Treatment effectiveness of Helicobacter pylori varies regionally and is decreasing worldwide, principally as a result of antibiotic resistant bacterium. Tetracycline is generally included in second line H. pylori eradication regimens. In Brazil, a high level of tetracycline resistance (TetR) is mainly associated with AGA926-928TTC 16S rDNA nucleotide substitutions. As H. pylori culture is fastidious, we investigated the primary occurrence of H. pylori 16S rDNA high level TetR genotype using a molecular approach directly on gastric biopsies of dyspeptic patients attending consecutively at Hospital das Clinicas of Marilia, São Paulo, Brazil.
Gastric biopsy specimens of 68 peptic ulcer disease (PUD) and 327 chronic gastritis (CG) patients with a positive histological diagnosis of H. pylori were investigated for TetR 16S rDNA genotype through a molecular assay based on amplification of a 16S rDNA 545bp fragment by polymerase chain reaction and HinfI restriction fragment length polymorphism (PCR/RFLP). Through this assay, AGA926-928TTC 16S rDNA TetR genotype resulted in a three DNA fragment restriction pattern (281, 227 and 37bp) and its absence originated two DNA fragments (264 and 281bp) due to a 16S rDNA conserved Hinf I restriction site.
The 545bp 16S rDNA PCR fragment was amplified from 90% of gastric biopsies from histological H. pylori positive patients. HinfI RFLP revealed absence of the AGA926–928TTC H. pylori genotype and PCR products of two patients showed absence of the conserved 16S rDNA HinfI restriction site. BLASTN sequence analysis of four amplicons (two conserved and two with an unpredicted HinfI restriction pattern) revealed a 99% homology to H. pylori 16S rDNA from African, North and South American bacterial isolates. A nucleotide substitution abolished the conserved HinfI restriction site in the two PCR fragments with unpredicted HinfI RFLP, resulting in an EcoRI restriction site.
H. pylori AGA926-928TTC 16S rDNA gene substitutions were not found in our population. More research is required to investigate if H. pylori TetR has a different genetic background in our region and if the nucleotide substitutions of the uncultured H. pylori 16S rRNA partial sequences have biological significance.
Keywords: Helicobacter pylori, Tetracycline resistance, Helicobacter pylori 16S rDNA, Nucleic acid based diagnosis, Helicobacter pylori 16S rDNA polymorphism