Study On Multi-drug Resistance In Raccoon-derived Escherichia Coli In Northeast China

In recent years,the raccoon dog breeding industry has developed faster and faster in China and has become an industry that cannot be ignored in China.The economic value of raccoon fur is very high,which also makes raccoon fur occupy a pivotal position in the fur industry.Escherichia coli(E.coli)is a Gram-negative bacterium commonly found in nature.It is a very important and common intestinal microorganism in humans and animals.It is also an important symbiotic bacterium.The resistance of E.coli is usually used as an indicator of antibiotic stress in the environment and a potential reservoir of drug resistance genes.With the in-depth study of antibacterial drugs,antibacterial drugs gradually began to be used in animal husbandry.Antibiotics are generally used for the treatment of clinical bacterial infectious diseases,but they are also used as additives in animal feed to prevent diseases.With the increasing use of antibiotics,the resistance of E.coli has also begun to increase,so that there has been a multi-drug resistance.This not only has a great impact on the raccoon breeding industry,but also poses a potential threat to humans.In this study,155 fecal samples were collected from healthy raccoon dogs from five raccoon dog farms in Northeast China.A total of 150 E.coli strains were isolated and identified from the fecal samples,and the isolation rate was 96.77%.The K-B disk diffusion method recommended by the World Health Organization(WHO)was used to test the in vitro antimicrobial susceptibility of 150 strains of E.coli from raccoon dogs.The results showed that150 strains of E.coli showed high resistance to Ampicillin(AMP)and Tetracyclines(TE),92%and 84.67%,respectively.The resistance rate to Amikacin(AK)was 0,while the sensitivity rates to Amikacin(AK)and Imipenem(IPM)were 92.67% and 89.33%,respectively.The number of multidrug-resistant strains reached 83.99%.Integron is a potential mobile genetic element in E.coli,carrying antibiotic resistance determinants,which can capture exogenous gene cassettes and integrate into their own genomes.The detection rate of type I integrase gene int I1 was 72% in 150 strains.The detection rate of type II integrase gene int I2 was 9.33%.Type III integrase gene int I3 was not found.The number of positive results of type I integron was 51,accounting for 34% of the total strains,of which 27 carried gene cassettes,accounting for 18% of the total strains.The detection rate of empty integron was 28.67%,and no positive strain of type II integron was detected.The integron type I carried 8 gene cassettes with 5 different combinations,namely aad A2,dfr A5,dfr A1-aad A1,dfr A17-aad A5,lnu(F)-aad A.In this study,PCR was used to detect the corresponding resistance genes of Beta-lactams,aminoglycosides,sulfonamides,tetracyclines,quinolones,chloramphenicol and polymyxins in150 strains of E.coli from raccoon dogs.Beta-lactam: OXA resistance gene positive rate was8%;TEM resistance gene positive rate was 32.67%;CTX-M resistance gene positive rate was15.33%.No positive samples were detected for SHV gene.Aminoglycosides: aac(3)-II resistance gene positive rate was 14.67 %;aac(3)-IV resistance gene positive rate was 9.33%;aph A1 resistance gene positive rate was 42%.Sulfonamides: sul1 resistance gene positive rate was 29.33%;sul2 resistance gene positive rate was 19.33%;sul3 resistance gene positive rate was 18.67%.Tetracyclines: tet A resistance gene positive rate was 41.33%;tet B resistance gene positive rate was 27.33%;tet C resistance gene positive rate was 3.33%;tet M resistance gene positive rate was 2.67%.No positive samples were detected for tet D and tet E genes.Quinolones: qnr B resistance gene positive rate was 0.67%;qnr S resistance gene positive rate was 24%;oqx A resistance gene positive rate was 26.67%;oqx B resistance gene positive rate was 40%;aac(6’)-Ib-cr resistance gene positive rate was 46%;Gyr A resistance gene positive rate was 11.33%;Gyr B resistance gene positive rate was 60%;Par C resistance gene positive rate was 36.67%;Par E resistance gene positive rate was 61.33%.No positive samples were detected for qnr A and qep A genes.Chloramphenicols: cml A resistance gene positive rate was13.33%;flo R resistance gene positive rate was 17.33%;cat resistance gene positive rate was10.67%.Polymyxins: mcr-1 resistance gene positive strain was 1.Gyr A gene mutation was Ser83→Leu,Asp87→Asn;the mutations of Par C gene were Thr178→Ala,Lys200→Arg and Asn304→Ser.There was no mutation in Gyr B and Par E genes.PFGE typing technology is often used to analyze the correlation betwen strains.It is the golden method of bacterial typing.In this experiment,10 of the 12 E.coli samples had typing ability.Among them,the homology coefficients of four strains were greater than 85%,and the others were less than 85%.Ten strains had 8 PFGE types,indicating that there were complex genotypes among the test strains.In this experiment,the drug resistance phenotype,PFGE molecular typing,drug resistance genes,integron-gene box system and QRDR gene mutation of E.coli from raccoon dogs were studied.The purpose of this study was to provide theoretical basis and basic research for controlling the spread of drug resistance genes,to provide data reference for reducing and preventing drug resistance of E.coli,and to provide reference and guidance for daily medication of raccoon dog farms in Northeast China.