Formation Mechanism Of Multidrug-resistant Fusion Plasmids In Escherichia Coli Isolated From Pets

Escherichia coli(E.coli)is the most commonly reported zoonoses strain in China and abroad.Prolonged use of antimicrobials leads to the continuous emergence and widespread spread of resistant strains.As the companion animals of human beings,the common living environment and long-term direct contact make bacteria isolated from pets become an important medium for the spread of antibiotic resistance genes between human beings and the environment.The plasmid is usually used as the vector of resistance gene in the propagation of bacteria and ertical propagation and horizontal transfer between different strains.Some conjugable plasmids in the strain can be used as conjugative helper plasmids to facilitate the transfer of non-conjugable plasmids at the same time of self-transfer.During conjugation,the accumulation of resistance genes and the evolution of plasmids may occur,which makes the transmission of resistance genes more rapid and brings problems to clinical treatment.The purpose of this study was to elucidate the mechanism of plasmid fusion in one strain of Escherichia coli isolated from pets with multi-drug resistance(MDR),and to provide a theoretical basis for reducing plasmid fusion and resistance gene transmission in clinic.106 strains of Escherichia coli were collected from three pet hospitals in Zhengzhou,and the drug sensitivity test was carried out with multiple antibiotics.A total of 44 strains with multiple antibiotic resistance were selected,among which cefotaxime resistance was the most common.One multi-drug resistant strain HB42 was selected for further study based on PCR amplification results.HB42 was highly resistant to ampicillin,cefotaxime,flufenicol,amikacin,gentamicin and colistin.In the preliminary resistance gene test,HB42 was detected to carry resistance genes bla CTX-M-14,bla CTX-M-55,rmt B,flo R and mcr-1.According to chromosome sequence analysis,no resistance genes were found on the chromosomes,so it was judged to be located on the plasmids of the strain.Whole genome sequencing revealed that HB42 carried five plasmids of different sizes.Two fusion plasmids with different resistance genes of different sizes were found in HB42 by conjugation tests.Southern hybridization test proved that the flo R-positive non-conjugative plasmid p HB42-3 fused with the bla CTX-M-55 positive conjugative plasmid pHB42-2 and the mcr-1 positive conjugative plasmid pHB42-4,respectively.Further whole genome sequencing and PCR verification showed that the fusion site was formed by recombinant plasmid p HB42-2 and p HB42-4,which were attacked by IS26 carried by plasmid p HB42-3.The biological characteristics showed that the fusion plasmids were stable in the antibiotic-resistant environment for 15 days.The fusion frequency of p HB42-3with p HB42-2 or p HB42-4 was 1.94×10-6 and 1.26×10-8,respectively.And p HB42-F1 and p HB42-F2 were further transferred to the recipient strain E.coli C600 at a high frequency of2.7×10-4and 5.7×10-3.The high stability and transmissibility of fusion plasmids may promote the stable existence and rapid propagation of antibiotic-resistant genes in non-conjugative plasmids under drug selection pressure.rec A deletion did not affect the fusion frequency of p HB42-2 and p HB42-3(from7.3×10-5 to 4.3×10-5),indicating that the factor affecting the fusion frequency was mainly located in the daughter plasmids.The recombinant sites where the daughter plasmids formed the fusion plasmids were studied.It was found that the daughter plasmids p HB42-3 and p HB42-2 had other plasmid fusion modes.200 transconjugants were randomly selected on agar plates containing florfenicol and cefotaxime,and PCR amplification was performed on200 transconjugants using fusion site primers and predictive primers of p HB42-F1.Another four different fusion modes were found,but only 29.5% of the transconjugants had confirmed fusion mode.The formation of cointegrates from p HB42-2 and p HB42-3 were mediated by multiple IS26 or Tn As1 in p HB42-3 attacking on different target sites including8 bp nucleotide sequences or IS26 or Tn As1 in p HB42-2.To our knowledge,this work was the first to demonstrate the role of Tn As1 in plasmid reorganization and IS26-mediated multi-pattern plasmid fusion in a single strain.In conclusion,the formation of fusion plasmids contributes to the transfer and propagation of drug-resistant nonconjugative plasmids,and accelerates the rapid spread of drug-resistant genes and plasmid evolution.Antibiotics should be used prudently and rationally in clinical practice to avoid or slow down the generation and spread of multidrug-resistant bacteria.