Genetic Environments Of Bla_NDM-1 Gene And Gene Deletion Mutant Construction And Analysis In Carbapenem-resistant Enterobacter Cloacae

Objectives:10 NDM-1-producing Enterobacter cloacae isolates which were screened in the early stage of the study were analyzed for environments of blaNDM-1gene,and initially verified and explained the rule that blaNDM-1 gene was mainly transmitted by plasmids at molecular level.E.cloacaeT2 were selected to carry out gene knockout and construction of blaNDM-1 gene deletion mutant,to explore the effects of blaNDM-1 gene deletion on the antimicrobial susceptibility,growth curves and in vitro competitiveness in E.cloacae,so as to provide theoretical foundation for further research on the relationship between blaNDM-1 gene and virulence,pathogenicity,as well as the prevention and control of drug-resistant bacteria infection.Methods:①The upstream and downstream structures of blaNDM-1 gene in E.cloacaeT1 were sequenced by primer walking,and designing primers accord to the sequencing results to carry out PCR mapping to study the structural characteristics of the upstream and downstream sequences of the remaining 9 E.cloacae isolates;All sequencing sequences were spliced by DNAMAN Version 9,compared and analyzed by BLAST,and the splicing results were submitted to GenBank by sequin software and obtained the accession number② The blaNDM-1 gene deletion mutant of E.cloacaeT2 was constructed by Red homologous recombination technology:First,due to E.cloacaeT2 is resistant to ampicillin,tetracycline resistance gene fragment is inserted into plasmid pKD46 by using DNA clone technology to perform resistance modification;Second,PCR amplified homologous recombination fragments with flanked homologous arms of blaNDM-1 gene and kanamycin resistance gene in the middle,and electroporated into E.cloacaeT2;Thrid,E.cloacaeT2 containing plasmid pKD46-Tet expressed Red homologous recombinase induced by L-arabinose.The blaNDM-1 gene was replaced by a kanamycin-resistant fragment carrying homologous arms of the blaNDM-1 gene through the Red homologous recombinase and the kanamycin resistance gene was further deleted by FLP;Finally,E.cloacaeT2 and E.cloacaeT2 blaNDM-1 gene deletion mutant were verified by PCR,DNA sequencing and RT-qPCR.③Antimicrobial susceptibility for the original strain and the blaNDM-1 gene deletion mutant were analyzed by VIKET-2 automatic identification system and E-test.④In LB liquid medium,the original strain and the blaND-1 gene deletion mutant was cultured,and the bacterial density was detected by ultraviolet spectrophotometer.The growth curves of the two strains were observed continuously for 24h.The original strain and the blaNDM-1 gene deletion mutant was mixed in a ratio of 1:1.and strains were inoculated on an LB plate without antibiotics and imipenem-containing LB plate overnight at 37℃ to obtain CFU of total bacteria and CFU of E.cloacaeT2,and get the competition index.Results:①The blaNDM-1 gene environments of 10 E.cloacae isolates was analyzed.9 E.cloacae isolates have same blaNDM-1 gene structures of upstream and downstream,the △ISAba125 truncated by ISEc33 element and the bleo followed by a△trpF and ISSen4 was located immediately upstream and downstream of the blaNDM-1 gene in 9 E.cloacae isolates;Another E.cloacae T10,the△ISAba125 and the bleo followed by a △trpF was located immediately upstream and downstream respectively;The sequence was submitted to Genbank and obtained the accession number MF927777.②The tetracycline resistance gene fragment was successfully inserted into the pKD46 sequence,so that it had tetracycline resistance,and the tetracycline anti-plasmid pKD46-Tet was successfully constructed.PCR,DNAsequencing and RT-qPCR showed that the blaNDM-1 gene deletion mutant was successfully constructed,and the blaNDM-1 gene sequence was deleted without kanamycin resistance gene,the deletion mutant was named E.cloacae T2△NDM-1.③Antimicrobial susceptibility test showed that E.cloacaeT2 and deletion mutant showed similar drug resistance spectrum to penicillins,cephalosporins,aminoglycosides,tetracycline and quinolones,but showed different drug sensitivity result to carbapenem antibiotics.E.cloacae T2 was resistant to imipenem,meropenem and ertapenem,while the deletion mutant was sensitive to all.④The E.cloacae T2 and deletion mutant had similar growth curves in Luria-Bertani liquid medium;In vitro competition experiment revealed that the competitive index of them was 0.69.Conclusions:①The upstream and downstream sequences of the blaNDM-1 gene often contain transposase or insertion element.②Red homologous recombinant technology was successfully used to knock out blaNDM-1 gene.in E.cloacae.③blaNDM-1 gene could affect the antimicrobial sensitivity of E.cloacae for carbapenems and in vitro competitiveness,but did not affect the growth trend and growth rate of E.cloacae.