CTX-M and mcr-1.Coexistence of plasmids pLWY24Jmcr-1(IncI2)and pLWY24J-3(IncF?)improved the conjugation transfer and stability of pLWY24J-3.