Mutations in 23S rRNA gene associated with clarithromycin resistance in Helicobacter pylori clinical isolates from Saint-Petersburg

Objective.

To identify point mutations in 23S rRNA gene associated with phenotypic drug resistance to clarithromycin (CLR) in clinical isolates of H. pylori.

Materials and Methods.

A total of 50 H. pylori clinical isolates (2014–2022) from adult patients with chronic gastritis (n = 32), duodenal ulcer (n = 11) and gastric cancer (n = 1) were included in this study. Of 50 isolates, 30 were obtained from newly diagnosed patients, 20 – from previously treated patients after eradication failure. All isolates were cultured on supplemented Columbia agar and incubated under microaerophilic conditions (GasPak 100). Antibiotic susceptibility testing was performed by disc diffusion method. The PCR products (1402 bp) of the 23S rRNA gene were sequenced by Sanger approach. The DNA sequences were compared to the H. pylori 26695 reference using Unipro UGENE v.38.1.

Results.

A total of 30 H. pylori isolates were determined as CLR-resistant and 20 isolates as CLR-susceptible. The A2147G point mutation was detected in 17 (56,7%) CLR-resistant isolates and one (5.0%) CLR-sensitive isolate (p = 0,0002). The other point mutation A2146G was found exclusively in 13.3% (⁴⁄₃₀) of CLR-resistant isolates (p = 0,14). None of the CLR-resistant isolates carried two A2146G/A2147G mutations simultaneously, whereas 9 (30.0%) had neither of them. The agreement between genotypic and phenotypic susceptibility to CLR based on both A2146G + A2147G mutations was 70.0%. All clinical isolates obtained from previously treated patients (n = 20) were assigned to CLR-resistant group. Of these, 16 (80.0%) carried either A2146G or A2147G mutations, while among newly diagnosed patients (n = 30) only 5 isolates (16.7%) had one of two mutations (p = 0,003). The other point mutations out of 2146–2147 positions identified were G1567T, C1568A/T, A1825G, G1830A, T1834C, T2186C, but with no association with phenotypic drug resistance (p > 0.05).

Conclusions.

Our study showed that among all variants of nucleotide substitutions in 23S rRNA gene, the only one (A2147G) is significantly associated with phenotypic resistance of H. pylori to CLR. Despite the low frequency of A2146G point mutation, combination of A2146G/A2147G mutations can be used as a predictor of phenotypic resistance of Russian H. pylori clinical isolates.