| The dramatic increase in multi-drug resistant bacteria has limited the options for treatment with antimicrobial agents.Howerver,colistin,as an"old"antibiotic,are reintroduced to clinic because of its good antibacterial activity against the multidrug resistant Gram-negative bacilli in clinical settings.In 2015,a new mobile colistin resistance gene,mcr-1 was discoverred and was attracted the attention worldwide.China’s Ministry of Agriculture has claimed that colistin will be banned from being added as a growth promoter in feed since April 30,2017.It is expected to limite the use of colistin to reduce the selection pressure and control the spread of colistin resistance.Howover the effect of this measure has not been reported in China.Therefore,this study conducted a successive survey of colistin resistance for a pig farm in Jiangxi by four-years from 2015 to 2018,The aim is to monitor the change of colistin resistance before and after colistin was officially enforced,the prevalence of mcr-1 gene distribution and study on the mechanism of transmission.In this study,352 strains of Enterobacteriaceae isolated from a pig farm in Jiangxi Province from August 2015 to May 2018 were tested for colistin MIC and screened for mcr-1 from the drug-resistant strains.A total of 109 strains of colistin-resistant strains bacteria were obtained.The colistin-resistant rate was 30.96%,among which the resistance rates in2015,2016,2017 and 2018 were 39.75%,30.77%,33.78%and 23.08%,respectively.The detection rates of mcr-1 in colistin-resistant strains was 22.72%（80/352）,of which the detection rates in 2015,2016,2017 and 2018 were 30.12%,23.08%,21.62%and 17.95%,respectively,the identification of Enterobacteriaceae carrying mcr-1 by MALDI-TOF MS were E.coli.The sensitivity of 80 strains of mcr-1 positive E.coli to 12 antibiotics was determined by agar dilution method.The results showed that it was highly resistant to ampicillin,florfenicol,sulfadiazine/trimethoprim and chlortetracycline;Gentamicin,ciprofloxacin,fosfomycin,and cefotaxime showed varying degrees of resistance;and sensitive to other antibiotics.According to the MIC results combined with PCR amplification method,several common drug resistance genes were detected.The results showed that the detection rate of flo R was 95%,which was the highest;the second was oqx AB,the detection rate is 73.75%;and the detection rates of qnr S、bla CTX-M-9G、fos A3、aac-（6′）Ib-cr、bla CTX-M-1G were 55%,27.5%,15%,6.25%,2.5%,respectively.Pulsed-field gel electrophoresis（PFGE）was applied to analyze the genetic relatedness of 80 mcr-1-positive E.coli isolates.The results showed that 63 differentially distinct spectra were obtained.Then,all the mcr-1-positive strains were subjected to carry out conjugation and transfer,a total of 43 strains were successfully joined.The horizontal propagation and plasmid characteristics of mcr-1 were analyzed by replicon typing,S1-PFGE and Southern blot hybridization.The results showed that among the 43 strains of E.coli,the replicon type was mainly Inc HI2（n=6）、Inc X4（n=19）、Inc I2（n=16）、Inc FIB（n=5）and Inc FII（n=5）,and there are two kinds of plasmids coexisting and the mcr-1 gene was located on plasmid with the size of 33 kb to 320 kb approximately,and mainly based on Inc X4（～33kb）and Inc I2（～60kb）plasmids.The results of PCR showed that co-transferred with oqx AB、flo R、qnr S、bla CTX-M-9G and other resistance genes could occur during conjugation.32 strains of E.coli carrying mcr-1 gene isolated from different years were randomly selected for genome sequencing.A total of 15 ST types were obtained,of which ST10（n=8）was the most popular,followed by ST101（n=3）and ST2705（n=3）.By analyzing the contig carrying mcr-1,further analysis revealed that the mcr-1 gene can be located on the chromosome and the Inc X4,Inc I2,Inc HI2,Inc FII,Inc FIB type plasmid.Through the search of drug resistance genes,32 strains of mcr-1 positive strains were found to carry 42 antibiotic resistance genes to different degrees.In summary,this study conducted a four-year monitoring of colistin resistance in a pig farm in Jiangxi,the resistance rate and mcr-1 positive detection rate showed a downward trend,but the colistin resistance rate and the prevalence of mcr-1 is still high in 2017 and2018.which may be that E.coli carrying mcr-1 often carries multiple drug resistance genes,exhibiting a multi-drug resistant phenotype,and mainly locates on the plasmid and can be co-transferred with other drug resistance genes,which provides the basis for assessing the inhibiting colistin sulfate as an animal growth-promoting to the effect on drug resistance. |
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