| Background and objective:Helicobacter pylori(H.pylori)is an infectious disease that has long been colonized in the stomach and is closely related to various gastrointestinal diseases.H.pylori infection can cause chronic gastritis,gastric ulcer,and gastric mucosa-associated lymphoma,even stomach cancer.Therefore,the eradication of H.pylori has a very important role in the prevention and treatment of disease-related diseases and prevention of gastric cancer.At present,the international and interal consensus recommended treatment is a proton pump inhibitor and a bismuth combined with two antibiotics.However,due to the widespread use of antibiotics,the increasing resistance of H.pylori to antibiotics has greatly reduced its eradication rate and makes a serious challenge to clinical treatment.The AcrAB-TolC efflux pump is the predominant efflux system for multidrug resistance in Gram-negative bacteria,with the hefA,hefD and hefG genes encoding the outer membrane channel proteins.Clarithromycin is a macrolide antibiotic,and the antibacterial mechanism is combined with 23 S rRNA on ribosomes to achieve antibacterial action by interfering with protein synthesis.It is a common antibiotic to eradicate H.pylori.However,the resistance rate of H.pylori to clarithromycin is as high as 20%i in China,which seriously affects the eradication of H.pylori.It has been reported that efflux pumps participate in the resistance process of metronidazole and levofloxacin in H.pylori,but whether it is involved in clarithromycin resistance is not clear.Our previous study showed that the expression of AcrAB-TolC efflux pump-related genes in clinically isolated multi-drug resistant strains was higher than that of sensitive strains and single-drug resistant strains.The administration of efflux pump inhibitor CCCP could improve the sensitivity of some strains to clarithromycin.However,the mechanism and the ability to prevent resistance H.pylori by which efflux pump genes remains to be further explored.Therefore,we designed this study to knockout efflux pump genes hefA,hefD,and hefG.To investigate whether the knockout pump gene can increase the sensitivity of H.pylori to clarithromycin,and whether can prevent or reverse the resistance of H.pylori to clarithromycin? As well,the effects of knockout pump gene on the morphology,growth and toxicity of H.pylori were observed.Methods:(1)Construction and identification of AcrAB-TolC efflux pump-related genes hefA,hefD and hefG knockout strains in H.pylori1.1 Resuscitation,culture,identification,passage and cryopresrvation of the experimental strain Hp 26695.1.2 The construction of target vectors PILL570-ΔhefA,PILL570-ΔhefD and PILL570-ΔhefG in the experimental strain Hp 26695.1.3 Construction of the ΔhefA,ΔhefD and ΔhefG gene knockout strains in the experimental strain Hp 26695.1.4 PCR,DNA sequencing and fluorescence efflux test to verify whether the knockout was successful.(2)The effect of knocking out AcrAB-TolC efflux pump related genes on H.pylori resistance in clarithromycin:2.1 Knockout of AcrAB-TolC efflux pump related genes on the morphology,growth and toxicity of H.pylori.2.1.1 Scanning and transmission electron microscopy to detect the ultrastructure of knockout strains ΔhefA,ΔhefD,ΔhefG and wild strain Hp 26695.2.1.2 Detecting the growth of knockout strains ΔhefA,ΔhefD,ΔhefG and wild strain Hp 26695.2.1.3 Real-time labeled cell function analysis system detected the effects of knockout strains ΔhefA,ΔhefD,ΔhefG and wild strain Hp 26695 on the proliferation of gastric epithelial cells AGS and GES-1.2.2 Effect of knockout AcrAB-TolC efflux pump related genes on H.pylori sensitivity to clarithromycin.2.2.1 E-test detects the minimum inhibitory concentration of knockout strains ΔhefA,ΔhefD,ΔhefG and Hp 26695 against clarithromycin.2.2.2 The growth inhibition of clarithromycin on knockout strains ΔhefA,ΔhefD,ΔhefG and wild strain Hp 26695 were detected.2.2.3 Confocal microscopy to detect the survival of knockout strains ΔhefA,ΔhefD,ΔhefG and wild strain Hp 26695 under the role of clarithromycin.2.3 Knockout AcrAB-TolC efflux pump related genes in the prevention and reversal the effect of H.pylori on clarithromycin resistance2.3.1.Analysis of the role of prevention in antibiotic resistance: The parental strain Hp 26695 and its efflux pump knockout strains ΔhefA,ΔhefD,ΔhefG were induced to resist drug resistance by clarithromycin,and the genomic DNA of the strain was collected,sequencing 23 S rRNA gene,observe whether the knockout pump-related gene could prevent the 23 S rRNA gene mutation.2.3.2 Analysis of the role of reversal in antibiotic resistance: We use the clinical isolated single clarithromycin-resistant strains 184 CH and 100 CH,construct the progeny strains of the knockout pump genes hefA,hefD and hefG respectively,and the genomic DNA of the strain was collected,sequencing 23 S rRNA gene,detecting the change of MIC of to clarithromycin and 23 S rRNA gene mutations.Result:(1)Construction of AcrAB-TolC efflux pump-related genes hefA,hefD and hefG knockout strains in H.pylori:1.1 The results of gel electrophoresis showed that PCR amplification,enzyme digestion and ligation results were correct.1.2 PCR and sequencing verificated that constructing targeting vectors PILL570-ΔhefA,PILL570-ΔhefD and PILL570-ΔhefG successfully.1.3 PCR,sequencing and fluorescence efflux function experiments confirmed that the construction of Hp 26695 ΔhefA,ΔhefD and ΔhefG knockout strains was effective.(2)The effect of knocking out AcrAB-TolC efflux pump related genes on H.pylori resistance in clarithromycin:2.1 knockout of AcrAB-TolC efflux pump related genes on the ultrastructure,growth and toxicity of H.pylori.2.1.1 Effect of knockout AcrAB-TolC efflux pump related genes on the ultrastructure of H.pylori:The results of scanning electron microscopy showed that the knockout strains were morphologically spherical and atrophiccompared compared with the wild strain.The results of transmission electron microscopy showed that the cell membrane of knockout strains ΔhefA,ΔhefD and ΔhefG were damaged and contents of some strains were sparse compared with the wild strain Hp 26695,suggesting that the morphology of the knockout strain was damaged.2.1.2 Effect of knockout AcrAB-TolC efflux pump related genes on the growth of H.pylori:The growth of knockout strains ΔhefA,ΔhefD,and ΔhefG was basically the same as that of wild strain Hp 26695,and there was no significant difference(P>0.05),indicating that knockout pump-related genes had no effect on the growth ability of the strains.2.1.3 Effect of knockout of AcrAB-TolC efflux pump-related genes on H.pylori cytotoxicity.The growth ability of AGS and GES-1 cells under the role of knockout strains ΔhefA,ΔhefD,ΔhefG and wild strain Hp 26695 was basically same,there was no significant difference(P>0.05),indicating that the knockout pump-related genes did not affect the virulence on strains.2.2 Effect of knockout of AcrAB-TolC efflux pump-related genes on the sensitivity of H.pylori to clarithromycin.2.2.1 Effect of knockout of AcrAB-TolC efflux pump-related genes on the MIC of clarithromycin to H.pylori.The E-test results showed that the MIC of clarithromycin in knockout strains ΔhefA,ΔhefD,and ΔhefG were significantly reduced to 1/4 to 1/2 compared with the wild strain Hp 26695.It indicated that the strains were more sensitive to clarithromycin after knocking out the pump-related genes.2.2.2 Effect of knock out AcrAB-TolC efflux pump-related genes on the inhibition in H.pylori growth under clarithromycin.Under the pressure of clarithromycin,the growth of knockout strains ΔhefA,ΔhefD and ΔhefG were significantly inhibited compared with the wild strain Hp 26695,and the results were statistically different(P<0.05).2.2.3 Effect of knockout of AcrAB-TolC efflux pump-related genes on the survival of H.pylori under clarithromycin pressure.After treatment with clarithromycin,the knockout strains ΔhefA,ΔhefD and ΔhefG showed more damage and death than wild strains compared with wild strain Hp 26695.It indicated that the strain had a decreased tolerance to clarithromycin after knocking out the pump-related genes.2.3 Knockout of AcrAB-TolC efflux pump-related genes in the prevention and reversal of H.pylori resistance to clarithromycin.2.3.1.Knockout of the role of AcrAB-TolC efflux pump-related genes in preventing H.pylori resistance to clarithromycin.The results of clarithromycin-induced drug resistance showed that wild strain Hp 26695 had resistance gene mutations in the 1×MIC,and the knockout strains ΔhefD and ΔhefG also had resistance gene mutations in the 1/2×MIC,while knockout strain ΔhefA gene mutation occurred in 4×MIC,suggesting that hefA knockout is beneficial to prevent the occurrence of 23 S rRNA gene mutation in clarithromycin resistance,while hefD and hefG genes have no such effect.2.3.2 Knockout of AcrAB-TolC efflux pump-related genes in reversing the resistance of H.pylori to clarithromycin.After 184 CH pump gene knockout,the MIC of ΔhefA and ΔhefD became 1/16 of of the parental t,and the strain became sensitive from resistant strain.The MIC of ΔhefD and ΔhefG becomes1/8 and 2/3 of of the parental,respectively.After the 100 CH knockout pump gene,the MIC of ΔhefA,ΔhefD and ΔhefG became 1/4/~1/8 of the parental,respectively,and the knock strains from the resistant strains to the sensitive strains.Before and after the 184 CH and 100 CH knockout pump genes,the mutation site of clarithromycin 23 S rRNA did not change,It is suggested that knocking out the pump-related genes can reverse the resistance of some clinical strains to clarithromycin.However,mutations in its clarithromycin-specific resistance gene failed to respond.Conclusion:1.After knocking out the pump genes hefA,hefD and hefG,the morphological structure of H.pylori was destroyed,but it had no significant effect on its growth and bacterial toxicity.2.Knockout of pumping genes hefA,hefD and hefG can increase the sensitivity of H.pylori to clarithromycin and reverse the resistance of some clinically resistant strains to clarithromycin.3.Knocking out the pump gene hefA can delay the mutation of H.pylori 23 S rRNA resistance gene and prevent H.pylori resistance to clarithromycin. |
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