rdxA Gene Research Articles

Aspirin increases susceptibility of Helicobacter pylori to metronidazole by augmenting endocellular concentrations of antimicrobials

To investigate the mechanisms of aspirin increasing the susceptibility of Helicobacter pylori (H pylori) to metronidazole. H pylori reference strain 26695 and two metronidazole-resistant isolates of H pylori were included in this study. Strains were incubated in Brucella broth with or without aspirin (1 mmol/L). The rdxA gene of H pylori was amplified by PCR and sequenced. The permeability of H pylori to antimicrobials was determined by analyzing the endocellular radioactivity of the cells after incubated with [7-(3)H]-tetracycline. The outer membrane proteins (OMPs) of H pylori 26695 were depurated and analyzed by SDS-PAGE. The expression of 5 porins (hopA, hopB, hopC, hopD and hopE) and the putative RND efflux system (hefABC) of H pylori were analyzed using real-time quantitative PCR. The mutations in rdxA gene did not change in metronidazole resistant isolates treated with aspirin. The radioactivity of H pylori increased when treated with aspirin, indicating that aspirin improved the permeability of the outer membrane of H pylori. However, the expression of two OMP bands between 55 kDa and 72 kDa altered in the presence of aspirin. The expression of the mRNA of hopA, hopB, hopC, hopD, hopE and hefA, hefB, hefC of H pylori did not change when treated with aspirin. Although aspirin increases the susceptibility of H pylori to metronidazole, it has no effect on the mutations of rdxA gene of H pylori. Aspirin increases endocellular concentrations of antimicrobials probably by altering the OMP expression.

헬리코박터 파일로리에서 fdxA 유전자에 의한 메트로니다졸 내성 조절 기전 연구

본 연구는 H. pylori에서 metronidazole내성에 관여하는 유전자를 발견하고 이들 유전자들의 상호 조절 기전을 밝힘으로서 위장질환의 원인균인 H. pylori를 퇴치하기 위한 기본바탕을 마련하고자 수행되었다. 우선적으로 metronidazole 내성을 조절하는 유전자인 fdxA(ferredoxin)에 의한 metronidazole 내성 조절 기전을 밝히기 위하여 다음의 연구를 수행하였다. Type I 균주인 26695균주의 fdxA 유전자에 chloramphenicol 내성 유전자를 삽입하여 결손돌연변이주를 구축하였다. fdxA의 비활성화에 의한 rdxA 및 frxA 유전자의 발현을 알아보기 위하여 2-D electrophoresis와 MALDI-TOP-MS을 이용하여 fdxA 유전자의 비활성화에 의해 over-expressed protein과 under-expressed protein을 검색하였다. 본 실험의 결과로 type I 균주인 26695에서 fdxA 유전자를 비활성화시킨 결과 frxA 유전자의 발현양이 증가함을 northern으로 확인하였으며, 또한 fdxA유전자의 downstream에 위치한 유전자들이 H. pylori의 생존에 중요한 역할은 한다는 것을 알 수 있었다. 또한 2-D electrophoresis와 MALDI-TOP-MS을 이용하여 fdxA 유전자의 inactivation에 의해 over-expressed protein으로 nifU-like protein(HP0221), frxA(HP0642), nonheme ferritin(HP0653)와 아직 기능이 밝혀지지 않은 hypothetical protein(HP0902) 등이 발견되었다. 그리고 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase(HP0089), (3R)-hydroxymyristoyl ACP dehydratase(HP1376)과 thioredoxin(HP1458)등이 under-expressed protein으로 발견되었다. Resistance to metronidazole in Helicobacter pylori results from inactivation of rdxA and frxA, the chromosomal genes for a nitroreductase that normally converts metronidazole from prodrug to bactericidal agent. Two types of metronidazole susceptible strains had been found distinguishable by their apparent levels of frxA expression. Most common in the populations we had studied were strains that required only rdxA inactivation to become resistant to moderate levels of metronidazole(type I strains). The second strain type required inactivation of both frxA and rdxA to become resistance to metronidazole(type II strains): this was linked to a relatively high level of frxA gene transcription in the type II strains. The fdxA gene regulated fdxA as well as rdxA gene. Thus, to study the function of fdxA as a regulatory gene we constructed a null mutant of fdxA in H. pylori genome and identified over-and under-expressed proteins by fdxA using two-dimensional(2-D) electrophoresis and MALDI-TOP-MS. There were four over-expressed proteins in fdxA mutant; nifU-like protein(HP0221), frxA(HP0642), nonheme ferritin(HP0653), and hypothetical protein(HP0902). Three under-expressed proteins were also identified in fdxA mutant, including 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (HP0089), (3R)-hydroxymyristoyl ACP dehydratase(HP1376), and thioredoxin(HP1458).

Could frameshift mutations in the frxA and rdxA genes of Helicobacter pylori be a marker for metronidazole resistance?

SummaryBackgroundAlthough resistance of Helicobacter pylori to metronidazole had been reported to be associated with mutations in the rdxA or the frxA gene, recent studies have indicated that they may contribute little to metronidazole resistance.AimTo clarify the roles of these genes in metronidazole resistance, we examined them in strains that were not eradicated by first‐line eradication therapy.Patients and methodsA total of 132 patients (92 males, 40 females, average age: 53.8 years old) who underwent upper gastrointestinal endoscopy after unsuccessful first‐line H. pylori eradication were enrolled. Antibiotic susceptibility to metronidazole was investigated using the agar dilution method. The H. pylorirdxA and frxA genes were then sequenced.ResultsIn the metronidazole‐resistant strains, the sensitivity of detection of frameshift mutations in the rdxA gene was 44%. A significant association between the presence of frameshift mutations in the rdxA gene and resistance to metronidazole was observed. No significant association between frameshift mutations in the frxA gene and resistance to metronidazole was observed.ConclusionMutation in the rdxA gene was sufficient, but not necessary to confer resistance to metronidazole in H. pylori. Mutation in the frxA gene was not necessary for resistance to metronidazole in H. pylori.

Alterations in rdxA and frxA genes and their upstream regions in metronidazole-resistant Helicobacter pylori isolates

Metronidazole resistance among Helicobacter pylori strains has been related to alterations in gene products having metronidazole nitroreductase activities. RdxA and FrxA proteins are the two major contributing factors. In this investigation, the rdxA and frxA genes and their upstream regions were analyzed in 19 H. pylori isolates, 8 of which were metronidazole-sensitive (MIC ≤ 8 μg/mL) and 11 of which were metronidazole-resistant (MIC ≥ 8 μg/mL), as determined by the E-test. Among the metronidazole-resistant isolates, three contained both RdxA and FrxA proteins with premature truncation caused by gene nonsense mutations or frameshift mutations, while three contained only stop mutations in FrxA and two only in RdxA. Substitutions of amino acids occurred in other RdxA (5/6) and FrxA (4/5) proteins from metronidazole resistant isolates as compared with those from metronidazole-sensitive ones. All metronidazole-resistant isolates had alterations in RdxA and/or FrxA proteins. Moreover, the upstream regions (−1 to −35) of rdxA and frxA genes in some metronidazole-resistant isolates varied by nucleotide insertion and/or deletion or substitution. The patterns of variation in both genes and their upstream regions were highly diversified. Alterations in rdxA and frxA genes and their upstream regions may be involved in the development of metronidazole resistance in H. pylori.

High prevalence and level of resistance to metronidazole, but lack of resistance to other antimicrobials in Helicobacter pylori, isolated from a multiracial population in Kuwait

The primary treatment regimen for Helicobacter pylori infection for Kuwaitis does not contain metronidazole, but that for expatriates does. There is also increasing failure of antimicrobial therapy. To determine the susceptibility of H. pylori from upper gastrointestinal biopsies of Kuwaitis and non-Kuwaitis to find out if differences existed in the susceptibilities of the isolates from the two different populations. The susceptibilities of 96 H. pylori isolates were tested against metronidazole, amoxicillin, clarithromycin and tetracycline by the E test. The rdxA gene was analysed from selected metronidazole-susceptible and metronidazole-resistant strains to find out polymorphism and the basis of metronidazole resistance. Approximately, 70% of isolates from both populations were metronidazole resistant with 65% isolates showing high minimum inhibitory concentration values of >256 mug/mL. No resistance to the other three antimicrobials was found. There were novel nonsense and missense mutations with no deletion in the rdxA gene by insertion of mini-IS605. The prevalence and level of metronidazole resistance in H. pylori in the two populations was high with no difference, in spite of different treatment regimens. Metronidazole resistance in this transitional country appeared to be independent of prior metronidazole use for treatment of H. pylori infection.

Could frameshift mutations in the frxA and rdxA genes of Helicobacter pylori be a marker for metronidazole resistance?

SummaryBackgroundAlthough resistance of Helicobacter pylori to metronidazole had been reported to be associated with mutations in the rdxA or the frxA gene, recent studies have indicated that they may contribute little to metronidazole resistance.AimTo clarify the roles of these genes in metronidazole resistance, we examined them in strains that were not eradicated by first‐line eradication therapy.Patients and methodsA total of 132 patients (92 males, 40 females, average age: 53.8 years old) who underwent upper gastrointestinal endoscopy after unsuccessful first‐line H. pylori eradication were enrolled. Antibiotic susceptibility to metronidazole was investigated using the agar dilution method. The H. pylorirdxA and frxA genes were then sequenced.ResultsIn the metronidazole‐resistant strains, the sensitivity of detection of frameshift mutations in the rdxA gene was 44%. A significant association between the presence of frameshift mutations in the rdxA gene and resistance to metronidazole was observed. No significant association between frameshift mutations in the frxA gene and resistance to metronidazole was observed.ConclusionMutation in the rdxA gene was sufficient, but not necessary to confer resistance to metronidazole in H. pylori. Mutation in the frxA gene was not necessary for resistance to metronidazole in H. pylori.

DNA sequence analysis of rdxA and frxA from paired metronidazole-sensitive and -resistant Helicobacter pylori isolates obtained from patients with heteroresistance

Genomic variations of rdxA and frxA genes in eight pairs of metronidazole (MTZ)-sensitive and -resistant isolates of Helicobacter pylori were investigated. The paired strains from each single biopsy had identical lspA- glmM restriction fragment length polymorphism profiles, and the differences in MTZ susceptibility were mostly accounted for by mutations in the rdxA gene. Truncation of RdxA is associated with a minimum inhibitory concentration of 64–128 mg/L. Early truncation of FrxA was observed both in susceptible and resistant isolates of seven paired strains. Inactivation of frxA might play a significant role only in one low-level MTZ-resistant isolate where a missense mutation was found in the rdxA gene.

Isogenic Variation of Helicobacter pylori Strain Resulting in Heteroresistant Antibacterial Phenotypes in a Single Host In Vivo

Antibiotic-susceptible and -resistant Helicobacter pylori can be present simultaneously in the same host. The aim of this study was to evaluate the genomic diversity of H. pylori strains resulting in heteroresistant antibacterial phenotypes. Twenty-one pairs of H. pylori strains isolated from the antrum and body displaying heteroresistant antibacterial phenotypes were included. We compared the genotypes of paired-isolates by random arbitrarily primed polymerase chain reaction (PCR), flagella gene PCR-based restriction fragment length polymorphism, and flaA gene sequencing. In metronidazole-heteroresistant isolates, the sequence variation of rdxA and frxA genes was analyzed using phylogenetic analysis. The DNA fingerprinting patterns of the paired isolates revealed that 12 pairs (57.1%) were identical, whereas one pair (3.8%) was different. The remaining eight pairs (38.1%) of isolates showed minor heterogenecity in fingerprinting patterns. In flaA gene sequencing, these identical and similar isolates showed close sequence similarity between the antrum and body, whereas different isolate showed 31 points of different nucleotide sequences. Phylogenetic analysis of the metronidazole-heteroresistant pairs showed consistent genetic relatedness of each paired isolates despite the sequence variation of the rdxA or frxA genes in five pairs (71.4%). These results suggest that continuing genomic diversities in the same strain may play an important role in modulating the antibiotic-heteroresistant H. pylori in vivo.

In vitro induction of resistance to metronidazole, and analysis of mutations in rdxA and frxA genes from Helicobacter pylori isolates

In clinical Helicobacter pylori isolates, metronidazole resistance has been associated with mutations in the rdxA and frxA genes. The aim of this study was to examine the role of the rdxA and frxA genes after the in vitro induction of metronidazole resistance. A total of five suscep-tible H. pylori isolates were initially exposed to different subinhibitory metronidazole concentrations to induce in vitro resistance to metronidazole. Susceptible and resistant strains after the in vitro induction of resistance were examined to evaluate mutations of the rdxA and frxA genes by sequence analysis. After the in vitro induction of resistance, analysis revealed that two and four susceptible strains developed resistance when cultured with 0.3 microg/ml and 0.6 microg/ml of metronidazole, respectively. Before and after the induction of resistance, none of the susceptible strains that developed low and moderate levels of resistance presented any mutation in either of the evaluated genes, whereas strains with high-level metronidazole resistance contained a simple mutation of the frxA gene, but no specific changes in the rdxA gene. Strains with moderate-level resistance contained both single and multiple mutations of rdxA and frxA, respectively, and the low-level-metronidazole-resistant strain contained a single mutation in the frxA gene, without any significant change in the rdxA gene. In this study, the strains that developed resistance were mainly associated with mutations of the frxA gene, suggesting the possibility that inactivation of this gene could originate metronidazole resistance. The results after the in vitro induction of resistance to metronidazole suggested the presence of additional metronidazole resistance mechanisms, other than mutations of the rdxA and/or frxA genes.

Helicobacter pyloriantibiotic resistance in Iran

To examine the frequency of antibiotic resistance in Iranian Helicobacter pylori (H pylori) strains isolated from two major hospitals in Tehran. Examination of antibiotic resistance was performed on 120 strains by modified disc diffusion test and PCR-RFLP methods. In addition, in order to identify the possible causes of the therapeutic failure in Iran, we also determined the resistance of these strains to the most commonly used antibiotics (metronidazole, amoxicillin, and tetracycline) by modified disc diffusion test. According to modified disc diffusion test, 1.6% of the studied strains were resistant to amoxicillin, 16.7% to clarithromycin, 57.5% to metronidazole, and there was no resistance to tetracycline. Of the clarithromycin resistant strains, 73.68% had the A2143G mutation in the 23S rRNA gene, 21.05% A2142C, and 5.26% A2142G. None of the sensitive strains were positive for any of the three point mutations. Of the metronidazole resistant strains, deletion in rdxA gene was studied and detected in only 6 (5%) of the antibiogram-based resistant strains. None of the metronidazole sensitive strains possessed rdxA gene deletion. These data show that despite the fact that clarithromycin has not yet been introduced to the Iranian drug market as a generic drug, nearly 20% rate of resistance alerts toward the frequency of macrolide resistance strains, which may be due to the widespread prescription of erythromycin in Iran. rdxA gene inactivation, if present in Iranian H pylori strains, may be due to other genetic defects rather than gene deletion.

Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori

Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.

Prevalence of Helicobacter pylori resistance to clarithromycin and metronidazole determined by 23S ribosomal RNA and rdxA gene analyses in Hiroshima, Japan.

Resistance to antibiotics in Helicobacter pylori is increasing and becoming a serious problem in eradication treatment of H. pylori. The prevalence of H. pylori infections that are resistant to clarithromycin, metronidazole, or both were determined in H. pylori isolates in Hiroshima, Japan. Sixty Japanese patients with H. pylori infection were collected between 1999 and 2000. To detect the resistance to clarithromycin and metronidazole, mutations of the 23S ribosomal RNA (rRNA) and rdxA genes that are responsible for resistance in H. pylori, were examined by direct sequencing analysis. Resistance to clarithromycin and metronidazole was detected in 12 (20.0%) and nine (15.0%) of the patients, respectively. Dual resistance to clarithromycin and metronidazole was detected in five (8.3%) patients. These results indicate that the relatively high prevalence of the dual resistance in H. pylori isolates may need special attention and new therapeutic approaches in Japan.

Molecular patchwork: Chromosomal recombination between two Helicobacter pylori strains during natural colonization.

Genetic analysis of two Helicobacter pylori strains isolated from a single gastric biopsy showed evidence of extensive horizontal gene transfer. Several large recombinations were identified in the rdxA gene, which is involved in metronidazole resistance.

Mutations in Helicobacter pylori rdxA gene sequences may not contribute to metronidazole resistance.

Metronidazole resistance in Helicobacter pylori reportedly occurs by mutational inactivation of the oxygen-insensitive nitroreductase gene rdxA. Nucleotide sequences of rdxA were determined in a set of 46 isolates from 19 dyspeptic patients from the UK. The study set comprised matched isolates that were either metronidazole susceptible (four) or mixed metronidazole susceptible and metronidazole resistant (15) before therapy and metronidazole resistant post-therapy (10) in the 11 patients that were followed up. Various mutation types were identified in rdxA of metronidazole-resistant strains (post-treatment) that were absent in matched metronidazole-susceptible strains (pre-treatment). However, rdxA sequences from pre-treatment metronidazole-resistant and metronidazole-susceptible subpopulations were identical in 11 of 15 patients. Thus, mutations in rdxA may not always be essential for metronidazole resistance. Future examination of rdxA expression at the transcription and translational level may provide further insight into the role of this locus in metronidazole action and resistance in H. pylori.

Analysis of the rdxA gene in high-level metronidazole-resistant clinical isolates confirms a limited use of rdxA mutations as a marker for prediction of metronidazole resistance in Helicobacter pylori

Metronidazole (Mtz) resistance in the gastric pathogen Helicobacter pylori is closely associated with inactivation of the nitroreductase gene rdxA. In order to identify respective mutations for diagnostic purposes we analyzed the rdxA gene in a collection of high-level Mtz-resistant clinical H. pylori isolates. Size alterations in the rdxA gene region were found in only two out of 45 and one out of 40 isolates showing lower-level (minimal inhibitory concentrations (MICs) 32–192 μg ml −1) and high-level (MIC≥256 μg ml −1) Mtz resistance, respectively. Point mutations that interrupt the rdxA reading frame were detected in two out of eight high-level resistant isolates (MICs≥256 μg ml −1). Most remarkably, the rdxA gene sequence was found to be identical in four out of five high-level Mtz-resistant and -susceptible paired H. pylori isolates from the same patients each. Taken together, these results demonstrate that although some isolates carry classical resistance-associated rdxA mutations, as described earlier, the use of rdxA mutations as a marker for prediction of Mtz resistance is limited.

Characterization of the genes rdxA and frxA involved in metronidazole resistance in Helicobacter pylori

Metronidazole (Mtz) resistance in Helicobacter pylori has been found to be associated with mutations in rdxA, a gene encoding an oxygen-insensitive NADPH nitroreductase, and enhanced by mutations in frxA, a gene encoding a NAD(P)H-flavin oxidoreductase. The roles of these two genes in Mtz resistance in H. pylori were examined in this study. The rdxA and frxA genes were sequenced in nine pairs of strains isolated from biopsies obtained from patients before and after failed eradication treatments which included Mtz and resulted in the appearance of resistant strains. Metronidazole resistance could be explained in seven of these pairs of strains by mutations in rdxA and frxA. However, in one pair of strains, rdxA was identical in the susceptible and resistant strains, and only changes in frxA were observed; and in another pair, neither rdxA nor frxA were different in the susceptible and resistant strains. Sequencing of the upstream region of frxA and of the recA gene in the latter pair of strains did not reveal any mutations. To establish whether mutations in frxA alone could be involved in Mtz resistance, a resistant Escherichia coli strain transformed with the frxA of a Mtz susceptible H. pylori strain was rendered susceptible, and transformation with a mutated H. pylori frxA gene under the same conditions did not change the resistant E. coli phenotype. The results suggested that a Mtz resistance phenotype may arise in H. pylori without mutations in rdxA or frxA, or with mutations only in frxA.

Correlation of rdxA gene mutation and metronidazole resistance of Helicobacter pylori

To demonstrate the correlation of rdxA gene mutation and metronidazole (MTZ) resistance of H.pylori isolates in the local area. Clinical 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. MTZ 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. The rdxA gene mutation may play an important role in MTZ resistance of H.pylori.

Production of the RdxA protein in metronidazole-susceptible and -resistant isolates of Helicobacter pylori cultured from treated mice.

The objective of this study was to use immunoblotting with RdxA antisera to examine the production of the RdxA protein in mouse-derived metronidazole-susceptible and -resistant isolates of Helicobacter pylori. A 24 kDa immunoreactive band corresponding to RdxA was observed in all 15 metronidazole-susceptible and five of 50 metronidazole-resistant isolates. The rdxA gene of these five isolates contained missense mutations and transformation experiments confirmed that these mutations were associated with inactivation of the rdxA gene. No RdxA protein was produced in the other 45 metronidazole-resistant strains, including one in which the nucleotide sequence of the rdxA gene was unchanged. These results demonstrate a high correlation between production of the RdxA protein and susceptibility of H. pylori to metronidazole. Testing for the absence of the RdxA protein identifies the majority of strains that will respond poorly to metronidazole-containing eradication regimens.

Metronidazole resistance in Helicobacter pylori is caused by a single amino acid substitution in the rdxA gene.

Metronidazole resistance in Helicobacter pylori is caused by a single amino acid substitution in the rdxA gene.

Resistance of Helicobacter pylori to antibiotics and its impact on treatment options.

The treatment of Helicobacter pylori infection is jeopardized by resistance to the antibiotics used, which turns out to be the main risk factor for failure. Resistance is due to point mutations. For clarithromycin only two sites in the 23S rRNA sequence are concerned and can be easily detected by molecular methods, while for metronidazole several mutations on rdxA and other genes can be responsible and so do not allow such detection. The situation for the rare cases of amoxicillin resistance is not fully determined. The impact of resistance on the clinical outcome is dramatic for clarithromycin while it only decreases the success by 20% for metronidazole.