Real-time PCR detection and frequency of 16S rDNA mutations associated with resistance and reduced susceptibility to tetracycline in Helicobacter pylori from England and Wales

Objectives: To investigate the occurrence of 16S rDNA mutations associated with resistance or reduced susceptibility to tetracycline in Helicobacter pylori isolated in England and Wales, and to develop a real-time PCR assay to detect these DNA polymorphisms from culture and gastric biopsies. Methods: Tetracycline susceptibility was determined by disc diffusion. The MIC of isolates with reduced susceptibility was determined by Etest and agar dilution methods. The 16S rDNA of these isolates was sequencedandresistance-associatedmutationsidentified.ALightCyclerassaydevelopedtodetectthese mutations was applied to DNA extracted from culture and gastric biopsies. Results: From 1006 isolates of H. pylori examined, 18 showed reduced susceptibility to tetracycline. Of these, three were resistant (‡4 mg/L). Mutations in 16S rDNA were detected in 10 of the reduced susceptibility isolates: one double base mutation of A926T/A928C, one A926C, one A928C; and seven A926G. The first two polymorphisms were novel and had not been reported from clinical isolates previously. The LightCycler assay identified each of the 10 isolates with 16S rDNA mutations, but did not detect polymorphisms in 100 tetracycline-susceptible H. pylori isolates. The assay correctly determined the tetracycline susceptibility of H. pylori in 20 gastric biopsy samples. Conclusions:Mutationsin16SrDNAweredetectedinH.pyloriisolatedinEnglandandWaleswithreduced susceptibility to tetracycline, but resistance to this antibiotic was uncommon. We show molecular-based susceptibility testing for tetracycline is possible direct from biopsy material.