| Colistin has been added to animal feed as a growth promoter for a long time in animal production,which not only increases economic benefits,but also brings hidden dangers to public health and safety problems.The continuous use of antibiotics promotes and accelerates the emergence of multi-drug-resistant bacteria.Due to the discovery and wide spread of plasmid-mediated colistin resistance gene mcr-1,colistin has been banned from being added to feed as a growth-promoting agent in China since April 30,2017.The purpose of this study was to monitor the changes of drug resistance of Escherichia coli from livestock and poultry before and after the implementation of this policy and to analyze the transmission characteristics of mcr gene.In this study,373 fecal samples collected from three livestock and poultry farms in Changchun,Jilin Province in 2017 and 2020.Coloration medium ECC and Mc Conkey medium with colistin resistance（2μg/m L）were used for selective culture.Escherichia coli was identified by specific 16sr DNA specific primer PCR of Escherichia coli.929strains of colistin resistant Escherichia coli were obtained.After excluding clones by ERIC fingerprinting,546 strains of colistin-resistant Escherichia coli were obtained,of which 141 strains were isolated from 107 samples in 2017 and 405 strains from 266samples in 2020.A total of 34 mcr-1 positive strains were identified by PCR detection of drug resistance gene mcr1-9.In 2017,the detection rates of mcr-1 positive strains in three farms were 39.02%,5.00%and 5.00%.In 2020,the detection rates of mcr-1 positive strains in three farms were 4.85%,0.00%and 3.68%.The detection rate of mcr-1positive strains decreased at two time points in the same farm.The results showed that the policy of banning colistin as feed additive was effective in the three farms in Changchun and had a great impact on reducing the prevalence of mcr-1.The sensitivity of the strains to colistin was determined by microbroth dilution method.The results showed that the resistance level of the isolated strains to colistin was between 0.25μg/m L and 8μg/m L,indicating that the colistin resistance in this area was still at a moderate level.Taking MIC≥2μg/m L as the judging point of drug resistance,the isolation rate of colistin-resistant strains in 2020（18.02%）was lower than that of colistin-resistant strains in 2017（26.24%）.The isolation rate of colistin-resistant strains decreased,indicating the effectiveness of colistin prohibition policy.The sensitivity of 546 strains of Escherichia coli to 16 antibiotics and whether they were ESBLs strains were detected by BD PhoenixTM-100 System.The results showed that they were most resistant to tetracycline,with high resistance to ampicillin,piperacillin,chloramphenicol and ciprofloxacin,and mild resistance to amikacin,imipenem,meropenem and ceftazidime.Compared with the drug resistance of strains in2017 and 2020,except for the increase of cefepime and chloramphenicol,the drug resistance of other antibiotics decreased,but the phenomenon of multiple drug resistance was serious,with more than 60%of multiple drug resistant strains and the most triple drug resistance.ESBLs strains were detected,of which 5 strains were mcr-1gene positive strains.A total of 34 mcr-1 gene positive strains were genotyped by PBRT kit.12 types of plasmid replicators were identified,mainly Inc FII,and each strain carried two or more plasmids.A total of 11 plasmids were detected in 21 strains of mcr-1-positive Escherichia coli in 2017,mainly Inc FII（80.95%）.In 2020,a total of 7 plasmids were detected in 13 strains of mcr-1 gene positive colistin-resistant Escherichia coli,mainly Inc FII and Inc FIB（92.31%）.Compared with 2017,the number of plasmid replicator types decreased in 2020.Through the conjugation transfer test,22 successful zygotes were obtained,and the conjugation rate was 64.71%.The transfer efficiency of Inc X4 and Inc I2 plasmids was the highest,followed by Inc FIB and Inc FII plasmids.The zygotes also showed partial resistance to gentamicin,tetracycline,cefazolin,cefotaxime,ampicillin,piperacillin,chloramphenicol and moxifloxacin.And two conjugates were ESBLs strains,which proved that the plasmid carrying mcr-1 gene also carriedβ-lactam antimicrobial resistance gene,and the drug resistance gene was co-transferred with the conjugated plasmid.The whole genome of 34 mcr-1 gene positive strains were sequenced.The sequencing results showed that these strains belonged to 15 ST types,of which ST29was the most popular,followed by ST20 and ST88.The MLST sequence typing of mcr-1 gene positive strains isolated in farm 2 and farm 3 every year is basically the same,but in the farm 1,the results of mcr-1 gene positive strains MLST sequence typing in 2017 are complex and diverse,which may be the reason why there is still a high level of mcr-1 gene positive strains in the farm nearly three years after colistin was banned.Analysis of the whole genome sequencing results showed that 34 mcr-1 gene positive strains carried other drug resistance genes in varying degrees,all strains carried four or more drug resistance genes,all of them were multidrug resistant strains,among which the detection rates of Oqx A,Oqx B,sul and tet A genes were higher,followed by drf A and cml A genes.bla CMY-2gene was detected in 2 mcr-1 positive strains isolated from chicken feces.Bla CMY-2 gene belongs toβ-lactam antibiotic resistance gene,and the emergence of mcr-1 is related to the use ofβ-lactam antibiotics.At the same time,mdf A gene was detected in 10 mcr-1 positive strains,which is one of the important reasons for multiple drug resistance.To sum up,through the comparison before and after the implementation of the policy of banning colistin as a feed additive,we understood the drug resistance characteristics of Escherichia coli isolated from three livestock and poultry farms in Changchun,Jilin Province.The rate of colistin resistance and the positive rate of mcr-1gene decreased from 2017 to 2020,but the multiple drug resistance of colistin-resistant Escherichia coli carrying mcr-1 gene was serious.The complexity and diversity of plasmid replicon and MLST sequence typing and the co-transfer of some drug resistance genes may be the reasons for the long existence of mcr-1 gene in livestock and poultry breeding environment.This study provides basic data for evaluating the effect of forbidden colistin as a feed additive on drug resistance,It also provides a scientific basis for the rational use of drugs in livestock and poultry farms. |
PDF Full Text Request