The Prevalence And Spread Mechanism Of Colistin Resistance Gene In Swine Farms

In recent years,due to the widespread and unreasonable use of antibiotics,bacterial resistance has become more and more serious.Colistin has become the last line of defense against the infection caused by multi-drug resistant Gram-negative bacteria in humans and animals.Since the first report of the plasmid-mediated colistin-resistant gene mcr-1 in China in 2016,the gene has been detected in bacteria of different species from different sources around the world.The other plasmid-mediated colistin resistance genes were also detected in both human and animal-derived bacteria.The emergence and spread of plasmid-mediated colistin resistance genes poses a serious threat to human health and public safety.The aim of this study was to understand the drug resistance of Escherichia coli in Henan pig farms and to study the emergence and spread of mcr-like genes,so as to provide a scientific basis for rational drug use in pig farms.In October 2017,309 pig fecal swabs samples were collected from three pig farms in Henan province.302 Escherichia coli strains were isolated and identified.Agar dilution method was used to detect 11 kinds of antibacterial agents,including ampicillin,cefotaxime,gentamycin,kanamycin,Amikacin,doxycycline,florfenicol,polymyxin,fosfomycin,ciprofloxacin and mequarine.The results showed that the strains showed different degrees of resistance to 11 drugs,the resistance rate was between 4.30%～87.75%,and the highest resistance rate to ampicillin was 87.75%,followed by florfenicol,the resistance rate was 80.79%,the resistance rate to colistin was the lowest,4.30%,and the resistance rate to Amikacin and fosfomycin was low,about 7%.Multidrug resistance was serious,80.1%of the strains were resistant to three or more drugs(n=242).The trend of resistance to 11 antibiotics was similar in three pig farms,among which ampicillin resistance rate was higher,ranging from 82%to 91%.The resistance rates of the strains from Yanshi pig farm and Xiping pig farm to ciprofloxacin and cefotaxime were significantly different.The resistance rates of the strains from Yanshi pig farm to ciprofloxacin and cefotaxime were twice as high as that of the strains from Xiping pig farm(17.9%).The resistance of the strains from Yanshi pig farm to ciprofloxacin and cefotaxime was the most serious.Thirteen Escherichia coli strains with colistin-resistant phenotype were det ected by PCR and sequencing.The results showed that 13 strains carried mcr-1 gene and no mcr-2～8 genes were detected.One strain also carried blaNDM-1,four strains carried fasA3 and seven strains carried blaCTX-M,including blaCTX-M-55(n=4),blaCTX-M-27(n=1)and blaCTX-M-14(n=2).Two of them carried mcr-1,fosA3 and blaVTX-M.The genetic context of mcr-1 was identified by PCR and sequencing.ISApl1 was detected in 1 strain upstream and downstream of me r-1,and the circular intermediate ISApl1-mcr-1-pap2 was formed.ISApll was not detected in 7 strains upstream and downstream of mcr-1.ISPl1 was dete cted upstream of mcr-1 gene in 4 strains,ISApl1 was detected upstream of m cr-I,and truncated ISApll was detected downstream of mcr-1.The transferability of mcr-1 was studied by conjugation experiment.The results showed that the mcr-1 in 5 strains were transferred successfully.The replicon typing of plasmids in transconjugants showed that there were three types of plasmids including IncF-IncN,IncI2 and IncHI2.WGS of the transconjugant D72C and the parental strain D72 showed that the chromosome of the strain D72 carried macrolide resistance gene mdf(A)and virulence gene gad encoding glutamate decarboxylase.The plasmid contained mucin resistance genes mcr-1,beta-lactam resistance genes blaTEM-238 arid blaCTX-M-55,aminoglycoside resistance genes aph(3’)-Ia,qnrS2 and floR were the main genes for quinolone resistance.BlaTEM-238 is a new subtype of TEM β-lactamase.According to the results of S1-PFGE,D72 carried four plasmids of different sizes,of which mcr-1 is located on the 96,320 bp plasmid pD72-mcrl.The plasmid is a phage-like plasmid,and the replicon type of the other three plasmids are IncN-F33:A-:B-,IncX1 and IncP,respectively.The plasmid pD72-mcr1 in D72C is the fusion of two plasmids from the parental strain.To summary,the resistance of Escherichia coli strains isolated from swine in pig farms in some areas of Henan,is serious.The different habits of different farms may lead to different resistance patterns.The pig farms are still mainly prevalent in mcr-1,and no other subtypes of colistin resistance genes are detected.The mcr-1 can be transmittedthrough transposable elements ISApl1 and conjugative plasmids,even by plasmid fusion.The mode of transmission will facilitate the transmission of the gene and pose a threat to animal husbandry and human health.