| The continuous emergence of drug resistance in chicken Escherichia coli is closely related to the horizontal transmission of drug resistance genes.Plasmids,as vectors for capturing resistant genes and gene horizontal transfer,play an important role in the emergence and dissemination of antibiotic resistance.The formation of larger plasmids from conjunctive plasmid and multidrug-resistence(MDR)non-conjunctive plasmid exacerbated the rapid spread of antibiotic resistance genes and the evolution of plasmids.The study was aimed at providing theoretical basis for retarding emergence and spread of large MDR fusion plasmids in clinic by investigating the formation mechanism and biological characteristics of fusion plasmids in MDR E.coli from chicken.370 E.coli strains were isolated and identified by morphology and 16 S r RNA sequences from 430 anal swab samples of healthy chickens and one liver sample from dead chicken collected from four chicken farms in China.The results of analysis of antimicrobial resistance of 370 E.coli strains by AGAR dilution method showed that370 E.coli strains showed different degrees of drug resistance to 6 tested drugs,among which the drug resistance rate of cefotaxime was the highest,61.08%(226/370),and the drug resistance rate of cefoxitin,colistin,tigecycline,amikacin and meropenem ranged from 24.05%(89/370)to 1.35%(5/370).Fourteen tet(X4)positive E.coli strains were detected by PCR and sequencing from 26 tegecycline resistant strains,among which 3 strains carried mcr-1,rmt B and blaCTX-M-64 genes,1 strain carried rmt B gene.E.coli C21 was identified as rmt B and blaCTX-M-55 positive in liver samples of dead chickens.Through multi-locus sequence typing,15 E.coli strains were divided into 3 ST types,including 10 ST641 strains,4 ST2973 strains and 1 ST156 strains.A total of 24 conjugants were obtained from 15 E.coli strains by conjugation test.Four different fusion plasmids were found by conjugation test,replicon typing,S1-PFGE and Southern blot hybridization.Two of them were found in 14 tet(X4)positive E.coli strains and their transconjugants,respectively tet(X4)and rmt B co-positive Inc HI1_Inc N fusion plasmids and tet(X4)positive Inc HI1_Inc FII hybrid plasmids.Two fusion plasmids were found in the two transconjugants of C21,which were rmt B and blaCTX-M-55 co-positive transconjugants and rmt B-carrying transconjugants,respectively.The formation of this fusion plasmids may also be related to the transfer of rmt B.The formation mechanism and biological characteristics of these fusion plasmids need further investigate.In addition,all the 14 strains of tet(X4)positive bacteria were located on the conjugation of temperature-sensitive Inc HI plasmids.The conjugation frequencies of these plasmids were significantly different at different temperatures.In order to analyze the formation mechanism of fusion plasmid in E.coli C21,The fusion plasmid was formed by the fusion of rmt B-carring non-conjugative plasmid p C21-2(Inc N)with conjugative plasmids p C21-1(Inc I2)and p C21-3(Inc I1)in the parent strain C21 by conjugation test,S1-PFGE and Southern blot hybridization.Further whole genome sequencing(WGS)and PCR showed that the fusion site was formed by recombination of IS1294 carried by plasmid p C21-2 with IS1294 region of plasmid p C21-1 and p C21-3,respectively.The biological characteristics of the fusion plasmid showed that the fusion plasmid was quite stable in 15 consecutive days of subculture,and the conjugation frequency of the fusion plasmid in the transconjugant was significantly higher than that of the parent strain.In conclusion,the formation of fusion plasmids contributes to the transfer and transmission of drug-resistant non-conjugative plasmids,and accelerates the rapid transmission of resistance genes and plasmid evolution.Clinical use of drug combination should be cautious and rational,so as not to accelerate the generation and spread of multidrug-resistant bacteria. |
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