| Background and objectives:Helicobacter pylori(H.pylori)is a global human pathogen that plays important role in gastrointestinal diseases,H.pylori eradication could significantly improve patients'life quality and reduce the occurrence rate of gastric cancer.The standard triple therapy constitutes with PPI and two antibiotics used to achieved good eradication rates,while with the increasing of antibiotic resistance rate,the eradication rates for several kinds of therapies have decreased a lot,which bring huge difficult to clinical cure H.pylori infection.For microorganism,there are a range of mechanisms that can be used to develop antibiotic resistance.Some of the mechanisms include the production of drug metabolic enzymes that inactivate drugs,the conformational change of the drug culminating in its inactivation,alteration of the drug binding site and reduce cell membrane permeability and efflux pumps which pump intracellular drugs such as antibiotic that do harms to the bacterial out of the cell.Alteration of the drug binding site is the main mechanism for bacterial resistance to fluoroquinolones,fluoroquinolones mainly produce antibacterial activity by inhibiting rotational and topological isomerase ?,which means the bacteria can be resistance to fluoroquinolones when the DNA rotational enzyme and topological isomerase IV have some specific mutation that change the binding site of fluoroquinolones.Efflux pump pumping intracellular drugs out of the bacteria is another mechanism that play an important role in the development of antibiotic resistance,among several kinds of efflux pump that have been identify in microorganism the resistance nodulation division(RND)family transporter is the most important one in gram-negative bacteria.There are three homologous genes encoded protein that belong to RND family transporter in H.pylori,named hefA/B/C,hefD/E/F,hefG/H/I,respectively.The hefA,hefD and hefG encode the outer membrane channel protein.In recent years,there are several studies focus on the relationship between efflux pump gene expression and antibiotic resistance,but the conclusion does not reach a consensus.It used to believe that the expression of efflux pump is mainly related to the occurrence of multidrug resistance.While some researchers found the efflux pump gene hefA is highly expressed in metronidazole resistance strain and amoxicillin resistance strains,using efflux pump inhibitor or knockout efflux pump gene hefA could increase the sensitivity of some metronidazole and amoxicillin in some strains,regain hefA gene after knockout this gene could reduce the sensitivity of metronidazole and amoxicillin again.But in some other investigations,knockout efflux gene hefC have no significant effect on antibiotic sensitivity change.In our previous research,it was found that the expression of hefA and hefD in multidrug resistance strains was significantly higher than antibiotic susceptibility strains and single antibiotic resistant strains,and there was no significant difference for the expression of hefG gene in strains with different antibiotic resistance background.What's the function of RND family genes hefA,hefD and hefG in the process of production antibiotic resistance of H.pylori,were there any relationship between efflux pump gene expression and resistance specific gene mutation,was it possible to prevent the occurrence of specific gene mutation through knockout efflux pump gene,to explore these questions we perform this experiment.Material and Methods:1.the changes of efflux pump gene expression and antibiotic resistance gene mutation in the process of antibiotic induced resistance(a)select seven strains that were susceptibility to amoxicillin,clarithromycin,metronidazole,levofloxacin,tetracycline and rifampicin from clinical isolated strains we stored before(b)use agar dilution method to confirm the minimal inhibitory concentration(MIC)of levofloxacin and metronidazole in these seven H.pylori strains that were selected.(c)Induced these seven H.pylori strains from susceptibility to resistance state by gradually elevate antibiotic concentration in H.pylori cultured medium.(d)The genomic DNA of these strains during each antibiotic gradient induction process was collected,the levofloxacin specific resistance gene gyrA and metronidazole specific resistance gene rdxA were amplified with PCR method and the gene sequences were obtained by sequencing.(e)the total RNA of these strains during each antibiotic gradient induction process were collected and the expression of efflux pump gene were detected by fluorescence quantitative PCR.2.the effects of antibiotic resistance gene mutation on efflux pump gene.(a)6 different amino acid mutations of gyrA gene were obtained during antibiotic gradient induction resistance process,transferred these mutations into four strains(one standard strain named 26695 and 3 clinical isolated strain used in first part,these were susceptibility to 6 antibiotic we detected)to construct levofloxacin resistance mutant strains.(b)the genomic DNA of these parental strains and gyrA mutation transferred strains were collected to confirm gyrA gene mutation sites was correct and detect their MIC for levofloxacin.(c)the change of efflux pump function before and after the transfer of gyrA mutations in H.pylori were observed with Hoechst 33342 accumulation assay.(d)3 different amino acid mutations of rdxA gene were obtained during antibiotic gradient induction resistance process,transferred these mutations into four strains(one standard strain named 26695 and 3 clinical isolated strain)to construct metronidazole resistance mutant strains.(e)the genomic DNA of these parental strains and rdxA mutation transferred strains were collected to confirm rdxA gene mutation sites was correct,and detect their MIC for metronidazole.(f)the change of efflux pump function before and after the transfer of rdxA mutations in H.pylori were observed with Hoechst 33342 accumulation assay.3.the role of efflux pump gene hefA,hefD and hefG in prevent and reverse antibiotic resistance gene mutations.(a)use the standard strain 26695.,clinical isolated strain 92MDR,F38MDR,639LE,298LE,23MZ to construct efflux pump gene hefA,hefD and hefG knockout strain.(b)use growth inhibition curves to detect the changes of antibiotic tolerance in 26695 parental strain and efflux pump knockout strains.(c)Induced 26695 strain and its efflux pump knockout strains from susceptibility to resistance status by gradually elevate antibiotic concentration in H.pylori cultured medium,and the genomic DNA of these strains were collected to observe whether knockout efflux gene could prevent the occurrence of antibiotic resistant mutations.(d)Compare the change of MIC value in parental strains and their efflux pump knockout stains,collect genomic DNA from the parental strains and their efflux pump mutant strains to observe whether the efflux pump mutation could reverse levofloxacin and metronidazole resistance to susceptibility.(e)with or without antibiotic stimulation,compare the gene expression of hefA,hefD and heft in parental and their efflux pump mutant strains.Results:1.the changes of efflux pump gene expression and antibiotic resistance gene mutation in the process of antibiotic induced resistance(1)antibiotic induced resistance:seven susceptibility strains were successfully induced to be resistance with levofloxacin and metronidazole,respectively.(2)the changes of efflux pump gene expression and antibiotic resistance genea)mutation in the process of levofloxacin induced resistance:compared with the mutations of levofloxacin resistance gene gyrA in each induction stage,the mutations of the 91st aspartic acid and the 87th aspartic amide in the gyrA gene were detected in 7 induction series,and the mutations of the 85th glycine and 88th Alanine appeared in a few strains.The mutation of the 91st aspartic acid act as first mutation detected in the induction process of 6 strains,the mutation of the 88th alanine act as first mutation detected in 1 strain,and the 91st amino acid mutations could be reverse when the 87th aspartic amide mutated.b)efflux pump gene expression:Comparing the gene expression of the hefA,hefD and hefG of the efflux pump in each induction stage of levofloxacin,in the process of induction,most strains showed higher gene expression of hefA,hefD and hefG in the 1/2*MIC concentration induction stage,However,in the process of further induction,the expression of these efflux pump gene showed some fluctuations.c)the correlation of efflux pump gene expression:based on the gene expression of efflux pump gene hefA,hefD and hefG in each induction stage,we do some correlation analysis,the result showed high correlation between her A and hefD gene expression in the process of levofloxacin resistance induction(Pearson Correlation=0.713,p<0.0001),while hefA and hefG(Pearson Correlation=-0.232,p=0.085),hefG and hefD(Pearson Correlation=-0.084,p<0.539)didn t show significant correlation in the process of levofloxacin induction.hefA,hefD may play a role in synergy,and hefG in this process plays a different role from hefA,hefD.(3)the changes of efflux pump gene expression and antibiotic resistance gene mutation in the process of metronidazole induced resistance.a)mutation in the process of metronidazole induced resistance:compared with the mutations of metronidazole resistance gene rdxA in each induction stage,it was found that there were great variations in base sequence and amimo acid sequences between different strains before and during induction.Besides,some strain cannot occur any base mutations during the process of metronidazole induced resistance.b)efflux pump gene expression:in the initial stage of metronidazole induction,some efflux pump gene hefA,hefD and hefG show significant increase in the induction stage of 1/2*MIC,but the expression of these genes showed some fluctuation in the process of continuous induction.c)the correlation of efflux pump gene expression:based on the gene expression of efflux pump gene hefA,hefD and hefG in each induction stage,we do some correlation analysis,the result showed no significant correlation between hefA and hefD gene expression in the process of metronidazole resistance induction(Pearson Correlation=-0.048,p=0.706),also no significant correlation between hefA and hefG gene expression(Pearson Correlation=-0.204,p=0.109),no significant correlation between hefD and hefG gene expression(Correlatio=0.154,p=0.229).It was suggested that there was no significant correlation between the gene expression of hefA,hefD and hefG in the process of metronidazole induced antibiotic resistance.3.the effects of antibiotic resistance gene mutation on efflux pump gene(1)construct gyrA gene mutant strain:six kinds of levofloxacin resistance gene gyrA mutations were transformed by using susceptibility H.pylori standard strain 26695 and clinical isolates strain J5A,R28 and 39 as receptor strains.6 amino acid mutations(87N/91N,87K/91N,87K/91G,87N/91G,87K/91D and 87N/91Y)of the gyrA gene were successfully constructed on 4 receptor strains.(2)the influence of gyrA mutation on strains'MIC:Detected the MIC of parental strains and the mutant strain that transformed the above 6 amino acid mutations,the results showed that compare with parental strain,the gyrA gene mutant strains have higher MIC value for levofloxacin,parental strains showed their MIC value were between 0.03-0.25 mg/l,strains that occur gyrA gene mutation at 87N/91N,87N/91G,87N/91Y showed their MIC value between 0.38-1.5mg/L,strains that occur gyrA gene mutation at 87K/91N,87K/91G,87K/91D mutation strain MIC between 1.0-32mg/L.(3)the effect of gyrA mutation on efflux pump function:comparing the efflux pump function between parental strains and their gyrA gene mutant strain,the result show that gyrA gene mutant with 87K/91D,87K/91G or 87N/91Y would increase the efflux pump function,while gyrA mutant with 87N/91N,87K/91N or 87N/91G do not have significant change.(4)construct rdxA gene mutant strain:three kinds of metronidazole resistance gene rdxA mutations were transformed by using susceptibility H.pylori standard strain 26695 and clinical isolates strain J5A,R28 and 39 as receptor strains.Only mutation of rdxA gene 194insertA was successfully constructed on 4 strains of receptor strain,and the remaining two amino acid mutations were not successfully screened,suggesting that 194insertA in rdxA gene was an effective metronidazole resistance mutation.(5)the influence of rdxA mutation on strains'MIC:Detected the MIC of parental strains and the mutant strain that transformed the 194 insert A mutations,the results showed that compare with parental strain,the rdxA gene mutant strains have higher MIC value for metronidazole,the MIC value of parental strains range from 0.5-2,and the MIC value of rdxA mutant strains range from 8-16.(6)the effect of rdxA mutation on efflux pump function:comparing the efflux pump function between parental strains and their rdxA mutant strains,the result show that rdxA mutant strain have higher efflux function.4.the role of efflux pump gene hefA,hefD and hefG in prevent and reverse antibiotic resistance gene mutations.(1)construct efflux pump gene knockout stain:use susceptibility strain 26695,clinical isolated strain 92MDR,F38MDR,M10MDR,639LE.,298LE act as parental strain to construct efflux pump knockout strain,we successfully constructed efflux pump mutant strain named 26695_?hefA,26695_,hefD and 26695_?hefG.(2)the effect of efflux pump gene knockout on efflux pump function:Hoechst 33342 accumulation assay showed that compared with parental strain 26691,efflux pump mutant strain 26695_?hefA,26695_?hefD and 26695_?hefG have lower efflux pump function.(3)the effect of efflux pump gene knockout on tolerance to antibiotic:growth inhibition curves show that compared with parental strain 26695,efflux pump mutant strain 26695_?hefA,26695_?hefD and 26695_?hefG's tolerance to levofloxacin and metronidazole decreased.And the difference was statistically significant.(4)efflux pump gene knockout prevents antibiotic resistance gene mutation:The results of levofloxacin resistance induction of parental strain 26695 and knockout strain 26695_?hefA,26695_?hefD,26695_?hefG showed that parental strain 26695 could detected gyrA gene 272 base mutations in the 2*MIC induction stage,knockout strains 26695_?hefD and 26695_?hefG could detected gyrA gene 273 base mutation in the 2*MIC induction stage.On the other hands,in the induction process,the knockout strain 26695_?hefA could detected the gyrA gene 271 base mutation in the 32*MIC induction stage;The results of the use of metronidazole induced antibiotic resistance showed that the parental strain 26695 could detected rdxA gene mutation in 8*MIC induction stage,the knockout strain 26695_?hefD could detected the rdxA gene mutation in the 64*MIC induction stage,26695_?hefG could detected the rdxA gene mutation in the 16*MIC induction stage,while the knockout strain 26695_AhefA did not detected rdxA gene mutation in the in the induction process.It is suggested that hefA gene knockout is beneficial to prevent the occurrence of levofloxacin and metronidazole resistant mutation.(5)efflux pump gene knockout reverse antibiotic resistance gene mutation:Detected the MIC value of parental strains and their efflux pump mutant strains,the result show that compared with parental strains M10MDR and 23MZ,mutant strains M10MDR_?hefA,M10MDR_AhefD,MIOMDR?AhefG,23MZ AhefA,23MZ_hefD become susceptibility to Metronidazole.Other strains had mild or no decrease in the MIC value of metronidazole.The MIC of levofloxacin with parental strains and mutant strains was detected,and no strain were changes from levofloxacin resistance to sensitivity.The changes of rdxA gene mutation could be detected in MIOMDR_?hefA,MIOMDR_?hefD,MIOMDR_?hefG,23MZ_?hefA and 23MZ_?hefD strains.(6)The interaction of efflux pump gene hefA,hefD and hefG:the previous part of the study suggested that in the process of levofLoxacin resistance induction,the expression of efflux pump gene hefA and hefD have a significant correlation.Therefore,we further compared the expression of hefA,hefD and hefG under levofloxacin pressure,and the results showed that:the expression of hefD in hefA knockout strain decreased compared with that of wild-type strains(p<0.05),The expression of hefA in the hefD knockout strain decreased compared with that of wild-type strains(p<0.05),It is suggested that efflux pump genes hefA and hefD may play a synergistic role under the condition of levofloxacin antibiotic pressure,and the expression of hefG in hefA knockout cannot be increased significantly compared with that of wild strains,and the expression of hefA in hefG knockout strain is lower than that of wild-type strains(p<0.05),It is suggested that the expression of hefA may require the participation of hefG,and the expression of hefG is not entirely influenced by hefA gene expression,and the expression of hefD in hefG knockout strain can be increased,decreased or not significantly compared with that of wild-type strains.The expression of hefd in hefG knockout strain decreased compared with that of wild-type strains(p<0.05),It is suggested that the expression of hefD may require the participation of hefG,and the expression of hefG is not entirely influenced by hefD gene expression.The previous part of the study suggested that all susceptibility H.pylori strains had no correlation between hefA,hefD and hefG gene expression during metronidazole resistance induction,so the correlation analysis of hefA,hefD and hefG gene mutations did not carried out.Conclusion:1.gyrA gene mutations are prevalent in levofloxacin resistance strain,and mutation in the 87th and 91st amimo acids of the gyrA gene are more important for levofloxacin resistance.rdxA gene mutations have greater diversity among different metronidazole resistance strains,and its clinical value in indicating metronidazole resistance needs to be strengthened.2.Bacterial efflux pumps increase significantly in the early stages of antibiotic stimulation,but do not remain at high levels as antibiotic pressure increases during subsequent antibiotic resistance induction.3.knockout efflux pump gene can prevent the drug resistance of levofloxacin and metronidazole in some degree and can reverse the resistance of metronidazole resistant in some strains.4.The efflux pump gene her A and hefD may play a synergistic role in coping with the pressure of levofloxacin antibiotics,hefG expression and other two efflux pump gene expression were less correlated in the response to antibiotic pressure. |
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