| Antibiotics are widely used in the livestock as growth promoters and therapeutic drugs.Due to the misuse and abuse of drugs,bacterial resistance genes are produced and transmitted to human through food chain.The purpose of this project is to study the antibiotic resistance of meat ducks and laying ducks in waiting areas of slaughterhouses,and to explore the horizontal transmission mechanism of plasmid-mediated quinolone resistance genes.(1)The mixed feces and sewage samples from the meat duck waiting area and laying duck waiting area of the slaughterhouse were collected respectively.The microbial structure and microbial resistance of samples from meat duck and laying duck were analyzed from the overall perspective by metagenome sequencing.In terms of microbial structure distribution,bacteria,fungi,archaea and virus were all detected,of which bacteria were the main component(more than 90%).At the taxonomic level of bacterial genera,the main bacterial genera of meat duck were Psychrophilus,Acinetobacter,Acidobacterium,Corynebacterium and Pseudomonas,while the main bacterial genera of laying duck were Corynebacterium,Psychrophilus and Acinetobacter.In terms of microbial resistance,the metagenome analysis matched a total of 320 antibiotic resistant genes corresponding to 78 antibiotics.287 kinds of antibiotic resistance genes were found in both of meat duck microorganism and laying duck microorganism,corresponding to 71 kinds of antibiotics.The first four antibiotic resistance types were macrolides,vancomycin,lincosamide,and bacitracin,and the major antibiotic resistance genes were macrolide efflux pump gene mac B and bacitracin efflux pump gene bcr A,and the proportion of other antibiotic resistance genes in total ARGs was less than 0.1%.In addition,microorganisms from meat duck contained 21unique resistance genes corresponding to 8 types of antibiotic,while microorganisms from laying duck contained 12 unique resistance genes corresponding to 4 types of antibiotic.The results of metagenomic sequencing showed that there were more bacterial species with a microbial abundance higher than 5%in meat duck samples than in laying duck samples,and the antibiotic resistance genes of microorganisms in meat duck were more abundant than those in laying duck,while the total antibiotic resistance genes relative abundance of microorganisms in laying duck was higher.(2)K-B drug susceptibility test and PCR technology were used to explore the resistance phenotype and genotype distribution of Enterobacteriaceae,and to evaluate the difference of antibiotic resistance between strains from two waiting areas from a local perspective.Through 16S r RNA technology,70 and 192 strains of Enterobacteriaceae were identified from meat duck and laying duck waiting areas respectively.Among the Enterobacteriaceae,Escherichia coli was the dominant bacteria(more than 70%),and the rest were Klebsiella pneumoniae,Proteus mirabilis,Enterobacter cloacae and Shigella.The distribution of Enterobacteriaceae from meat duck and laying duck was similar.The K-B test showed that among the 24 antibiotics in 9 categories,the multidrug resistant bacteria had the highest resistance rate to oxacillin and amoxicillin(more than 98%),and the lowest resistance rate to meropenem,minocycline,and polymyxin(less than 6%).In addition,the proportion of severely resistant bacteria(bacteria resistant to more than 16 kinds of antibiotics used in the experiment)from laying duck was 6.2%,which was higher than that from meat duck(1.5%),so the situation of antibiotic resistance was more serious in bacteria from laying duck.The results of PCR showed that sul1(sulfonamides),fos X(fosfomycins),and aac(6')-Ib-cr(aminoglycosides),mcr-1(polymyxins),cat A3(chloramphenicols),tet A(tetracyclines),erm A(macrolides),bla TEM(extended-spectrum?-lactamases)and qnr S(quinolones)were the most resistant genes.In each category of antibiotic resistance genes,the ratio of the number of bacteria containing this category of antibiotic resistance genes to the number of Enterobacteria from meat duck and the number of Enterobacteria from laying duck was calculated respectively.The results showed that the detection rates of aac(6')-Ib-cr,mcr-1 and flo R were significantly higher in bacteria from laying duck than those from meat duck(P<0.05),while bla CTX-M was significantly higher in bacteria from meat duck(P<0.05).K-B test and PCR test showed that the drug resistance situation of bacteria from laying ducks was more severe.(3)qnrB-positive strain isolated from the laying duck waiting area was used to explore the transmission mechanism of quinolone resistance genes by bioinformatics analysis of plasmid.The conjugative transfer experiment verified that the qnrB gene was located on the plasmid and could be transferred horizontally.The plasmid p2008-qnrB extracted from the zygote was Inc FIIk plasmid,whose backbone was interrupted by accessory regions,which carried antibiotic resistance genes such as aac(3)-IId,bla TEM-1b,and flo R,tet(A),arr3,dfr A27,aac(6')-Ib-cr,aad A16,sul1,qnrB2,mph(A)and aph(3')-Ia.In plasmid p2008-qnrB,qnrB2 was introduced by ISCR1-qnrB2-sul1-IS6100 transposable unit.IS6100 could promote the transfer of qnrB2 between bacterial chromosomes and plasmids,thus spreading resistance.In addition,the upstream of the ISCR1-qnrB2-sul1-IS6100 transposable unit was the integron In1021,which could coexist and co-transfer with qnrB2 to promote the spread of qnrB2.The aac(6')-Ib-cr resistance gene carried by In1021 could co-express with qnrB2,increasing the resistance to quinolone antibiotics.In this study,we found that the antibiotic resistance of bacteria from laying ducks was more serious.Bioinformatics analysis of the plasmid P2008-qnrB from laying ducks showed that the qnrB resistance gene was carried by plasmid.Mobile elements such as In1021,ISCR1,IS6100 promoted the spread of qnrB and bacterial drug resistance.This study provided a reference for the rational use of drugs in duck industry,and had an important significance for delaying the spread of bacterial resistance and ensuring food safety. |
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