| Background and purpose:Helicobacter pylori(H.pylori)infection is a major cause of chronic gastritis,peptic ulcer disease,gastric mucosa-associated lymphoid tissue(MALT)lymphoma and closely associated with gastric cancer.H.pylori is classified as a group I carcinogen by the International Agency for Research on Cancer and the World Health Organization.Therefore,effective eradication of H.pylori is necessary for the prevention and treatment of these diseases.Currently,antibiotics commonly used in clinical for the eradication of H.pylori include metronidazole,clarithromycin,levofloxacin,amoxicillin,tetracycline,and furazolidone and so on.The triple therapy regimen,composed of a proton pump inhibitor or bismuth-containing quadruple is the most commonly used clinical therapeutic regimen.In recent years,with the widespread use of antibiotics,the problem of resistance to H.pylori antibiotics has become more and more serious.In particular,the resistance rate to metronidazole,clarithromycin,and levofloxacin has been increasing,and dual and multidrug resistance have emerged MDR strains,resulting in a decrease of eradication rate of H.pylori.It brought great difficulties to clinical treatment.Therefore,it is critical to identify the mechanism of H.pylori resistance and to find effective H.pylori eradication programs.Resistance gene mutations and high expression of efflux pump are the main mechanisms of bacterial resistance.The main drug resistance genes of metronidazole,clarithromycin,and levofloxacin are rdxA gene,23S rRNA gene,and gyrA gene.The AcrAB-TolC efflux pump in the RND family is mainly found in Gram-negative bacteria and plays an important role in H.pylori resistance.Changes on the expression and function of the efflux pump together with mutations in the drug-specific target gene result in secondary resistance to the bacteria.Based on the above research status,in order to further explore the relationship between the changes of efflux pumps and the mutations of drug-specific target genes,we detected the transcription level of the genes encoding hefA,hefD,and the related genes of the AcrAB-TolC efflux pump of the different resistant H.pylori strains,and the mRNA expression of hefG and the corresponding mutation of antibiotic-specific drug resistance genes;and further examined the expression of hefA,hefD and hefG genes encoding AcrAB-TolC efflux pump-related proteins during the artificial induction of H.pylori resistance by clarithromycin mRNA expression and mutation of clarithromycin-specific drug resistance genes.Finally,the effect of efflux pumps on inhibition of efflux pump expression by carbonylcyanide-m-chlorophenylhydrazo-ne(CCCP)and antibacterial activity against H.pylori were also observed.This study provides new ideas for solving the problem of resistance to H.pylori and other pathogens,and it can also provide effective guidance for the rational use of antibiotics in clinical treatment.Methods:1.Selection of H.pylori strain:Eight strains being all-sensitive strains(sensitive group)for eight antibiotics metronidazole(MZ),clarithromycin(CH),azithromycin(AZ),levofloxacin(LE),moxifloxacin(MX),amoxicillin(AM),tetracycline(TC),rifampicin(RI)were screened out in 600 clinical isolates of H.pylori stored in our laboratory.Furthermore,five multidrug resistant strains(multi-resistance group),six strains with metronidazoles resistance(metronidazole group),two strains with clarithromycin resistance(clarithromycin group),and five strains with levofloxacin resistance(levofloxacin group)were also screened out.They were then subjected to resuscitation,culture,and identification.2.Sensitivity of H.pylori to antibiotics:E-test was used to detect the minimum inhibitory concentration(MIC)of antibiotics against H.pylori,and H.pylori international standard strain ATCC43504 was used as a quality control according to the American Committee of Clinical Laboratory Standards(NCCLS)standard.Bacteria are regarded as resistant or sensitive when:MIC≥8 μg/ml as metronidazole resistance;MIC≥1 μg/ml as clarithromycin,azithromycin,levofloxacin,moxifloxacin,amoxicillin,Tetracycline,rifampicin resistance.3.Detecting mRNA level of hefA,hefD and hefG mRNAs of AcrAB-TolC efflux pump-related proteins in H.pylori strains.Here RT-PCR method was applied and 16S rRNA was used as a reference gene4.Mutation detection of H.pylori strains corresponding to antibiotic-specific drug-resistance genes:Genomic DNA was extracted and was then sequenced after PCR amplification.The DNA sequencing software was used to analyze the sequencing results and compared with the sequence control of H.pylori 26695 strain.5.In vitro induction test of clarithromycin-induced H.pylori resistance:Six strains of eight strains were selected for S.aureus,S12a,S28a,S39a,S59a,and S96a.They were added for the first time by liquid culture.The MIC concentration of 1/2 times the clarithromycin concentration of the sensitive strain began,the concentration gradually increased by two times,namely 1/2 ×MIC,then gradually induced to high concentrations of 1×MIC,2×MIC,until 256×MIC or even higher.MICs,efflux protein-related protein-encoding genes,hefA,hefD,and hefG,and 23S rRNA gene mutations were detected in each step.6.Effects of efflux pump inhibitor CCCP on H.pylori strains:CCCP was used to detect the expression of efflux pump genes after H.pylori standard strain ATCC43504 and clarithromycin-induced drug strain S12a-2×MIC.CCCP of the same concentration Role of efflux pump gene expression at different time points(0 h,3 h,6 h,until 48h)of H.pylori standard strain ATCC43504 and clarithromycin-induced resistant strain S12a-2×MIC;efflux pump inhibitor CCCP against antibiotic MIC Impact.Changes in the MICs of antibiotics against H.pylori 4 or more times after addition of CCCP inhibitors are considered to be significantly different.Results:1.The E-test method confirmed that the selected strain is the expected strain,including 8 strains which are all sensitive to 8 antibiotics,5 multi-drug resistant strains,6 single metronidazole-resistant strains,2 strains Single clarithromycin-resistant strain,5 strains of single levofloxacin-resistant strains.2.The mRNA expressions of hefA,hefD and hefG genes encoding AcrAB-TolC efflux pump related proteins in different H.pylori strains were as follows:The expression of hefA and hefD mRNA in the multi-drug resistance group was significantly higher than that in the sensitive group and single metronidazole.In the drug-resistant group,single clarithromycin-resistant group,and single levofloxacin-resistant group(P<0.001),the relative expression levels of hefA and hefD mRNA in each single-drug-resistant group were different,but the difference was not statistically significant(P>0.05).HefG mRNA expression in multidrug resistance group was higher than that of sensitive group,single metronidazole resistance group,single clarithromycin resistance group and single levofloxacin resistance group,but the difference was not statistically significant(P>0.05).There was a difference in the relative expression of hefG mRNA among the single drug-resistant groups,but the difference was not statistically significant(P>0.05).Based on the above results,the mRNA of the gene encoding the AcrAB-TolC-associated protein was highly expressed in the multidrug-resistant H.pylori strain,whereas no single strain of the antimicrobial-resistant strain showed high expression.3.Mutation of drug resistance genes:There were rdxA gene mutations in 5 multidrug resistant strains,and the mutation sites were dispersed.There were also gyrA gene mutations,the main mutation sites were T260C,C302T,and T308C.There were 23 S rRNA gene mutations in the drug-resistant strains and the two clarithromycin-resistant strains.The main mutation site was A2143G,and some strains showed G2212A.Based on the above results,there is a corresponding resistance gene mutation in multidrug resistance and clarithromycin-resistant H.pylori strains.4.The expression of AcrAB-TolC gene and the mutation of the corresponding drug resistance gene in the process of H.pylori resistance induced by clarithromycin:a comprehensive analysis of the changes of 6 sensitive H.pylori strains in clarithromycin-induced drug resistance resulted in the following results:After the same passage of clarithromycin-inducible strains,the gene encoding the efflux pump-related protein was continuously low expressed,and there was no mutation in the drug resistance gene.After clarithromycin induction,all strains were resistant to 2×MIC concentrations,with 1 strain at 2×MIC concentration,2 strains at 1×MIC concentration,and 3 strains at 1/2×MIC concentration.At the time of drug resistance,the resistance of S5a strains that developed resistance at 2×MIC occurred before the high expression of efflux pump genes and the mutation of drug resistance genes.Highly-expressed or drug-resistant gene mutations;3 strains of these 5 strains(S28a,S39a,S96a)produced resistance at 1/2×MIC concentrations.High expression of efflux pump genes in H.pylori strains occurred in drug resistance before the gene mutation,especially the hefG gene.Only when the efflux pump gene was highly expressed,the resistance of H.pylori to clarithromycin was low.When the MIC of clarithromycin to H.pylori was more than 64 μg/ml,there was a mutation in the drug resistance gene and the mutation was detected.Persistence,although mutation sites may vary.During the induction process,the expression of the efflux pump gene will fluctuate,especially after the mutation of the drug resistance gene,the high expression of the efflux pump gene is no longer important for the maintenance of H.pylori resistance.5.Effect of efflux pump inhibitor CCCP on H.pylori strain:① For H.pylori standard strain ATCC43504 being sensitive to antibiotics,CCCP concentration in the range of 0.02-0.3 μg/ml,the inhibitory effect of efflux pump gene expression is not significant,When the concentration was 0.6 μg/ml,the expression of efflux pump gene was significantly inhibited(P<0.05).For clarithromycin-induced S12a-2×MIC strain was produced,efflux pump gene expression was significantly inhibited when the CCCP concentration reached 0.04 μg/ml(P<0.05).②the CCCP concentration of 0.6 μg/ml revealed the best inhibitory effect on efflux pump gene expression in H.pylori standard strain ATCC43504 at 9-24h,and showed the best inhibitory effect for the S12a-2×MIC strain at 3-15h.③The MIC of the 3 strains of metronidazole in the 5 MDR strains was 4 or more times higher than that of the efflux pump inhibitor CCCP.The MICs of the two clarithromycin bacteria showed a 4 or 4 fold increase,and the MICs of 2 strains of levofloxacin were 4 or more times higher.In sensitive strains,CCCP presented no significant effect on antibiotic MICs.Conclusion:1.Drug resistant gene mutations were found in clinically isolated clarithromycin-resistant H.pylori strains.The efflux pump gene expression was not significantly increased,Drainage pump gene expression was preceded by drug resistance mutation,and efflux pump gene expression could be decreased after drug resistance mutation in some of the bacteria during the H.pylori resistance induced by clarithromycin.This suggests that the efflux pump may be highly expressed in the early stage of the development of resistance to H.pylori and may have adaptive protection against H.pylori in the presence of antibiotics,giving H.pylori time to produce specific drug resistance mutations when the mutation occurs.Bacteria no longer need the adaptive protection of the efflux pump,and the expression of the efflux pump is restored and no longer plays a role in H.pylori resistance.2.AcrAB-TolC efflux pump-related protein encoding genes,hefA and hefD,were more highly expressed in clinical isolates of H.pylori MDR strains than susceptible strains and single drug-resistant strains.Efflux pump inhibitors could reduce some antibiotics against H.pylori and result in the decrease of MIC value in the MDR strain,suggesting that the AcrAB-TolC efflux pump plays a role in H.pylori multidrug resistance.The resistance status of all H.pylori MDR strains was consistent with the mutation of drug resistance genes,suggesting that AcrAB-TolC efflux pump and specific resistance mutations mediate multidrug resistance of H.pylori MDR. |
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