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<it>Objectives</it>: To investigate the occurrence of 16S rDNA mutations associated with resistance or reduced susceptibility to tetracycline in <it>Helicobacter pylori</it> isolated in England and Wales, and to develop a real-time PCR assay to detect these DNA polymorphisms from culture and gastric biopsies. <it>Methods</it>: 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 sequenced and resistance-associated mutations identified. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture and gastric biopsies. <it>Results</it>: From 1006 isolates of <it>H. pylori</it> 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 <it>H. pylori</it> isolates. The assay correctly determined the tetracycline susceptibility of <it>H. pylori</it> in 20 gastric biopsy samples. <it>Conclusions</it>: Mutations in 16S rDNA were detected in <it>H. pylori</it> isolated in England and Wales with reduced susceptibility to tetracycline, but resistance to this antibiotic was uncommon. We show molecular-based susceptibility testing for tetracycline is possible direct from biopsy material.

<it>Objective</it>: Surveillance data on <it>Helicobacter pylori</it> antibiotic susceptibilities in Wales are limited, despite resistance being a key factor in treatment failure. A single-centre survey was undertaken over 3 years to determine local antibiotic resistance rates on isolates from dyspeptic patients in Bangor, Gwynedd (North Wales). <it>Methods</it>: Susceptibilities were determined for 363 isolates by disc diffusion and the Etest. Isolates were also genotyped (<it>cagA</it> presence and <it>vacA</it> allelic types). <it>Results</it>: Overall <it>in vitro</it> resistance rates were 24% for metronidazole and 7% for clarithromycin, with 4% resistant to both antibiotics. Resistant strains typically had high MICs of >256 mg/L. Tetracycline resistance was identified in only one isolate whereas no isolates showed resistance to amoxicillin. There was a two-fold increase in resistance over the study period. No gender and age associations with resistance were detected. Resistant and susceptible isolates were genotypically diverse with respect to <it>cagA</it>/<it>vacA</it> type but the <it>vacA</it> s1m2 form was a feature of all clarithromycin-resistant isolates compared with 56% of the susceptible isolates. <it>Conclusion</it>: Although the overall antibiotic resistance rates of <it>H. pylori</it> from North Wales were low compared with many other regions in Europe, continued surveillance, particularly of high-level resistance (MIC >256 mg/L), is recommended to monitor the effects of the ‘test and treat’ strategy for <it>H. pylori</it> eradication.