In Vitro Induction Of Resistance To Metronidazole, Clarithromycin, Amoxicillin In Helicobacter Pylori Strains Isolated From Children

Helicobacter pylori (H.pylori) infection is accepted as an most important causative agent of chronic active gastritis in children, and also associated with the occurrence, progress, recurrence of peptic ulcer. Currently the most effective treatments for H.pylori combine a proton pump inhibitor and two antibiotics. Amoxicillin, clarithromycin and metronidazole are three of the most frequently used antibiotics in H.pylori strains from children.With antibiotic-resistant H.pylori strains increasing, in vivo efficacy of anti-H.pylori eradication therapy have been decreased. Lots of clinical studies indicated that H.pylori strains rarely acquired resistance to amoxicillin but easily to clarithromycin and metronidazole. However, it was unclear whether the antibiotic resistance of H.pylori strains was induced or passively selected during long-term or frequent treatment with amoxicillin, clarithromycin and metronidazole. To compare the propensity of acquired resistance to antibiotics, H.pylori strains were exposed to amoxicillin, clarithromycin and metronidazole in vitro.All H.pylori strains were clinical isolates, derived from biopsy specimens of patients submitted to endoscopy in Affiliated Children's Hospital, Zhejiang University school of medicine from December 2004 to July 2005. Gastric biopsies were homogenated with tissue homogenizers, and incubated on selective medium plates enriched with sheep blood at 37 ℃ for 3 to 5 days under a microaerophilic (5% O₂ 10% CO₂ 85% N₂) atmosphere. The identity of H.pylori were confirmed by colony appearance, microscopic examination after Gram staining and positive biochemical tests for urease, oxidase and catalase activities. To seek metronidazole-susceptible strains(MTZ^S), the minimum inhibitory concentrations(MICs) of metronidazole was determined by using Epsilometer test (E-test) method. 13 strains were randomly selected from the metronidazole susceptible strains(MTZ^R), before the MICs of amoxicillin and clarithromycin were determined by using E-test method. 7 out of 13 strains were found susceptible to all of the antibiotics. When antibiotic initial MICs (iMICs) of these 7 strains were determined by a routine agar dilution technique, in vitro selection of amoxicillin, clarithromycin and metronidazole resistant H.pylori strains from children were processed. Isolates were also transferred at least three times on antimicrobial agent-free medium, followed by a redetermination of the final MICs (fMICs) to assess the stability of the selected resistance. Finally, the propensity of three antibiotic groups was compared. H.pylori strains were considered resistant to clarithromycin when the MIC ^ l(j.g/mL, resistant to amoxicillin when the MIC>0.5ug/mL and resistant to metronidazole when the MIC>8^g/mL.In this study, 7 and 3 strains respectively acquired resistance to metronidazole and clarithromycin after serial passage to antibiotic plates, while none of the strains were resistant to amoxicillin. The inductive folds were calculated by the ratios of fMICs and iMICs, which were 2¹6 folds in amoxicillin group compared with 1 > 128 and 8^>64 folds in clarithromycin group and metronidazole group respectively. Looking through the whole process of selection, the MICs arised rapidly on the first stage but became steady on the second stage in amoxicillin groups, and the MICs remained unchanged after reaching 0.5j!g/mL. In contrast, the MICs of clarithromycin remained stable on the first stage but increased sharply on the second stage duringserial passages. After three transfers on antimicrobial agent-free medium, the MICs decreased for 6 strains that were isolated on amoxicillin plates and for 2 strains that were isolated on clarithromycin plates. For resistant strains that were isolated with metronidazole, the MICs remained stable.From all the outcomes above, we could conclude: the resistance was easily selected to metronidazole and could be selected to clarithromycin but could not be selected to amoxicillin after in vitro induction for H.pylori isolated from children;the amoxicillin MICs for H.pylori strains that were isolated by serial passage technique were unstable, since they rapidly declined to baseline after three transfers on antimicrobial agent-free medium, but the MICs of clarithromycin and metronidazole for H.pylori were stable.