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Hsiang Tso Huang,1 Hsin-Ming Wang,1 Shih-Cheng Yang,1 Wei-Chen Tai,1 Chih-Ming Liang,1 Keng-Liang Wu,1 Chen-Hsiang Lee,2 Seng-Kee Chuah1 1Division of Hepato-Gastroenterology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Niao-Song District, Kaohsiung 833, Taiwan; 2Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Niao-Song District, Kaohsiung 833, Taiwan Purpose: To assess the efficacy of amoxicillin, tetracycline, high-dose metronidazole, and a proton-pump inhibitor for third-line Helicobacter pylori eradication. Methods: We enrolled 70 consecutive patients who had registered, failed to respond to two rounds of H. pylori eradication, and undergone endoscopy for H. pylori culture. Seven patients were lost to follow-up. Patients were treated according to the results of antibiotic-susceptibility testing reports (cultured group, n=39). Those who failed the H. pylori culture were prescribed 14-day quadruple therapy containing esomeprazole 40 mg twice daily, amoxicillin 1 g twice daily, tetracycline 500 mg four times daily, and metronidazole 500 mg three times daily (empirical group, n=24). A follow-up urea breath test was performed 8 weeks later. Results: Antibiotic-resistance rates were 79.5% (clarithromycin), 94.9% (levofloxacin), 66.7% (metronidazole), 2.6% (amoxicillin), and 0 (tetracycline). Eradication rates attained by the cultured and empirical group were 89.7% (95% CI 72.7%–97.1%) and 58.3% (95% CI 36.6%–77.9%) in per-protocol analysis (P=0.004) and 81.4% (95% CI 66.6%–91.6%) and 51.8% (95% CI 31.9%–71.3%) in intention-to-treat analysis (P=0.014), respectively. Culture-guided therapy was the only clinical factor influencing the efficacy of H. pylori eradication (OR 0.16, 95% CI 0.04–0.60; P=0.006). Despite the high metronidazole-resistance rate (66.7%) after two treatment failures, the eradication rate in patients with this condition was 84%. Conclusion: Empirical 14-day modified quadruple therapy is not acceptable as an alternative third-line rescue H. pylori treatment. The success rate of third-line susceptibility-guided treatment was near 90%. This report is valuable as a reminder to medical practitioners that rather than a try-and-see approach, susceptibility-guided therapy should always be considered whenever possible for patients who have undergone several treatment failures. Keywords: amoxicillin-resistance, metronidazole-resistance, empirical quadruple therapy, culture-guided therapy