| Helicobacter pylori (H.pylori) infection is one of the most common chronic bacterial infection in the world and has been established as a major cause of gastritis, peptic ulcer disease, and has also been considered a risk factor in the development of gastric carcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. Multiple studies have demonstrated that eradication of H.pylori may cure of the peptic ulcer disease, reduce the risk of gastric adenocarcinoma and has been associated with regression of MALT lymphoma. Currently the most effective treatments for H.pylori combine a proton pump and two antibiotics. The macrolide clarithromycin has emerged as the most important antibiotic in combined therapy for eradication of H.pylori infection. However, concerns about increasing clarithromycin resistance to H.pylori and its impact on the efficacy of eradication therapy have been raised since its widespread acceptance in H.pylori therapy. No documented data is available concerning associated with clarithromycin resistance in strains from Chinese childhood patients. The purpose of this study was to analyze the prevalence and mechanism of clarithromycin resistance in H.pylori strains from children.From October 2002 to January 2004, 108 strains of H.pylori isolated from both antrum and corpus biopsy specimens of 108 patients submitted to endoscopy in Affiliated Children Hospital of Zhejiang University Medical College. Gastric biopsies were ground with a tissue homogenizer, and incubated on selective medium platesenriched with fresh sheep blood at 37 癈 for 3 to 5 days under a microaerophilic (5% O2 10% CO2 85% N2 ) atmosphere. H.pylori isolates were identified by Gram staining and biochemical tests for urease, oxidase and catalase activities. The susceptibility of H.pylori isolates to clarithromycin was determined by using Epsilometer test (E-test) and two-fold agar dilution test. A strain was considered resistant to clarithromycin when the MIC lg/ml. Genome DNAs of the 108 isolates were extracted and prepared for PCR to detect the corresponding gene in the V domain of the 23S rRNA. The amplified fragments of all sensitive and resistant strains were digested with the restriction enzyme and analyzed by restriction fragment length polymorphism (RFLP) when an additional restriction site was created by the mutation. The restriction enzyme Bbsl and Bsal allowed a discrimination between the wild-type genotype (AAAGACC, from position 2143 to position 2149) and two mutant genotypes described : Bbsl for A2143G and Bsal for A2144G. The restriction products were analyzed on a 1.5% agarose gel stained with ethidium bromide. Some of the target amplification products were sequenced after purification. The sequences of resistant H.pylori were compared with the reported sequences form H.pylori 26695 and H.pylori J99 and sensitive isolates in order to find out the difference between them. Statistical analysis: The prevalence rate (%) of clarithromycin was calculated for the total isolates. Analysis was performed by using SPSS program (version 11.5) with a Bivariate to derive Spearman nonparametric correlations between mutations at positions 2143 and 2144 with respect to the MIC. P values of < 0.05 was considered statistically significant.Sixteen of 108 isolates of H.pylori were categorized as resistant to clarithromycin by the agar dilution method and E-test method, and the resistance rate was 14.8% (16/108) . The MICs of clarithromycin for resistant isolates varied from 1 to 128g/ml and included MICs of lug/ml (n=l), 2g/ml (n=3), 8g/ml (n=2), 16g/ml (n=4), 32g/ml (n=l), 64g/ml (n=2), and 128g/ml (n=3). Comparison of susceptibility and resistance determined by both methods showed a perfect category correlation. The target fragment 425bp in length containing 23S rRNA corresponding gene wassuccessful amplified. An A2144G mutation digested with Bsal was detected in 13 resistant isolates, but an A2143G mutation digested with Bbsl in only 3 among all 16 clarithromycin resistant strains. None of the sensitive isolates w...
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