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Background  Clarithromycin (CLR) is the most commonly recommended antibiotic in  Helicobacter pylori  eradication regimens, but the prevalence of CLR-resistant  H .  pylori  is increasing. CLR resistance is associated with mutations in the 23S rRNA gene. However,  H. pylori  eradication can still be achieved with triple therapy, and an additive effect may occur with multiple antibiotics.    Methods  Twenty-six CLR-resistant strains were examined. The MIC of clarithromycin was determined by agar-dilution-testing on Columbia agar, as described elsewhere. The conserved region of the H. pylori 23S rRNA gene between nucleotide positions 1445 and 2846 [GenBank: U27270] was amplified. RFLP and sequence analysis were performed with the 1402-bp PCR product. Synergy between clarithromycin and amoxicillin was assessed using the agar dilution checkerboard technique. To confirm the correlation between mutation and synergistic effect with subinhibitory concentrations of AMX, site-directed mutagenesis was performed in four CLR-susceptible H. pylori isolates.    Results  Twenty-six clarithromycin-resistant strains were examined. The conserved region of the  H. pylori  23S rRNA gene was amplified, and the purified PCR product was checked for mutations by restriction fragment length polymorphism (RFLP) analysis and sequencing. A synergistic effect was found in only three of the 12  H. pylori  strains (25%) with the A2142G mutation and five of the 10  H. pylori  strains (50%) with the A2143G mutation (fractional inhibitory concentration: FIC < 0.5, minimal inhibitory concentration: MIC<2 mg/L) was found. Site-directed mutagenesis was performed in four CLR-susceptible  H. pylori  isolates.  Three of these isolates harboring a mutation in position A2143G grew under selection with CLR (MIC >16 mg/L), and all three strains showed the synergistic effect (FIC<0.5). In contrast, three of the same four strains transformed with DNA fragments with a mutation in position A2142G were resistant to CLR (MIC>16 mg/L) and showed no synergism with amoxicillin (FIC>2).    Conclusions  Here we demonstrate that in 100% of the  in vitro  transformed strains, a mutation at position A2143G leads to a synergistic effect between clarithromycin and amoxicillin, whereas a mutation at position at A2142G had no discernible effect.

Influence of a 23S ribosomal RNA mutation in Helicobacter pylori strains on the in vitro synergistic effect of clarithromycin and amoxicillin