| Summary: | Background: The emergence of multidrug-resistant <i>Helicobacter pylori</i> has undermined eradication strategies to prevent the development of gastric cancer. This study was conducted to estimate the prevalence of secondary antibiotic resistance of <i>H. pylori</i> in urban multicultural areas in Malaysia. Methods: From January 2017 to December 2021, gastric biopsies from 218 patients with a history of <i>H. pylori</i> eradication failure were sent to our laboratory for antibiotic susceptibility testing. A minimal inhibitory concentration was determined for six antibiotics, namely metronidazole, clarithromycin, levofloxacin, amoxicillin, tetracycline, and rifampicin using the E-test method. Two multidrug-resistant <i>H. pylori</i> strains identified in this study were subjected to whole genome sequencing. Results: Eradication failure was observed to be significantly higher in the Malaysian Chinese patients than in the Malaysian Indian and Malay patients. <i>H. pylori</i> were successfully isolated from 51 patients (23.4%). Overall, the antibiotic resistance rates of <i>H. pylori</i> to metronidazole, clarithromycin, levofloxacin, and amoxicillin were 82.4% (42/51), 72.5% (37/51), 52.9% (27/51), and 3.9% (2/51), respectively. Resistance to tetracycline and rifampicin were not observed during the study period. Resistance to more than one antibiotic was observed in 82.4% (42/51) of the isolates, of which 42.2% (19/42) were resistant to three antibiotic classes. Resistance to both clarithromycin and metronidazole were most frequently observed in isolates with dual resistance (56.5%; 13/23). Codon substitutions in penicillin-binding protein 1A (V346L, V374L, G595_V596InsG, Y604H, and N608S) were detected in amoxicillin-resistance <i>H. pylori</i> strains. Herein, we report amoxicillin resistance in <i>H. pylori</i> isolated from Malaysian patients, and its resistance mechanism, for the first time. Conclusion: Our results show the increase trend in secondary multidrug resistance in <i>H. pylori</i> isolates, which warrants continuous surveillance.
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