Pyrosequencing analysis for rapid and accurate detection of clarithromycin resistance-associated mutations in Iranian Helicobacter pylori isolates

Abstract

Abstract Background Treatment of Helicobacter pylori (H. pylori) infection has become challenging following the development of primary antibiotic resistance. A primary therapeutic regimen for H. pylori eradication includes clarithromycin; however, the presence of point mutations within the 23S rRNA sequence of H. pylori contributes to clarithromycin resistance and eradication failure. Thus, we aimed to develop a rapid and precise method to determine clarithromycin resistance-related point mutations using the pyrosequencing method. Methods and results H. pylori was isolated from 82 gastric biopsy samples and minimal inhibitory concentration (MIC) was evaluated using the agar dilution method. Clarithromycin resistance-associated point mutations were detected by Sanger sequencing, from which 11 isolates were chosen for pyrosequencing. Our results demonstrated a 43.9% (36/82) prevalence in resistance to clarithromycin. The A2143G mutation was detected in 8.3% (4/48) of H. pylori isolates followed by A2142G (6.2%), C2195T (4.1%), T2182C (4.1%), and C2288T (2%). Although the C2195T mutation was only detected by Sanger sequencing, the overall results from pyrosequencing and Sanger sequencing platforms were comparable. Conclusions Pyrosequencing could be used as a rapid and practical platform in clinical laboratories to determine the susceptibility profile of H. pylori isolates. This might pave the way for efficient H. pylori eradication upon detection.

Keywords

• Helicobacter pylori
• Antibiotic resistance
• Clarithromycin
• Gene mutation
• Pyrosequencing
• Sanger sequencing