OBJECTIVETo demonstrate the correlation of rdxA gene mutation and metronidazole (MTZ) resistance of H.pylori isolates in the local area.

METHODSClinical strains of H.pylori were isolated from gastric biopsy of patients. Resistance to metronidazole of the isolates was determined by using diffusion test and two fold dilution test. Genome DNAs of the isolates were prepared for PCR to detect rdxA gene. The target amplification products were sequenced after T-A cloning. The sequences were compared with the reported sequences from Hp26695 and 134 other strains of H.pylori.

RESULTSMTZ resistance rate was 76.1% in 21 clinical isolates. The target fragment 886 bp in length containing rdxA gene could be successfully amplified. In comparison with the reported corresponding sequence of H.pylori stain 26695, homologies of the nucleotide sequences from the amplification products were 90.1% approximate, equals 95.1%. Mutations caused by base insertion/deletion and substitution in the MTZ resistance isolates were found. Among these mutations, two types of insertion mutations have not been reported in literatures. No same mutations were present in the MTZ sensitive isolates.

CONCLUSIONThe rdxA gene mutation may play an important role in MTZ resistance of H.pylori.

Amino Acid Sequence ; Drug Resistance, Bacterial ; Helicobacter pylori ; drug effects ; genetics ; Metronidazole ; pharmacology ; Molecular Sequence Data ; Mutation ; Nitroreductases ; chemistry ; genetics

OBJECTIVETo investigate the relationship between the helicobacter pylori (HP) infection and the genetic instability of mitochondrial DNA (mtDNA) in human gastric adenocarcinoma epithelial cells (AGS).

METHODSAfter treated with extracts of HP11638 (CagA+, VacA+) or Hp11638 mutant strain (CagA+, VacA-), AGS cells were collected, and mitochondrial DNA was extracted and Cox-I, Cox-II, Cox-III, ATPase6, ATPase8 and Cytb genes and the D-Loop region were amplified by PCR and then sequenced.

RESULTSThe mutation rates of the mtDNA in AGS cells were correlated with the extracts of the two HP strains in a concentration- and time-dependent manner. But the mtDNA mutation rate in AGS cells treated with the HP11638 extract was higher than that treated with the Hp11638 mutant extract. Total of 616 mutations in D-Loop region were detected, including 489 point mutations, 81 insertions and 46 deletions. Among them, 70.9% (437/616) belonged to GC to AT and AT to GC transition. Seventeen out of 20 (85%) AGS cells treated with extract of HP had mutations in 303PolyC, 16184PolyC and 514CA regions of mtDNA D-Loop. No mutation was detected in Cox-I, Cox-II, Cox-III, ATPase6 and ATPase8 genes, three point mutations were found in the Cytb gene.

CONCLUSIONHP can cause the accumulation of mutations in mtDNA, in particular, in the D-Loop region, and the VacA participated in the process.

Antigens, Bacterial ; pharmacology ; Base Sequence ; DNA, Mitochondrial ; drug effects ; genetics ; Endothelial Cells ; drug effects ; pathology ; Helicobacter Infections ; complications ; Helicobacter pylori ; chemistry ; Humans ; Mutation ; Stomach ; pathology

OBJECTIVETo screen metronidazole (MTZ) -resistance associated gene fragments of Helicobacter pylori (H.pylori) by suppression subtractive hybridization(SSH).

METHODSThe suppression subtractive hybridization (SSH) was used to screen the different DNA fragments between MTZ-resistant and -susceptible clinical strains of H.pylori. The resistant strains specific gene fragments were obtained by SSH and identified by dot blotting.

RESULTAmong the 120 subtractive colonies which were randomly chosen, 37 DNA fragments were different (>or=2 times) in DNA-copy numbers between resistant and susceptible strains and 17 of them were significantly different (>or=3 times). These 17 DNA fragments were sequenced subsequently. Ten of them were new sequences and the other 7 were duplicated sequences. These sequences represented respectively: depeptide ABC transporter periplasmic dipeptide-binding protein (dppA), permease protein (dppB), et al.

CONCLUSIONGene fragments specific to MTZ-resistant H. pylori strains were obtained by SSH and these genes may associated with MTZ-resistance of H.pylori.

Anti-Infective Agents ; pharmacology ; Cloning, Molecular ; DNA, Bacterial ; chemistry ; genetics ; Drug Resistance, Bacterial ; genetics ; Helicobacter pylori ; drug effects ; genetics ; Humans ; Metronidazole ; pharmacology ; Nucleic Acid Hybridization ; methods ; Sequence Analysis, DNA

BACKGROUND/AIMS: A worldwide increase in amoxicillin resistance in Helicobacter pylori is having an adverse effect on eradication therapy. In this study, we investigated the mechanism of the amoxicillin resistance of H. pylori in terms of amino acid substitutions in penicillin-binding protein 1 (PBP1). METHODS: In total, 150 H. pylori strains were isolated from 144 patients with chronic gastritis, peptic ulcers, or stomach cancer. The minimum inhibitory concentrations (MICs) of the strains were determined with a serial 2-fold agar dilution method. The resistance breakpoint for amoxicillin was defined as >0.5 microg/mL. RESULTS: Nine of 150 H. pylori strains showed amoxicillin resistance (6%). The MIC values of the resistant strains ranged from 1 to 4 microg/mL. A PBP1 sequence analysis of the resistant strains revealed multiple amino acid substitutions: Val16-->Ile, Val45-->Ile, Ser414-->Arg, Asn562-->Tyr, Thr593-->Ala, Gly595-->Ser, and Ala599-->Thr. The natural transformation of these mutated genes into amoxicillin-sensitive strains was performed in two separate pbp1 gene segments. A moderate increase in the amoxicillin MIC was observed in the segment that contained the penicillin-binding motif of the C-terminal portion, the transpeptidase domain. CONCLUSIONS: pbp1 mutation affects the amoxicillin resistance of H. pylori through the transfer of the penicillin-binding motif.
Adult ; Amino Acid Sequence ; *Amino Acid Substitution ; Amoxicillin/*pharmacology ; Anti-Bacterial Agents/*pharmacology ; Female ; Helicobacter Infections/drug therapy ; Helicobacter pylori/*chemistry/*drug effects/genetics ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; *Penicillin Resistance/genetics ; Penicillin-Binding Proteins/*chemistry/genetics ; Republic of Korea ; Sequence Analysis, Protein ; Transformation, Genetic