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Absence of Helicobacter pylori high tetracycline resistant 16S rDNA AGA926-928TTC genotype in gastric biopsy specimens from dyspeptic patients of a city in the interior of São Paulo, Brazil

Abstract

BackgroundTreatment effectiveness of Helicobacter pylori varies regionally and is decreasingworldwide, principally as a result of antibiotic resistant bacterium. Tetracycline is generallyincluded in second line H. pylori eradication regimens. In Brazil, a high level of tetracyclineresistance (TetR) is mainly associated with AGA926-928TTC 16 S rDNA nucleotidesubstitutions. As H. pylori culture is fastidious, we investigated the primary occurrence of H.pylori 16 S rDNA high level TetR genotype using a molecular approach directly on gastricbiopsies of dyspeptic patients attending consecutively at Hospital das Clinicas of Marilia, SãoPaulo, Brazil.MethodsGastric 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 16 SrDNA genotype through a molecular assay based on amplification of a 16 S rDNA 545 bpfragment by polymerase chain reaction and HinfI restriction fragment length polymorphism(PCR/RFLP). Through this assay, AGA926-928TTC 16 S rDNA TetR genotype resulted in athree DNA fragment restriction pattern (281, 227 and 37 bp) and its absence originated twoDNA fragments (264 and 281 bp) due to a 16 S rDNA conserved Hinf I restriction site.ResultsThe 545 bp 16 S rDNA PCR fragment was amplified from 90% of gastric biopsies fromhistological H. pylori positive patients. HinfI RFLP revealed absence of the AGA926-928TTC H. pylori genotype and PCR products of two patients showed absence of theconserved 16 S 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 16 S rDNA from African, North and South American bacterialisolates. A nucleotide substitution abolished the conserved HinfI restriction site in the twoPCR fragments with unpredicted HinfI RFLP, resulting in an EcoRI restriction site.ConclusionsH. pylori AGA926-928TTC 16 S rDNA gene substitutions were not found in our population.More research is required to investigate if H. pylori TetR has a different genetic backgroundin our region and if the nucleotide substitutions of the uncultured H. pylori 16 S rRNA partialsequences have biological significance. (AU)

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VEICULO: TITULO (DATA)
VEICULO: TITULO (DATA)