The Characteristics Of Heteroresistance To Colistin And Ceftiofur In Escherichia Coli Isolates From Animals

Heteroresistance is the intermediate state of susceptible bacteria developing into clinical resistant bacteria,and it is also one of the reasons for the failure of clinical antibacterial treatment.Therefore,in order to effectively prevent and control the infection of E.coli from animal origin and delay the acquisition of resistance to colistin and ceftiofur,it is particularly important to deeply analyze the epidemiological characteristics of colistin/ceftiofur heteroresistance in Escherichia coli.The mechanisms of colistin resistance in Escherichia coli mainly include plasmid mediated mcr genes and chromosome mediated two-component signal transduction systems PmrAB and PhoPQ and their negative regulatory protein MgrB.In order to compare the role of mgrB mutation and mcr genes to colistin resistance in Escherichia coli isolates from swine,the susceptibility determinations were assayed by a broth microdilution method and K-B disk.PCR for the mcr-1?mcr-5 genes were performed on colistin resistance E.coli.Mutations in the mgrB gene and mgrB promoter regions were analysed.The results showed that 147 strains were resistant to colistin with the resistance rate of 83.1%(147/177).Among the 147 tested bacteria,146 strains were detected with mcr-],none of the mcr-2?mcr-5 genes were detected,and the mcr genes were not detected in EPF25 bacteria.PCR amplification and sequencing of the mgrB gene showed that 115 strains of mgrB were mutated,among which 104 strains were mutated in the promoter region of mgrB and 11 strains were mutated in the amino acid coding region of mgrB.Comparing the resistance phenotype of colistin to tested bacteria and the relationship between mcr-1 gene and mgrB mutation in resistant bacteria,it was found that the combined action of mgrB and mcr-1 was more likely to cause colistin resistance in Escherichia coli.K-B disk method was used to detect the susceptibility determinations of 177 strains of E.coli isolates from swine.It was found that 12 tested strains had scattered colonies in the inhibition zone of colistin,which was suspected to be colistin heteroresistance.The mechanisms of colistin heteroresistance(CHR)were assessed in 12 tested strains.CHR was investigated by population analysis profile(PAP)tests and CHR stability was studied.At the same time,mcr-1 gene,two-component signal transduction systems PmrAB,PhoPQ and its negative regulatory protein MgrB amino acid mutation were detected and verified by RT-qPCR.The results showed that 11 of the 12 tested strains were CHR strains,and each CHR strain contained more than one resistant subpopulation.At the same time,11 of the 17 subpopulations(64.71%)harbored point mutations in pmrB and/or phoQ,differing from their parental isolates.among which,only one stable resistant subpopulation(EPF42-4)was identified,it harbored an arginine-to-proline substitution at position 93(R93P)within the pmrB.Compared to the pmrB expression levels in the parental isolate EPF42 and E.coli K-12 MG 165 5,remarkable pmrB overexpression was observed in EPF42-4,which showed upregulated pmrA,pmrK,and pmrC expression.Structural analysis demonstrated that the R93P substitution promotes conformational changes in the HAMP domain,leading to an acceleration in its signal transduction ability and the activation of pmrB expression,which led to the EPF42-4 strain was resistant to colistin.In conclusion,point mutations in pmrB and/or phoQ were primarily associated with CHR.The R93P substitution resulted in the establishment of stable resistant subpopulations in E.coli showing CHR.?-lactams are commonly used in the treatment of bacterial infection in Enterobacteriaceae,while colistin is often used to treat the infection caused by Enterobacteriaceae,which is resistant to ?-lactams.Therefore,33 strains of E.coli were isolated from 110 samples collected from healthy poultry,and the HR were screened.At the same time,the formation reasons were explored.The results showed that 15 of 33 strains were susceptible to ceftiofur.Two strains of ceftiofur heteroresistance(number 5 and 10)were screened by PAP tests,and each of them contained a dominant subpopulation(5S and 10S)and a stable resistant subpopulation(5R and 10R).PFGE and MLST confirmed that the number 5 and 10 were polyclonal heteroresistance to ceftiofur.In vitro competition experiments showed that compared with the stable resistant subpopulations 5R and 10R,the 5S and 10S had significant competitive advantages.The serotypes of 5R and 10R were 025,and they carried tet(A),qnrs and blaCTX-M-15 genes.The results of I-Ceul-PFGE and southern blot showed that blaCTX-M-15 was located on the chromosome genome.The results of whole genome sequencing showed that the structure of the skeleton region of 10R chromosome was highly similar to that of E.coli 6409 from human excreta in Colombia.The main difference was that two multidrug-resistant regions with length of 3,055 bp and 16,998 bp were inserted into 10R chromosome.The formation of ceftiofur polyclonal heteroresistance is mainly due to the obvious competitive advantages of sensitive bacterias,and the insertion of multidrug-resistant regions is the main reason for stable resistant subpopulations to acquired resistance.In conclusion,colistin/ceftiofur heteroresistance have existed in E.coli isolates from animal.At the same time,point mutations in pmrB and/or phoQ were primarily associated with CHR.The R93P substitution resulted in the establishment of stable resistant subpopulation in E.coli showing CHR.At the same time,it is the first time to find ceftiofur polyclonal heteroresistance from Escherichia coli,but whether it has monoclonal heteroresistance need to be further studied.