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Open Access Research article

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

Türkan Sakinc12*, Barbara Baars2, Nicole Wüppenhorst3, Manfred Kist3, Johannes Huebner1 and Wolfgang Opferkuch2

Author Affiliations

1 Division of Infectious Diseases, University Hospital Freiburg, Hugstetter Strasse 55, Freiburg, D-79106, Germany

2 Department of Medical Microbiology, Ruhr-University Bochum, Universitätstraße 150, Bochum, 44801, Germany

3 Institute of Medical Microbiology and Hygiene, National Reference Centre for Helicobacter, University Medical Centre Freiburg, Freiburg, Germany

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BMC Research Notes 2012, 5:603  doi:10.1186/1756-0500-5-603

Published: 30 October 2012

Abstract

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.

Keywords:
Helicobacter pylori; Synergistic effect; 23S rRNA