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

Background: Tetracycline is an antibiotic widely used for the treatment of Helicobacter pylori infection, butits effectiveness is decreasing due to increasing bacterial resistance. The aim of this study was to investigatethe occurrence of 16S rRNA mutations associated with resistance or reduced susceptibility to tetracyclineofHelicobacter pylori by real-time PCR (RT-PCR) assays from culture. Materials and
Methods: Tetracyclinesusceptibility 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 104isolates of H. pylori examined, 11 showed resistance to tetracycline.
Results: LightCycler assay was applied toDNA extracted from 11 tetracycline-susceptible and 11 tetracycline resistance H. pylori isolates. In our studythe sequencing of the H. pylori wild types in 16 s rRNA gene were AGA 926-928 with MIC (0.016 to 0.5μg/ml),while the sequencing and MIC for resistant were GGA and AGC, (0.75 to 1.5μg/ml), respectively. Also we founda novel mutation in 2 strains with 84°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 H. pylori tetracyclineresistance related mutations that could be used directly on biopsy specimens.