OBJECTIVETo investigate the prevalence of Helicobacter pylori (H. pylori) resistance to clarithromycin (CLM) in children and to demonstrate the correlation of 23S rRNA gene mutation to clarithromycin resistance of Helicobacter pylori isolates.

METHODSTotally 108 clinical strains of H. pylori were isolated from gastric biopsy specimens obtained from children who underwent endoscopy during the period from October 2002 to January 2004 in Children's Hospital Affiliated to Medical College of Zhejiang University. H. pylori was identified by morphology and biochemical tests after culture. Clarithromycin susceptibility of H. pylori isolates was determined by both E-test and two-fold agar dilution method. A strain was considered resistant when the MIC was defined as >or= 1 microg/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 were recognized and analyzed by restriction fragment length polymorphism (RFLP) when an additional restriction site is created by the mutation. The PCR products of all sensitive and resistant strains were digested with restriction enzyme BbsI and BsaI and were analyzed on a 1.5% agarose gel to discriminate different kinds of mutant genotype.

RESULTSSixteen of 108 isolates of H. pylori were resistant to clarithromycin by the agar dilution method and E-test method in clinical isolates from children, and the CLM resistance rate was 14.8% (16/108) with MICs ranging from 1 microg/ml to 128 microg/ml. Comparison of results of the two methods showed that these two methods were quite consistent in determination of susceptibility and resistance. The target fragment 425 bp in length containing 23S rRNA corresponding gene was successfully amplified. An A2144G mutation digested with BsaI was detected in 13 resistant isolates, but an A2143G mutation digested with BbsI in only 3 among all 16 clarithromycin resistant strains. None of the sensitive isolates was cleaved by either BsaI or BbsI enzyme, indicating that there was no mutation on them. It was also found that all the fragments from the resistant strains were not completely digested, and 425 bp uncut fragments were also visible and showed three bands indicating that they were heterozygotic strains with a mixture of wild-types and A-->G genotypes. In addition, in this study, no statistically significant difference between mutations at positions 2143 and 2144 with respect to the MIC was observed (r = 0.035, P > 0.05).

CONCLUSIONA high prevalence of clarithromycin-resistant H. pylori strains were detected among strains isolated from Chinese children studied. The 23S rRNA gene mutation at positions A2143G and A2144G plays an important role in clarithromycin resistance of H. pylori and A2144G mutation is the predominant finding among the resistant strains.