| Since the discovery of plasmid-mediated colistin resistance gene mcr-1,then the mcr-1 has been reported in more than 40 countries on six continents,and its transmission mechanism is complex.However,to date,the prevalence,resistance and transmission mechanism of strains carrying mcr-1 in some areas are still unclear,and the corresponding research data are lacking.Therefore,aiming at this gap,this study selected 90 strains of Escherichia coli isolated from a dairy farm in Shanghai and 900 strains of Enterobacteriaceae isolated from the First People's Hospital of Shanghai for screening of mcr-1 gene,and further analysis the resistance of mcr-1 positive strains and propagation characteristics of mcr-1 gene,in order to provide a reference for studying the transmission mechanism of mcr-1 gene.The main research contents and results are as follows:?1?Isolation,identification and drug resistance analysis of an animal-derived Escherichia coli EC11 carrying mcr-1 gene.To understand the prevalence of mcr-1gene in livestock farms in Shanghai and the drug resistance of the mcr-1positive strain,in this study,macrogenomic samples of feces,soil and sewage from a dairy farm in Shanghai were collected and applied selective medium for preliminary screening of colistin-resistant E.coli,biochemical experiments and 16s rDNA were used for further verification.Finally,PCR method was used to detect the presence of mcr-1 in metagenomic samples and isolated strains,and the resistance phenotype and genotype of mcr-1 positive strains were analyzed.The results showed that mcr-1 gene was detected in cow feces,and a strain of E.coli EC11 carrying mcr-1 gene was isolated.Disk diffusion method and broth dilution method showed that the E.coli EC11 was sensitive to most antibiotics,and the MIC of colistin was 8?g/mL;and the EC11 not carry other resistant genes.This study is the first report that colistin resistance gene mcr-1 was found in a dairy farm in Shanghai.Although the drug resistance is at a low level,the emergence of mcr-1 in the livestock industry still needs to arouse domestic vigilance and attention.Detection of the mcr-1 gene in the livestock industry and the drug sensitivity of the strain carrying the gene should be continuously monitored and closely attention.?2?Study on the transmission characteristics of mcr-1 gene in E.coli EC11.In order to explore the transmission characteristics of mcr-1-positive E.coli EC11,the multilocus sequence typing?MLST?technique was used to typing EC11,in addition,the genetic and evolutionary relationships of 616 strains of mcr-1positive E.coli belonging to 245 different sequence types?STs?were analyzed;the transferability of mcr-1 gene was analyzed by plasmid conjugation,the potential transmission mechanism was further analyzed by whole genome sequencing?WGS?.The results showed that EC11belonged to ST278,which was the first reported mcr-1-positive E.coli sequence type.In addition,eBURST and Neighbor-Joining Tree analysis showed that the mcr-1-positive E.coli population had a high genetic diversity.WGS showed that EC11 contained an IncX4-type plasmid pEC11b carrying mcr-1,which contained putative conjugal transfer components,including an oriT-like region,relaxase,type IV coupling protein,and type IV secretion system;the upstream of mcr-1gene contains promoter sequences,all of the above elements contribute to the spread of mcr-1 gene.We were successful in transferring pEC11b to E.coli C600 with an average transconjugation efficiency of 4.6×10-5.The application of MLST and WGS technologies in this study to provide data and further understand the transmission mechanism of mcr-1 gene.?3?Screening,drug resistance and transmission characteristics of mcr-1 in clinical isolated of Enterobacteriaceae bacteria.To understand the prevalence and transmission mechanism of mcr-1 gene in clinical samples in Shanghai,the mcr-1 gene was screened by 900 strains of Enterobacteriaceae bacteria isolated from Shanghai First People's Hospital from 2011 to 2015 in this study.The drug resistance of mcr-1-positive bacteria was determined by broth dilution method;the transferability of mcr-1 gene was analyzed by plasmid conjugation;the type of mcr-1plasmid and its surrounding gene environment were determined by PCR;the sequence type and genetic evolution relationship of mcr-1-positive bacteria were determined by MLST.The results showed that 6 strains of E.coli carried mcr-1 gene,and the MIC of colistin was 4-8?g/mL,they all showed multiple resistance phenotypes and genotype;All the 6 strains of E.coli were successfully conjugated,and the frequency of conjugation transfer was 3.92×10-36.25×10-8,and all showed multiple drug-resistant phenotypes and genotypes.Compared with the donor bacteria,the transconjugants showed an increase or loss of drug-resistant phenotypes or genotypes;Plasmid replicants of 6 mcr-1positive transconjugants were classified into IncX4 type and IncI2 type,and their upstream and downstream gene environments were diverse,including the translocase gene tnpA,etc;the strains of 6 mcr-1 positive E.coli belong to different sequence types.In this study,the strains of 6 mcr-1 positive E.coli were screened from clinical samples in Shanghai,not only showed multi-drug resistance,but also had a more complicated transmission mechanism,which should be paid more attention.In conclusion,this paper screened the mcr-1 gene in the dairy environment samples and clinical samples in Shanghai,and finally detected one mcr-1 positive strain of E.coli in cow feces and six mcr-1 positive strains of E.coli in clinical samples.All 7 strains of E coli showed different levels of drug-resistant phenotypes and genotypes,but the drug resistance of clinical isolates was stronger.All the 7 strains of E.coli have different levels of conjugation and transfer ability,and have a variety of genetic environments.The results of this study laid a theoretical foundation for studying the resistance and transmission characteristics of mcr-1 gene. |
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