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The aim of this study was to elucidate the mechanism of clarithromycin (CAM) resistance in laboratory strains and clinical isolates of Helicobacter pylori. The CAM resistance in laboratory strains was induced in vitro by CAM exposure. The majority of CAM-resistant strains were highly resistant to CAM (MICs > 100 micrograms/ml). These CAM-resistant strains also showed cross resistance to azithromycin, rokitamycin and clindamycin. The sites of point mutations in these resistant strains were identified as follows; the conserved domain V of genes encoding 23S rRNA were amplified first by PCR and this PCR products (1.4 kb) were subsequently digested with BsaI and MboII and RFLP patterns were analyzed. 1.4 kb amplicons of CAM-susceptible strains yielded two DNA bands of 1000 bp and 400 bp when digested with BsaI but no digestion product was seen by MboII digestion. In contrast to this, two types of RFLP patterns were observed for the resistant strains induced in vitro by CAM; one was the formation of three bands (700 bp, 400 bp and 300 bp) after BsaI digestion, and the other was the formation of two bands (approximately 700 bp) by MboII digestion. RFLP patterns of CAM-susceptible and CAM-resistant clinical isolates obtained from patients before and after CAM medication were similar to those observed for the CAM-susceptible strains and CAM-resistant strains developed in the laboratory. These results strongly suggest that the CAM resistance of H. pylori was caused by point mutation of 23S rRNA.
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