Characteristic Of Mcr-1-bearing Plasmids In Escherichia Coli From Pigs

In recent years,as an antibiotic of last resort against multidrug-resistant infections,the increasing positive rate of colistin resistance gene mcr-1 in Enterobacteriaceae has become a public problem in the world.Mcr-1 mainly spreads between bacteria from different origins（human,animal and environment）through plasmids on five continents around the world.There were also 11 genetic variants of mcr-1 and other mcr-like genes（mcr-2,mcr-3,mcr-4 and mcr-5）.Currently,the IncI2,IncX4 and IncHI2 plamids are the most popular plasmid types carrying with mcr-1.Twenty-seven mcr-1-positive E.coli strains from a pig farm in Shanghai were selected as the research object in this study,which is used to evaluate the impact of diverse plasmids bearing colistin resistance gene mcr-1 on host fitness and the structural characteristics of IncI2,IncX4 and IncHI2 plamids carrying with mcr-1.And all these are further analysed by the plasmid sequencing,to provide a basis for controlling the rapid spread of the mcr-1 gene.Firstly,mcr-1-positive plasmids with diverse struction were selected for transformation experiments,and E.coli DH5αwas used as the recipient strain to obtain transformants carrying with plasmids pHNSHP45（IncI2）,pHNSHP23（IncX4）,pHNSHP45-2（IncHI2）,pHNSHP17（IncY）and pHNSHP24（IncFII）.Secondly,the stability of the plasmid was measured by serial transfer for 14 days.pHNSHP45,pHNSHP45-2,pHNSHP23,and pHNSHP17 maintains stable in E.coli in an antibiotic-free environment and plasmid loss occurred in transformant DH5α/pHNSHP24 from day 5.Then growth curve was determined by 12 h culture,no obvious difference in growth was observed between the transformants and the recipient strain after 12 h assessment.Finally,through competition experiments in vitro,it was obversed that fitness increased for the mcr-1-bearing IncI2 and IncX4 plasmid at 96 h（91%and 28%,respectively）,whereas the mcr-1-bearing IncHI2,IncY and IncFII plasmid presented fitness cost for its host.IncY and IncFII plasmid showed a slight fitness increase at the beginning（2% and 11%,respectively）,but the fitness was vanished following continuous growth.Compared with the IncHI2 and IncX4 plasmids,the Incl2 type plasmid was more beneficial to the host E.coli DH5α,while IncHI2 plasmid was relative to a significant competition disadvantage versus IncI2 or IncX4 plasmid.Mcr-1-positive IncI2（pHNSHP8,pHNSHP16 and pHNSHP50）,IncX4（pHNSHP10,pHNSHP23 and pHNSHP49）and IncHI2（pHNSHP26,pHNSHP41,pHNSHP47 and pHNSHP48）plasmids were randomly selected for sequencing analyse in this farm.The IncX4-type plasmids,pHNSHP10,pHNSHP23 and pHNSHP49,showed highly similar to the previousiy reported mcr-1-bearing IncX4 plasmids.pHNSHP8,pHNSHP16 and pHNSHP50 showed 99%coverage to our previous reported IncI2 plasmids pHNSHP45.pHNSHP45-2,pHNSHP26 and pHNSHP47,had same drug resistance regions and they contained numerous resistant genes.In addition to pHNSHP48 which did not contain bla_CTX-M-14TX-M-14 and fosA3,other resistant genes were consistent with above three IncHI2plasmids.Compared to others,part of the conjugative tranfer regions（htdZKLVT and trhBCELKV）was absent in pHNSHP48.In conclusion,stability of plasmids,competitive advantage,high engaging frequency or co-selection by other antimicrobials might contribute to the dissemination of the three epidemic mcr-1–positive plasmids（IncI2,IncX4 and IncHI2）in this farm.