Real-Time PCR Detection of 16S rRNA Novel Mutations Associated with Helicobacter pylori Tetracycline Resistance in Iran

Abstract

Background: Tetracycline is an antibiotic widely used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing due to increasing bacterial resistance. The aim of this study was to investigate the occurrence of 16S rRNA mutations associated with resistance or reduced susceptibility to tetracycline ofHelicobacter pylori by real-time PCR (RT-PCR) assays from culture. Materials and Methods: Tetracycline susceptibility and minimal inhibition concentration (MIC) was determined by the Epsilometer test (Etest) method. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture. The 16S rRNA of these isolates was sequenced and resistance-associated mutations were identified. From 104 isolates of Helicobacter pylori examined, 11 showed resistance to tetracycline. Results: LightCycler assay was applied to DNA extracted from 11 tetracycline-susceptible and 11 tetracycline resistance Helicobacter pylori isolates. In our study the sequencing of the Helicobacter pylori wild types in 16 s rRNA gene were AGA 926-928 with MIC (0.016 to 0.5 mu g/ml), while the sequencing and MIC for resistant were GGA and AGC, (0.75 to 1.5 mu g/ml), respectively. Also we found a novel mutation in 2 strains with 84 degrees C as their melting temperatures and exhibition of an A939C mutation. Conclusions: We conclude that real-time PCR is an excellent method for determination of Helicobacter pylori tetracycline resistance related mutations that could be used directly on biopsy specimens.