| Polymyxin was used to treat gram-negative bacterial infections in clinical treatment and animal husbandry m 1950s.But it was substituted by novel antibiotics due to its neurotoxicity and nephrotoxicity in 1980s.Polymyxin was emphasized again as ’the last defense for human to fight the infections of multi-antibiotic resistant gram-negative bacteria’,especially the carbapenems·resistant Enterobacteriaceae.The separation rate of mcr-1 gene from animal-derived bacteria was increased every year after the plasmid-mediated polymyxin resistance gene mcr-1,which located in an epidemic plasmid with high conjugate frequency,was reported in 2015,China.And all the reported mcr-1 positive strains were isolated from various sources.Furthermore,mcr-1 gene can proliferate through animals,human and environments by epidemic plasmids.The Escherichia coli was chosen in this study because of its extensive existence in environments,and six kinds of samples including nasal swabs,anal swabs,feces,water,dust and clay were collected from farm A and farm B in Jiangsu Province.83 mcr-1 positive E.coli strains were isolated from the samples.Their MICs of 8 antibiotics which belong to 8 different types were tested by plate dilution method while colistin was tested by micro broth dilution method and other 14 antibiotic resistance genes were detected by PCR to analyze the epidemic status,including mcr-2,mcr-3,mcr-4,mcr-5;fosfomycin resistance genes:fosA,fosA3,fosC2;tetracycline resistance genes:tet(A),tet(B),tet(C),tet(D),tet(M);carbapenems resistance gene:blaNDM-1 and florfenicol resistance gene:floR.Six types of plasmids including IncI2,IncHI2,IncX4,IncP,IncF and IncN were also detected by PCR along with the circular intermediate’ISApll-mcr-1-pap2’,PFGE and conjugation tests were performed to the 83 mcr-1 positive strains and another 56 mcr-1 positive E.coli stocked by our laboratory,in order to understand the genetic relationship between the mcr-1 positive strains and the transmission mechanisms of the mcr-1 gene.The antibiotic susceptibility results showed that the strains conferred extensive resistance to antibiotics which could propagate horizontally.The mcr-1 positive E.coli isolated from various samples showed different resistance rates against 8 types of antibiotics except meropenem.Both of the strains isolated from the two farms had the similar resistance rates of florfenicol and amoxicillin above 94%,while the resistance rates of streptomycin and enrofloxacin showed a significant difference.Combining multiple antibiotic resistance results,the antibiotic resistance status of farm B is lower than farm A.The results of PCR showed that the antibiotic resistant phenotypes of the isolates were basically the same as their genotypes.And we also found a strain carrying both the mcr-1 gene and the blaNDM-5 gene,which mediated low sensitivity to meropenem and the MIC is only 1μg/mL.The simultaneous presence of both genes indicated that the multidrug-resistant gram-negative bacteria resistant to carbapenems were likely to be resistant to polymyxin at the same time.The results of PCR detection of dominant plasmid types showed that 38 strains had IncI2 plasmid,6 strains had IncHI2 plasmid,6 strains had IncX4 plasmid and 3 strains had IncN plasmid,while the PCR results of IncP,IncF and ISApll-mcr-1-pap2 were negative.The PFGE results of the positive strains indicated that the mcr-1 gene could be propagated horizontally and clonally by E.coli,and the mcr-1 positive E.coli in the same farm was diverse,which greatly increased the probability of mcr-1’s transmission.It also provided favorable conditions for the variation and evolution of mcr-1.The conjugation success rate of mcr-1 gene was 34.5%,which demonstrated that the mcr-1 gene can be horizontally transferred through the dominant plasmid. |
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