| In recent years,antimicrobial agents are often used as animal food additives or clinical medication which can result in porcine E.coli resistance problem more serious,especially the emergence of multiple drug resistant strains,making for many years been abandoned with renal toxicity effect of colistin back into clinical use.But that didn't last long,and the line was successfully breached by bacterias in 2015.In this study,E.coli samples collected from seven provinces(Anhui,Henan,Hunan,Hubei,Jiangxi,Shanxi,Shanxi)from 2015-2016 were isolated and identified.The susceptibility tests of 13 antimicrobial agents of eight major classes of isolated strains were carried out by micro broth dilution method,and their drug-resistant genes were detected by PCR and sequenced.The cloning and horizontal transmission of mcr-1 positive strains were analyzed by ERIC typing,multilocus sequence typing and plasmid conjugation test.The plasmid sequence of mcr-1 positive key conjugate strain was sequenced,and the molecular characteristics of mcr-1 plasmid were analyzed,which provided the basis for the transmission control of mcr-1.The positive rate of mcr-1 in E.coli was 25.5%(78/306).Of the 102 strains isolated from sick pigs,46 were positive,accounting for 45.1%,32 were positive in 204 strains isolated from healthy pigs,accounting for 15.7%(P=0.000).At the same time,the former showed more severe resistance to colistin,ceftiofur,cefquinome,gentamicin,amikacin,doxycycline,florfenicol,enrofloxacin,olaquindox.This is similar to whether or not to carry mcr-1,except for amikacin and doxycycline.Moreover,the resistant phenotype of mcr-1 positive E.coli was more extensive than that of mcr-1 negative E.coli.The resistant rate of 306 strains to 13 antimicrobial agents was very serious,such as amoxicillin,oxytetracycline,sulfamethoxazole/trimethoprim(5/1),in proper order 100%,97.4%,96.1%,mequindox,amikacin,olaquindox resistance relatively low rate were 3.3%,4.3%,14.1%,the drug resistance rates of the beta-lactam medicament ceftiofur and the cefquinome are both 27.1%,The resistance rate of gentamicin which is aminoglycosides was 31.4%.The resistance rate of doxycycline which is tetracycline drugs reached 62.1%.The resistance rate of florfenicol,colistin,enrofloxacin were 71.6%?26.5%?33.0%;The results of PCR showed that the detection rate of tet A?flo R?sul was more than 60%,oqx A,oqx B,qnr D detection rate over 36.9%,and bla TEM?mcr-1?bla CTX detection rate over 24.8%,rmt B,aac(6')-Ib detection rate was 2.6%(8/306),5.2%(16/306);The results of conjugation test showed that 32.1%(25/78)of the mcr-1 positive strains could transfer mcr-1 resistance genes between genus of bacteria by plasmids.The drug sensitivity test of the conjugate showed that the conjugate had many kinds of antimicrobial resistance besides colistin resistance.Sequencing of the conjugate plasmid of mcr-1 positive strain EPF03,The results showed that the plasmid belonged to Inc FI2 type,There is a ISApl1 insertion sequence upstream of the mcr-1 gene,The plasmid also carries a variety of resistant genes,such as oqx AB-sul1-sul2-sul3-flo R-aac(3)-IVa-CTX-M-14-fos A3 resistance genes.The results of multilocus sequence typing showed that there was a clone transmission relationship among the mcr-1 positive strains of 44.9%(35/78),and they belonged to the ST10 clone complex.The results of MLST results showed that there was a clone propagation relationship between 44.9%(35/78)mcr-1 positive strains and ST10 clones.The E.coli of the seven provinces has a wide drug resistance,and the resistance genes are complex and diverse,ceftiofur,cefquinome,Amikacin,quinolones,quinoxalines can be used as the first choice drug in the clinic.mcr-1 resistance genes are mainly transmitted in both horizontal and clone ways. |
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