Analysis of gene mutations associated with antibiotic resistance in Helicobacter pylori isolated from Korean patients

Abstract

Helicobacter pylori is an etiologic agent of acute or chronic gastritis and can be a predisposing condition for peptic ulcer diseases, gastric cancer, and mucosa associated lymphoid tissue (MALT) lymphoma. Eradication of H. pylori is expected to cure gastritis and peptic ulcer disease and prevent atrophic gastritis and gastric cancer. To treat H. pylori infections, clarithromycin, amoxicillin, metronidazole, tetracycline, or fluoroquinolone are usually prescribed along with the proton pump inhibitor (PPI). Recently, however, the resistance to these antibiotic agents has been increased, which is regarded as the main cause of therapy failure. For this reason, it is necessary to choose proper antibiotics based on the antimicrobial susceptibility test results. But the antimicrobial susceptibility test is not widely used due to low H. pylori culture rate and lack of standard methods. In this study, minimum inhibitory concentration (MIC) on 5 antibiotics against 82 strains isolated at Yongin Severance Hospital in Korea was analyzed and then relationship between the resistance gene mutations and curative effects was investigated. The aims of this study are to identify the domestic pattern of resistance gene mutations and to ascertain whether the molecular methods are suitable ways to predict the antibiotics resistance or not. In the case of clarithromycin, resistant strain groups and susceptible strain groups were clearly manifested. In 23S rRNA gene in 5 strains resisting to clarithromycin, A2143G was observed to have a point mutation. Also, the bacteriostatic treatment in 2 patients ended in failure, which was resulted from clarithromycin resistance and amoxicillin sensitivity. In 4 strains (MIC 1-2 μg/mL) resisting to amoxicillin, S414 and N562 in penicillin-binding proteins (PBPs) gene was observed not to have mutations. However, a substitution occurred at N562Y in a

strain (MIC 2 μg/mL). Full-length nucleotide sequence of rdxA in 28 strains resisting to metronidazole was analyzed. Truncations was observed in 8 strains (28.5%), which was a result of the mutation caused by nucleotide deletion, insertion or stop codon. In MIC there was no strain resisting to tetracycline. 16S rRNA genes, including AGA926-928 of strains between 0.5 and 2 μg/mL of tetracyclin were analyzed, but no point mutations were observed. Eleven (57.8%) out of 19 strains resisting to levofloxacin showed amino acid substitutions in GyrA: N87K in 8 strains, N87I, A88V and D91N. In short, resistance gene mutation tests on 5 antibiotics were performed. Clarithromycin resistance gene observed at A2143G in 23S rRNA and levofloxacin resistance gene observed at N87, A88 and D91 have high correlation with MIC results. In the case of amoxicillin and tetracycline resistance, patterns of mutations were not diverse owing to a small number of resistance cases, but the mutation regions related to resistance could be detected. It is considered that multiple factors are leading to resistance to metronidazole. In conclusion, resistance mutation patterns related to antibiotics resistance in H. pylori strains isolated from Koreans are expected to be a basic data necessary in the development of the molecular-based method for detection of antibiotics resistance.