Clinical And Basic Study Of Metronidazole On Helicobacter Pylori Eradication

Aims:Helicobacter pylori(H.pylori)is the major pathogen of gastric disorders,its eradication would be of great significant.With an increasingly antibiotic resistance,H.pylori eradication rate is getting lower and lower.For those allergic to penicillin,one common practice has been to substitute metronidazole for amoxicillin.In clinical,metronidazole resistance could be overcome by increasing metronidazole doses.However,it remains to be seen whether high dose of metronidazole could overcome resistance in the absence of bismuth.There were unconsistencies between metronidazole phenotypic resistance and genotypic resistance,and the mechanism of metronidazole resistance still remains unclear.This study were aiming to explore the mechanism of metronidazole's resistance,so as to provide theoretical evidence and new ideas for H.pylori treatment.Methods:(1)66 penicillin allerge,treatment naive subjects were recruited and randomized(1:1)to 14-day esomeprazole(20 mg q12h),clarithromycin(500 mg q12h)and high dose of metronidazole(400 mg q6h)with(BECM group)or without(ECM group)bismuth(600 mg q12h).H.pylori strains were isolate and cultured from gastric mucosa.Eradication was confirmed by ¹³C-urea breath test 6 weeks after therapy.Metronidazole and clarithromycin susceptibility was assessed by agar-dilution.Adverse events were recorded.(2)65 clinical strains were selected randomly to confirm the metronidazole MIC by agar dilution method,then genome DNA were extracted,rdx A were amplified,sequenced and blasted with strain 26695 to figure out the relationships between rdx A mutation and MIC of metronidazole.(3)Induced metronidazole resistance of 24 strains(including 26695,J99 and 22 clinical susceptible isolates)in vitro,then extracted DNA,amplified,sequenced and blasted their rdx A to find the mutation site between parental and induced strains.Extract the total RNA of parental strain(S),metronidazole induced strain grown with(8ug/ml)(M8)or without metronidazole pressure(M0),analyze the m RNA expression of rdx A,frx A(HP0642)and sod B et al by Real-time PCR.Results:(1)Sixty-six subjects were randomized,four were lost to follow-up and eight violated the protocol.The eradication rates was 63.6%(95%CI 47.2%-80.0%)for ECM versus 84.8%(95%CI 72.6%-97.1%)(p=0.049)for BECM by intention-to-treat,67.7%(95%CI 51.3%-84.2%)versus 90.3%(95%CI 79.9%-100%)(p=0.029)by modified ITT and 70%(95%CI 53.6%-86.4%)versus 96%(95%CI 88.3%-100%)(p=0.033)by per-protocol.Metronidazole,clarithromycin and dual resistant rates were74.2%,24.2%and 18.2%,respectively.The cure rates were significantly improved by the addition of bismuth for both clarithromycin-resistant isolates(100%versus 25%,p=0.024)and metronidazole-resistant isolates(94.7%versus 63.6%,p=0.043).Adverse events were reported by 45.5%of subjects in ECM group and 48.5%in the BECM group(p=0.805).(2)65 clinical H.pylori strains including 12 metronidazole susceptible,53 resistant(MIC=16ug/ml,6 strains;MIC=32ug/ml,16 strains;MIC=64ug/ml,21 strains;MIC?128 ug/ml,10 strains)were sequenced.In the strains of MIC?16ug/ml,rdx A of 5.6%(1/18)strains contained advanced stop condon,while in the strains of?32ug/ml,44.7%(21/47)contained advanced stop condon(p=0.003).In MIC?32ug/ml strains,100%(5/5)advanced stop codons were caused by rdx A base substitution mutation,while in MIC?64 ug/ml strains,58.8%(10/17)were caused by frameshift mutation(p=0.004).(3)24 metronidazole susceptible strains including26695 and J99 were induced to high level metronidazole resistant,19 strains of which were sequenced successfully.4 induced strains developed large part delete mutation of rdx A,11 stains developed advanced stop codon(3 due to base substitution mutation,8due to frameshift mutation).m RNA expression of rdx A,frx A,sod B,HP0650,HP1084,HP1179,HP1159 were down-regulated in those induced strains(M0/M8 vs S),while somewhat up-regulated in the presence of metronidazole pressure(M8 vs M0).Conclusions:(1)This prospective trial demonstrated that while high-dose metronidazole could not completely overcome metronidazole resistance,bismuth was additive and improved the overall cure rates by 21%-26%.(2)Advanced stop codon caused truncated Rdx A were usually occurred in those strains with metronidazole MIC?32 ug/ml,while truncated Rdx A due to rdx A insert or delete mutation caused frameshift mutation were usually occurred in those with MIC?64 ug/ml.(3)It is easy to induce metronidazole resistance in vitro.Truncated Rdx A due to rdx A frameshift mutation caused advanced stop codon can predict high level phenotypic resistance of metronidazole.In the presence of metronidazole pressure,down-regulated expression of rdx A,sod B et al may be a reaction of H.pylori to adapt to the environment,which is an important evidence of induced phenotypic resistance.