Genetic determinants of tetracycline resistance in Helicobacter pylori isolates

Abstract

Background: Helicobacter pylori is a gram-negative, spiral-shaped bacterium that colonizes the stomach mucosa and causes gastritis, peptic ulcers, gastric adenocarcinoma, and MALT lymphoma. Globally, over 50% of people are infected, with rates reaching 70.1% in Africa and over 80% in Nigeria. Treatment typically involves triple therapy using two antibiotics and a proton pump inhibitor or bismuth, often including clarithromycin, amoxicillin, or metronidazole. Tetracycline is added in quadruple therapy for resistant cases. Tetracycline, a broad-spectrum antibiotic that inhibits protein synthesis by binding to the 30S ribosomal subunit, faces rising resistance due to triple base pair mutations in the H. pylori 16SrRNA gene. Objectives of the study: In this study, we determined the frequency of mutations in the 16S rRNA genes of H. pylori isolates that are phenotypically resistant to tetracycline, and also determined the proportion of tetracycline susceptible H. pylori isolates that harbor mutations in the 16S rRNA genes. We then compared the frequency of the mutations to the pre-determined MIC values of the H. pylori isolates. Methods: A conventional PCR and Sanger sequencing were performed to find alterations in 16S rRNA genes of H. pylori isolates linked to resistance to tetracycline. Results: Sequence analysis of the tetracycline binding site on the 16S rRNA gene of H. pylori fragment revealed multiple SNPs, primarily transition substitutions A-G, C-T accounting for over 90% of all mutations. The most recurrent substitutions included A247G, C252T, C255T, G265T, and T278A, which appeared in more than 80% of the resistant isolates. Downstream mutations beyond position 265 bp were associated with markedly elevated MICs. Conclusion: This study demonstrates that tetracycline resistance in H. pylori arises from cumulative single-nucleotide polymorphisms within the 16S rRNA gene, in the tetracycline binding region A/C. Mutation frequency and positional clustering show a direct relationship with MIC, making genotypic variation to translate into phenotypic resistance. Key words: Phenotypic resistance, MICs, Genetic determinants, Tetracycline, H. pylori, Mutations, Single nucleotide polymorphisms.