A Next-Generation Sequencing-Based Approach to Identify Genetic Determinants of Antibiotic Resistance in Cambodian Helicobacter pylori Clinical Isolates

Abstract

We evaluated the primary resistance of Helicobacter pylori (H. pylori) to routinely used antibiotics in Cambodia, an unexplored topic in the country, and assessed next-generation sequencing's (NGS) potential to discover genetic resistance determinants. Fifty-five H. pylori strains were successfully cultured and screened for antibiotic susceptibility using agar dilution. Genotypic analysis was performed using NGS data with a CLC genomic workbench. PlasmidSeeker was used to detect plasmids. The correlation between resistant genotypes and phenotypes was evaluated statistically. Resistances to metronidazole (MTZ), levofloxacin (LVX), clarithromycin (CLR), and amoxicillin (AMX) were 96.4%, 67.3%, 25.5%, and 9.1%, respectively. No resistance to tetracycline (TET) was observed. Multi-drug resistance affected 76.4% of strains. No plasmids were found, but genetic determinants of resistance to CLR, LVX, and AMX were 23S rRNA (A2146G and A2147G), GyrA (N87K and D91Y/N/G), and pbp1 (P473L), respectively. No determinants were genetically linked to MTZ or TET resistance. There was high concordance between resistant genotypes and phenotypes for AMX, LVX, and CLR. We observed high antibiotic resistance rates of CLR, MTZ, and LVX, emphasizing the need for periodic evaluation and alternative therapies in Cambodia. NGS showed high capability for detecting genetic resistance determinants and potential for implementation in local treatment policies.

Keywords: Helicobacter pylori; antibiotic resistance; genetic determinants; mutation; next-generation sequencing; whole genome sequence.

Figure 1

Antibiotic susceptibility testing based on agar dilution test from 55 isolates collected in Cho Ray Phnom Penh Hospital, Phnom Penh, Cambodia. Clinical breakpoints according to European Committee for Antimicrobial Susceptibility Testing (EUCAST) were drawn as black dashed line. R denotes resistance and S denotes susceptibility, respectively.

Figure 2

Genomic analysis of 53 H. pylori genomes sequenced in this study and the 26695 reference strain. Maximum likelihood phylogenetic tree based on whole genome sequences, the resistance patterns, and their corresponding genetic determinants for each antibiotic. The sensitive and resistant patterns are denoted by dark blue and red rectangles, respectively. The presence and absence of mutations are denoted by green and grey rectangles, respectively.

Figure 3

Distribution of minimum inhibitory concentrations (MICs) and corresponding genetic determinants of resistance. Dotted horizontal lines mark clinical breakpoints. (A) Distribution of MICs for clarithromycin (CLR) of all observed combinations of relevant antibiotic-resistant determinants. * indicates p < 0.05 compared to groups without mutation. (B) Distribution of MICs for levofloxacin (LVX)s for all observed combinations of relevant antibiotic-resistant determinants. ** indicates p < 0.05 compared to groups without mutation. *** indicates p < 0.05 compared to groups with double mutation at codons 87 and 91.