Localization And Validation Of Polymyxin-resistant Genes In Acinetobacter Baumannii Mutants

Acinetobacter baumannii just be put in the first place of "the strongest resistance TOP12" by WHO,which almost resistant to all categories of antibiotic.Polymyxin is considered the "last line of defense" for treating A.baumannii,while,there still have many problems for the treatment of A.baumannii infection by polymyxin.Although clinical isolated A.baumannii are sensitive to colistin,the failure rate to treatment of A.baumannii infection by polymyxin is as high as 35%,which indicate that the hospital treatment failure has nothing to do with the traditional drug-resistant bacteria and may be related to adaptive resistance of bacteria caused by insufficient polymyxin doses.Therefore,we suspect A.baumannii genome may has "suppressor gene drug resistance",under the condition of changing external environment,"resistant suppressor genes" are "silent" leads to the bacteria resistance.In order to verify the idea of drug-resistant suppressor genes,we use both mariner transfer technology and polymyxin to obtain some drug-resistant mutant strains,positioning and prove the existence of suppressor "resistance",which lay the foundation for subsequent resistance mechanism study.This study takes the polymyxin-resistant mutant strains as the research sample,which be constructed by mariner transposon mutagenesis system and polymyxin.First,part of mariner transposon insertion sites of polymyxin-resistant mutant strains are identified by reverse PCR technology,drug resistance related gene which is interrupted to be cloned into the covering carrier?pSUTetAB?,then transfer each covering plasmid into the corresponding mutant strains,the better resistance related gene be found by contrast the polymyxin minimal inhibitory concentration?MIC?of mutant strains with covering mutant strains.The gene is fixed-point knocked out with RecETab recombineering systems,furthermore,phenotypic verification test prove the knockout gene and resistant with high correlation.Finally,the mechanism of drug resistance to be preliminary explored by bacteria two-hybrid system.This study has successfully pinpoint six insertion sites of polymyxin-resistant mutant strains by reverse PCR technology,DJ41₁093?anti-anti-??locus is a good resistance related gene by contrast the polymyxin minimal inhibitory concentration?MIC?of mutant strains with covering mutant strains,the gene be fixed-point knocked out with RecETab recombineering systems and has high correlation with resistant.In addition,we find out a gene locus DJ41₈48?anti-??which possibly interact with DJ41₁093 locus,interaction relationship between two genes before and after codon optimization are identified by bacteria two-hybrid system,up to now,the experiment results show that this two genes without any correlation.