Study On Carbapenem-Resistant Mechanism Of Two Animal-Derived Enterobacter Strains

Enterobacteriaceae bacteria are one of the main clinical isolates in animal farms.At presen t,the prevention and control of these bacteria are still based on chemical drugs.In recent years,with the over-scope and over-use of antibiotics in clinical treatment,carbapenem-resistant Enter obacteria(CRE)has resulted in the emergence.Epidemiological studies show that the rate of CRE isolation is increasing year by year in the world.We isolated 500 samples of animal clinical collection(pig blood samples,duck anal swabs),strew culture on Mac Conkey medium,picked single colonies,and then the bacteria by 16 S rRNA universal primer PCR sequencing.A total of 575 strains were identified.Among them,49 carbapenem-resistant Enterobacter strains were selected.In the study,we selected two highly res istant Enterobacter strains(258E: Escherichia coli,93CP: Klebsiella pneumoniae)for in-depth st udy.Kirby-Bauer disc agar method results indicated that 258 E and 93 CP displayed multi-drug-re sistant profile,including rifamycin,cephalosporins,macrolides,aminoglycosides.Using genome high-throughput sequencing technology and bioinformatics analysis platform,we analyzed the information of chromosome and plasmids of 258 E and 93 CP.Multi-locus sequ ence typing showed that strain 258 E belonged to type ST602 of Escherichia coli and carries th ree plasmids,one of which contains multiple drug resistances genes,such as dfrA14,dfrA17,qnrs1,acc(6')-Ib,blaCTX-M-3,blaKPC-2,blaTem-1B,ARR-3,catA1,etc.The plasmid replicator type is IncN,and it is named pEC258 E.pEC258 E is similar to the structural sequence of pECN580 and pCRKP-1-KPC reported in the literature;the 93 CP strain belongs to the Klebsiella p neumonia ST101 type,and carries three plasmids,named p93-1,p93-2,and p93-3.Among whic h,plasmid p93-2 carries the bla KPC-2 gene only,and the plasmid replicon type is IncF,p93-2 has a very high genetic relationship with the plasmid p17-16-KPC reported in the literature be fore.The conjugation experiments proved that the drug-resistant plasmid carried by 258 E or 93 C P could increase the minimum inhibitory concentration of host bacteria to different antibiotics.S1-PFGE assay confirmed that the transferred plasmids of 258 E and 93 CP were pEC258 E and p93-2,respectively.Sequence analysis showed that there is some amino acid mutations in the outer membrane proteins OmpK36 / OmpK35 in 93 CP,further study proved that these mutations were not the major cause of carbapenem resistance.The high carbapenem resistance of 93 CP may be due to the existence of the resistant plasmid with bla KPC-2.In addition,using the prokaryotic expres sion system of Escherichia coli,we confirmed that 93 CP strain carries type ? Rel E toxin-antito xin system,which has a certain relationship with antibiotic resistance in 93 CP,and could be us ed as a target for the development of novel antimicrobial synergists and antimicrobial drugs.