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

Rodrigo Buzinaro Suzuki,Márcia Aparecida Sperança,Cristiane Maria Almeida

doi:10.1186/1471-230x-12-49

Rodrigo Buzinaro Suzuki, Márcia Aparecida Sperança + Show 1 more

Open Access

https://doi.org/10.1186/1471-230x-12-49

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Abstract

BackgroundTreatment 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 16 S rDNA nucleotide substitutions. 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 gastric biopsies of dyspeptic patients attending consecutively at Hospital das Clinicas of Marilia, São Paulo, 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 S rDNA genotype through a molecular assay based on amplification of a 16 S rDNA 545 bp fragment by polymerase chain reaction and HinfI restriction fragment length polymorphism (PCR/RFLP). Through this assay, AGA926-928TTC 16 S rDNA TetR genotype resulted in a three DNA fragment restriction pattern (281, 227 and 37 bp) and its absence originated two DNA 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 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 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 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.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 background in our region and if the nucleotide substitutions of the uncultured H. pylori 16 S rRNA partial sequences have biological significance.

Highlights

Treatment effectiveness of Helicobacter pylori varies regionally and is decreasing worldwide, principally as a result of antibiotic resistant bacterium
There is no standardized treatment regimen for H.pylori infection [2] and once the bacterium is detected in altered gastric mucosa, the indicated treatment consists of a triple antibiotic regimen including metronidazole, clarithromycin, amoxicillin, tinidazole, tetracycline and fluoroquinolones associated with a proton pump inhibitor such as omeprazole, lansoprazole or pantoprazole [3,4,5], according to antibiotic prescription policies for local medical care
Using a molecular approach based on a polymerase chain reaction associated with restriction fragment length polymorphism (PCR-Restriction fragment length polymorphism (RFLP)) assay, we investigated the primary incidence of H. pylori high level tetracycline resistance (TetR) directly in gastric biopsy specimens obtained from dyspeptic patients submitted to gastroscopy at Hospital das Clinicas of Marilia, São Paulo, Brazil, from January 2003 to July 2006

Summary

Introduction

Treatment effectiveness of Helicobacter pylori varies regionally and is decreasing worldwide, principally as a result of antibiotic resistant bacterium. There is no standardized treatment regimen for H.pylori infection [2] and once the bacterium is detected in altered gastric mucosa, the indicated treatment consists of a triple antibiotic regimen including metronidazole, clarithromycin, amoxicillin, tinidazole, tetracycline and fluoroquinolones associated with a proton pump inhibitor such as omeprazole, lansoprazole or pantoprazole [3,4,5], according to antibiotic prescription policies for local medical care. In Brazil, a country of continental dimensions, the majority of practicing clinicians include tetracycline in a second line treatment regimen after failure of the classical triple regimen composed of claritromycin, amoxicillin and a proton pump inhibitor for seven days to overcome H. pylori infection [12]

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