Study On The Mechanism Of Heteroresistance Of Acinetobacter Baumannii To Colistin

Background and purpose: Acinetobacter baumannii is one of the most important pathogens causing clinical infections in hospitals,and its resistance to antibiotics is becoming more and more serious.Its heteroresistance is an important cause of repeated infection.Although colistin is the "last line of defense" against Gram-negative pan-drug resistant bacteria,Acinetobacter baumannii also produces heteroresistance to colistin,and its mechanism of action is less studied.This thesis intends to study the heterogeneous mechanisms of Acinetobacter baumannii to colistin,and provide reference for the research of related drugs and clinical rational drug use.Methods: The minimum inhibitory concentration(MIC)was used to determine the sensitivity of Acinetobacter baumannii to five antibiotics.The heteroresistance to five antibiotics was determined by population analysis profiles(PAP).The stable high-resistance heterogeneous bacteria were screened by colistin and sequenced and genome-wide analyzed.The killing curve was used to investigate the formation of persistent bacteria by Acinetobacter baumannii under the action of five antibiotics.A homologous recombination double exchange technique was used to establish a system for Acinetobacter baumannii gene knockout.(p)ppGpp synthase gene(relA),UDP-glucose biosynthesis-related genes(pgm,algC-1,galU,cugP and icaA)and efflux pump genes(adeAB and acr)of Acinetobacter baumannii were investigated by crystal violet staining,PAP assay and killing curve.That is to explore the effects of biofilm formation,the number of heterogeneous colonies and formation of persistence bacteria in Acinetobacter baumannii to colistin.Scanning electron microscopy was used to observe the changes of Acinetobacter baumannii morphology.Results: Acinetobacter baumannii had heteroresistance under the action of 10 times MIC colistin,tetracycline,meropenem and ceftazidime,while gentamicin did not have heteroresistance.Under the action of colistin,a highly resistant strain with stable inheritance was screened.No base mutation was found in the pmrA/pmrB gene,and four local base mutations were analyzed by whole genome sequencing.At 10 times MIC concentration,gentamicin can kill all Acinetobacter baumannii for 24 h,but Acinetobacter baumannii has persistence for colistin and ceftazidime,and bacteria still survive at 24 h.For tetracycline and meropenem,Acinetobacter baumannii has restored growth.The Acinetobacter baumannii knockout strain was successfully constructed.(1)After relA knockout,the biofilm formation,the number of heterogeneous colonies and the production of persistent bacteria decreased significantly.(2)After pgm knockout,the biofilm formation,the number of heterogeneous colonies and the production of persistent bacteria decreased,the volume of the bacteria became larger and the contour shape was irregular.After algC-1,galU,cugP,icaA knockout,the biofilm formation,the number of heterogeneous colonies and persistence of bacteria were not significantly affected.(3)After adeAB,acr and double knocking,the biofilm formation,the number of heterogeneous colonies and the production of persistent bacteria showed a significant decrease,but the double knocking of genes did not differ from single knock.Conclusions: The bacterial stringent reaction(p)ppGpp synthase relA,pgm gene-mediated UDP-glucose biosynthesis pathway,and efflux pump gene(adeAB,acr)-regulated drug efflux were important factors affecting the heterogeneity of Acinetobacter baumannii to colistin.