Development of a High-Resolution Melting Method for the Detection of Clarithromycin-Resistant Helicobacter pylori in the Gastric Microbiome

<b>Background:</b> The issue of <i>Helicobacter pylori</i> (<i>H. pylori</i>) resistance to clarithromycin (CLR) has consistently posed challenges for clinical treatment. Hence, a rapid susceptibility testing (AST) method urgently needs to be developed. <b>Methods:</b> In the present study, 35 isolates of <i>H. pylori</i> were isolated from 203 gastritis patients of the Guangzhou cohort, and the antimicrobial resistance phenotypes were associated with their genomes to analyze the relevant mutations. Based on these mutations, a rapid detection system utilizing high-resolution melting (HRM) curve analysis was designed and verified by the Shenzhen cohort, which consisted of 38 <i>H. pylori</i> strains. <b>Results:</b> Genomic analysis identified the mutation of the 2143 allele from A to G (A2143G) of <i>23S rRNA</i> as the most relevant mutation with CLR resistance (<i>p</i> < 0.01). In the HRM system, the wild-type <i>H. pylori</i> showed a melting temperature (Tm) of 79.28 ± 0.01 °C, while the mutant type exhibited a Tm of 79.96 ± 0.01 °C. These differences enabled a rapid distinction between two types of <i>H. pylori</i> (<i>p</i> < 0.01). Verification examinations showed that this system could detect target DNA as low as 0.005 ng/μL in samples without being affected by other gastric microorganisms. The method also showed a good performance in the Shenzhen validation cohort, with 81.58% accuracy, and 100% specificity. <b>Conclusions:</b> We have developed an HRM system that can accurately and quickly detect CLR resistance in <i>H. pylori</i>. This method can be directly used for the detection of gastric microbiota samples and provides a new benchmark for the simple detection of <i>H. pylori</i> resistance.