Detection Of Antibiotic Resistance Genes In Faeces And Isolation And Identification Of Escherichia Coli Carrying Mcr-1 From Scale Duck Farms In China

At present,the prevalence of antibiotic resistance genes in large-scale duck breeding farms in China is still not clear.This study aimed at the different types of antibiotic resistance genes popular in large-scale duck breeding farms,including macrolides,vancomycins,sulfonamides,tetracyclines,chloramphenicol,16S rRNA methylases,?-lactams aminoglycosides,active efflux and polymyxins antibiotics.A total of 10 classes of 38 resistant genes were compared and the primers were designed.Then,the fecal samples of large-scale duck breeding farms in China was collected and the total DNA was extracted as a template.The prevalence of resistant genes was detected by PCR and sequencing.The detection results of antibiotic resistance genes showed that the detection rates of different antibiotic resistance genes were sulfonamides(90.93%),tetracyclines(74.38%),chloramphenicol(84.43%),and polymyxins(59.89%),active exclusion(58.16%),macrolides(76.47%),?-lactams(64.84%),16S rRNA methylases(17.58%),vancomycin(9.34%).The 10 kinds of antibiotic resistance genes were detected in samples MY,CQ,ZJ,AH,LN and Hu N.The detection of mcr-1 in drug-resistance genes is one of the most important antibiotic resistance mechanisms that mediate the discovery of polymyxin-resistant antibiotics,which has attracted worldwide attention.For the fecal samples positive for mcr-1 detected by PCR,the final concentration of 100?g/m L of polymyxin B in Mac Conkey culture medium was selected to obtain the E.coli isolate mcr-1 harbouring,and the genome and plasmid DNA were extracted.Whole genome sequencing was performed using Pac Bio RS II third-generation single molecule real-time sequencing and bioinformatics analysis.The results showed that the RCAD0514 genome of mcr-1 harbouring E.coli was 4,679,465bp,and the content of GC was50.76%.It contains 3 plasmids,the size of which is 123,510bp,87,297bp and 60,954bp,and the content of GC is 52.58%,51.02%,52.34%,respectively.There are 50 antibiotic resistance genes in the genome,and three plasmids contain 8,4 and 1 antibiotic resistance genes,respectively.Among them,mcr-1 is in plasmid pRCAD0514DY-3.The plasmid typing results showed that the plasmid pRCAD0514DY-3 is a FII plasmid.The results of antibiotic sensitivity test and MIC test show that the antibiotic resistance of mcr-1 harbouring E.coli RCAD0514 is very serious.The transformation experiment results showed that the plasmid pRCAD0514DY-3 mcr-1 harbouring can be transferred laterally into DH5?with a conversion rate of 3.49×10^-8 to 1.7×10^-7.In summary,the resistance genes of large-scale duck breeding sites in China are widely prevalent,and multidrug resistance is widespread.For the first time,E.coli containing mcr-1on plasmids has been isolated from commercial duck populations.