The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.Helicobacter pylori infection is chronic in nature, causes gastritis, and increases the risk of development of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer (for reviews, see references 13, 18, and 20). Increasing rates of resistance to the first-line antibiotic drugs (e.g., clarithromycin) are compromising the eradication of H. pylori and result in therapy failures (8). Thus, alternative treatments that include tetracyclines are recommended (1, 2, 14).Tetracyclines are bacteriostatic drugs which exert their antimicrobial effects by affecting the 30S subunit of the ribosome and block the binding of aminoacyl-tRNA, resulting in impaired protein biosynthesis (3,22). The resistance of H. pylori to tetracyclines is reported to be caused by mutations in the 16S rRNA. H. pylori isolates exhibiting AGA 926-928 3 TTC triple-base-pair mutations (4,5,22) show MICs higher than 4 mg/liter and probably represent the clinically most relevant strains, whereas single-or double-base-pair mutations were, rather, associated with MICs between 1 mg/liter and 4 mg/liter (3, 5).The susceptibility of H. pylori to tetracycline is routinely examined by agar diffusion (Etest) or agar dilution tests, which are accepted to be the "gold standards" (10). These methods are slow and time-consuming, and they sometimes fail due to a lack of growth of the infecting H. pylori strain or due to overgrowth with contaminating bacteria.It has already been shown that real-time PCR assays are elegant methods for prediction of resistance to clarithromycin (15, 16) or ciprofloxacin (7) in H. pylori. The major advantage of this technique over classical microbiological resistance testing is not in its enhanced speed and standardization but is mostly in the option to test for resistance in clinical specimens that no longer contain live bacteria.In this study, a real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline re...