Mutations In frxA Research Articles

Investigation of Metronidazole Resistance-Associated Mutations and Virulence Genotypes of Helicobacter pylori Isolated from Egyptian Population: A Cross-Sectional Study

IntroductionThis cross-sectional study assesses the prevalence of metronidazole resistance-associated mutations and virulence genotypes in Helicobacter pylori (H. pylori) strains isolated from the Egyptian population. H. pylori infection is a significant public health concern, with antibiotic resistance challenging its eradication. MethodsGastric biopsy samples were collected from symptomatic patients referred for upper gastrointestinal endoscopy at selected healthcare facilities. The study included 250 participants with symptoms suggestive of H. pylori infection and aged 18 years or older. Biopsy samples were obtained using standard endoscopic techniques, and H. pylori strains were isolated and identified in the laboratory. Antimicrobial susceptibility testing was conducted using standard methods. Molecular analysis, including polymerase chain reaction (PCR) and sequencing, was performed to identify metronidazole resistance-associated mutations (rdxA and frxA) and virulence genotypes (cagA and vacA). ResultsAntimicrobial susceptibility testing revealed that 43.6% of the isolates were resistant to metronidazole, while 11.8%, 4.5%, and 55.4% were resistant to clarithromycin, amoxicillin, and levofloxacin. Molecular analysis identified rdxA and frxA mutations in 36.3% and 31.8% of the isolates, respectively, indicating metronidazole resistance-associated mutations. Additionally, 60.0% of the isolates were positive for the cagA gene, and 80.0% had the vacA s1 type, both associated with increased virulence. A significant association was found between metronidazole resistance and the presence of cagA gene, vacA s1 type, rdxA mutation, and frxA mutation. Statistical analysis revealed associations between specific mutations and virulence genotypes with respective odds ratios, indicating higher likelihoods of metronidazole resistance in isolates exhibiting these genetic characteristics. ConclusionsThis study highlights the prevalence of metronidazole resistance and the association between specific mutations and virulence genotypes in H. pylori strains isolated from the Egyptian population. The findings underscore the importance of monitoring antibiotic resistance patterns and understanding the genetic determinants of virulence in H. pylori for effective management and treatment strategies.

An Updated Systematic Review and Meta-Analysis on the Helicobacter pylori Antibiotic Resistance in Iran (2010-2020).

This updated systematic review and meta-analysis followed two objectives: (1) to determine Helicobacter pylori antibiotic resistance in Iran during 2010-2020 and (2) to assess the trend of resistance from 1997 to 2020. A systematic search in multiple databases, including ISI Web of Knowledge, PubMed, Scopus, Google Scholar, and the Scientific Information Database (SID), was performed using MeSH-extracted keywords. Meta-analysis was done on extracted data from a total of 27 included citations published between 2010 and January 20, 2020. The overall mean prevalence of H. pylori resistance was 64.9% for metronidazole, 25.3% for clarithromycin, 20.7% for amoxicillin, 16.1% for tetracycline, 21.9% for levofloxacin, 22.8% for rifampicin, 27.2% for furazolidone, 32.3% for ciprofloxacin, and 38.7% for erythromycin. In addition, the prevalence of multidrug-resistant strains of H. pylori was 26.5% in Iran. The pooled prevalence of point mutations A2143G, A2142G, and A2142C associated with clarithromycin resistance were 46.6%, 37.2%, and 5.5%, respectively; mutations in frxA and rdxA genes associated with metronidazole resistance were 46.4% and 19.7%, respectively; gyrA and gyrB genes mutations among fluoroquinolone-resistant strains were 55.3% and 48.2%, respectively; and resistance associated with integrons was 47%. According to the present findings, resistance of H. pylori to antibiotics used for eradication therapy has reached an alarming level in Iran. Furthermore, the trend of H. pylori resistance has increased between 1997 and 2020. Hence, continuous surveillance on resistance patterns, logical prescription and appropriate consumption of antibiotics, and selecting effective therapeutic regimens in accordance with local resistance patterns are required to prevent further spread of resistance and ensuing treatment failures.

Study of rdxA and frxA genes mutations in metronidazole-resistant and -susceptible Helicobacter pylori clinical isolates from the central region of Portugal.

Helicobacter pylori is one of the most prevalent global pathogens colonising an estimated 50% of the world's population. Although metronidazole (MTZ) is an important antibiotic playing a relevant role in various H. pylori eradication therapies, its frequent consumption results in an increased frequency of resistance with a consequent negative impact on treatment efficacy. Mutations on genes encoding NADPH nitroreductases, commonly known as rdxA gene (oxygen-insensitive) and frxA gene (flavinreductase) have been associated to H. pylori resistance to metrodinazole. The aim of this study was to evaluate the mutation profile of rdxA and frxA genes in a population of 38 H. pylori isolates with phenotypic patterns of susceptibility and resistance to this antibiotic. Touchdown PCR with the purpose of amplifying rdxA and frxA genes in one PCR was used. Sequence data were made by pair-wise sequence alignment and were examined in terms of codons, and comparison was achieved regarding amino acids. Although repeated mutations occurred in positions 118, 131, 172 and 183 of rdxA and in positions 72, 73, 110, 126 and 193 of frxA, it must be highlighted that the mutations are widespread along these two genes in this population. Furthermore, six MTZ-resistant isolates did not present any mutation in the frxA gene. This work appears to confirm that mutations in rdxA and frxA alone are unable to explain MTZ resistance in H. pylori isolates and therefore additional mechanisms may exist and should be investigated.

RdxA, frxA, and efflux pump in metronidazole-resistant Helicobacter pylori: Their relation to clinical outcomes.

rdxA and frxA mutations and enhancement of efflux pump have been suggested as the cause of metronidazole resistance in Helicobacter pylori. This study was performed to investigate the resistance mechanisms related to clinical eradication outcome, and to examine direct involvement of hefA in metronidazole-resistant isolates with intact rdxA and frxA. A total of 53 H.pylori-positive patients who were treated with metronidazole-containing sequential or quadruple therapy from 2011 to 2015 were enrolled. The metronidazole susceptibility of H.pylori isolates was examined by agar dilution test. Mutations in rdxA and frxA, were analyzed with DNA sequencing, and impact of hefA on metronidazole resistance was examined with quantitative real-time reverse transcription polymerase chain reaction, knockout and genetic complementation test for hefA. Seven mutation types of rdxA and/or frxA were found in H.pylori isolated from non-eradicated subjects. rdxA mutation was associated with eradication failure (P=0.002), and nonsense mutation in rdxA reduced eradication efficacy (P=0.009). hefA expression was significantly higher in resistant isolates (P<0.001), especially in rdxA(-)frxA(-) as compared to rdxA(+)frxA(+) (P=0.027). Resistant isolates with no mutation in rdxA and frxA became susceptible after hefA knockout. Genetic complementation for hefA recovered metronidazole resistance in all of three hefA knockout mutants. These results suggest that rdxA mutations play a critical role in metronidazole resistance as well as the outcomes of eradication therapy. In addition, hefA seems to be directly involved in metronidazole resistance, which explains the resistance in clinical isolates with intact rdxA and frxA.

Clarithromycin-Based Triple Therapy is Still Useful as an Initial Treatment for Helicobacter pylori Infection in the Dominican Republic.

AbstractHelicobacter pylori antibiotic susceptibility in the Dominican Republic has not been monitored. We assessed H. pylori antibiotic susceptibility in the Dominican Republic, and analyzed H. pylori mutations associated with antibiotic resistance. We recruited 158 dyspeptic patients in Santo Domingo and used agar dilution to test susceptibility to five antibiotics. Polymerase chain reaction-based sequencing was used to assess gyrA, gyrB, rdxA, frxA, and 23S rRNA mutations; next-generation sequencing was used to identify other metronidazole resistance-associated genes. Among 64 H. pylori strains isolated, we identified two (3.1%), one (1.6%), and no strains with clarithromycin, amoxicillin, and tetracycline resistance, respectively. Moreover, high frequency of metronidazole resistance (53/64, 82.8%) was observed, whereas levofloxacin resistance is emerging (23/64, 35.9%). We identified many rdxA and frxA mutations in metronidazole-resistant strains, but no synergistic effect was apparent. We revealed novel mutations in dppA, dppB, fdxA, and fdxB, irrespective of rdxA and frxA mutations. Novel mutations at Ser-14 of trx1 and Arg-221 of dapF were associated with different levels of metronidazole resistance. Most levofloxacin-resistant strains had a substitution at Asn-87 of gyrA, including the strain with the highest levofloxacin resistance, whereas only three substitutions were found at Ser-479 of gyrB with no synergistic effect. Besides the 23S rRNA A2142G mutation, we observed another mutation at T1958G in both clarithromycin-resistant strains. We confirmed high metronidazole and levofloxacin resistance associated with genetic mutations in the Dominican Republic. However, prevalence of clarithromycin resistance was low, suggesting that standard clarithromycin-based triple therapy remains useful as initial treatment of H. pylori infection.

Emerging Helicobacter pylori levofloxacin resistance and novel genetic mutation in Nepal.

BackgroundThe prevalence of Helicobacter pylori antibiotic susceptibility in the Nepalese strains is untracked. We determined the antibiotic susceptibility for H. pylori and analyzed the presence of genetic mutations associated with antibiotic resistance in Nepalese strains.ResultsThis study included 146 consecutive patients who underwent gastroduodenal endoscopy in Kathmandu, Nepal. Among 42 isolated H. pylori, there was no resistance to amoxicillin and tetracycline. In contrast, similar with typical South Asian patterns; metronidazole resistance rate in Nepalese strains were extremely high (88.1 %, 37/42). Clarithromycin resistance rate in Nepalese strains were modestly high (21.4 %, 9/42). Most of metronidazole resistant strains had highly distributed rdxA and frxA mutations, but were relative coincidence without a synergistic effect to increase the minimum inhibitory concentration (MIC). Among strains with the high MIC, 63.6 % (7/11) were associated with frameshift mutation at position 18 of frxA with or without rdxA involvement. However, based on next generation sequencing data we found that one strain with the highest MIC value had a novel mutation in the form of amino acid substituted at Ala-212, Gln-382, Ile-485 of dppA and Leu-145, Thr-168, Glu-117, Val-121, Arg-221 in dapF aside from missense mutations in full-length rdxA. Mutations at Asn-87 and/or Asp-91 of the gyrA were predominantly in levofloxacin-resistant strains. The gyrB mutation had steady relationship with the gyrA 87–91 mutations. Although three (44.4 %) and two (22.2 %) of clarithromycin resistant strains had point mutation on A2143G and A2146G, we confirmed the involvement of rpl22 and infB in high MIC strains without an 23SrRNA mutation.ConclusionsThe rates of resistance to clarithromycin, metronidazole and levofloxacin were high in Nepalese strains, indicating that these antibiotics-based triple therapies are not useful as first-line treatment in Nepal. Bismuth or non-bismuth-based quadruple regimens, furazolidone-based triple therapy or rifabutin-based triple therapy may become alternative strategy in Nepal.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0873-6) contains supplementary material, which is available to authorized users.

Analysis of allelic variants of rdxA associated with metronidazole resistance in Helicobacter pylori: detection of common genotypes in rdxA by multiplex allele-specific polymerase chain reaction

Resistance to metronidazole (Mtz) in Helicobacter pylori is a major problem worldwide, especially in developing countries. Alterations in Mtz nitroreductase enzymes, such as oxygen-insensitive NADPH nitroreductase (RdxA) and NADPH flavin oxidoreductase (FrxA), are the major contributing factors for this resistance. In this study, rdxA and frxA were amplified, sequenced, and analyzed in 34 Mtz-resistant H. pylori isolates (MIC ≥ 8 μg/mL) using multiple allele-specific polymerase chain reaction (MAS-PCR); this method was developed to target the most common genotypes of rdxA in H. pylori. In this study, the rdxA and frxA genes in Mtz-resistant H. pylori strains displayed a large number of point mutations. The rdxA and frxA genes of Mtz-resistant clinical isolates showed a higher percentage of missense mutations (97.1 and 78.6%, respectively) compared to 26695 reference strains; additionally, missense mutations were more common than frameshift (20.6 and 32.1%) and nonsense mutations (8.8 and 10.7%, respectively) in these genes. The most common missense mutations in rdxA were D 59 N (94.1%), T 31 E (88.2%), and R 131 K (85.3%). The most common missense mutations in frxA were F 72 S (57.1%), G 73 S (57.1%), and C 193 S (53.6%). The developed MAS primers, specific to position 175 and 392 of rdxA, successfully amplified the common alleles and distinguished the variants. MAS-PCR could be a useful tool for epidemiological studies of H. pylori, associated with Mtz resistance. rdxA variants must be screened in order to ensure the effectiveness of Mtz-based H. pylori therapies in developing countries.

Search for novel candidate mutations for metronidazole resistance in Helicobacter pylori using next-generation sequencing.

Metronidazole resistance is a key factor associated with Helicobacter pylori treatment failure. Although this resistance is mainly associated with mutations in the rdxA and frxA genes, the question of whether metronidazole resistance is caused by the inactivation of frxA alone is still debated. Furthermore, it is unclear whether there are other mutations involved in addition to the two genes that are associated with resistance. A metronidazole-resistant strain was cultured from the metronidazole-susceptible H. pylori strain 26695-1 by exposure to low concentrations of metronidazole. The genome sequences of both susceptible and resistant H. pylori strains were determined by Illumina next-generation sequencing, from which putative candidate resistance mutations were identified. Natural transformation was used to introduce PCR products containing candidate mutations into the susceptible parent strain 26695-1, and the metronidazole MIC was determined for each strain. Mutations in frxA (hp0642), rdxA (hp0954), and rpsU (hp0562) were confirmed by the Sanger method. The mutated sequence in rdxA was successfully transformed into strain 26695-1, and the transformants showed resistance to metronidazole. The transformants containing a single mutation in rdxA showed a low MIC (16 mg/liter), while those containing mutations in both rdxA and frxA showed a higher MIC (48 mg/liter). No transformants containing a single mutation in frxA or rpsU were obtained. Next-generation sequencing was used to identify mutations related to drug resistance. We confirmed that the mutations in rdxA are mainly associated with metronidazole resistance, and mutations in frxA are able to enhance H. pylori resistance only in the presence of rdxA mutations. Moreover, mutations in rpsU may play a role in metronidazole resistance.

Genetic analysis of Helicobacter pylori clinical isolates suggests resistance to metronidazole can occur without the loss of functional rdxA

Resistance to metronidazole (MTZ) in Helicobacter pylori is associated with mutations in rdxA, encoding an oxygen-insensitive NADPH nitroreductase, and mutations in frxA, encoding a NAD(P)H-flavin oxidoreductase. Despite this association, the strict correlation of MTZ resistance with mutations in rdxA or frxA is still controversial. In this study, rdxA allelic replacement was used to distinguish resistance-associated nucleotide mutations from the natural genetic diversity of H. pylori. Replacement with truncated rdxA resulted in MTZ resistance, whereas replacement with missense-mutated rdxA from resistant clinical isolates failed to yield MTZ resistance. Thus, although truncation of rdxA confers MTZ resistance in G27 H. pylori, MTZ resistance found in other clinical isolates is not due to the identified amino-acid substitutions. Three of our MTZ-resistant clinical isolates expressed functional rdxA and two of these also encoded full-length frxA. Therefore, MTZ resistance can arise in H. pylori possessing functional rdxA, suggesting that other factors are involved in MTZ resistance.

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.

Frameshift mutations in frxA occur frequently and do not provide a reliable marker for metronidazole resistance in UK isolates of Helicobacter pylori.

Mutations in the NAD(P)H flavin oxidoreductase gene (frxA) are thought to contribute to the development of metronidazole resistance in Helicobacter pylori. To test this further, 44 frxA sequences in 18 patient isolate sets of H. pylori were examined including a unique collection comprising separated Mtz-sensitive (MtzS) and Mtz-resistant (MtzR) subpopulations pre-treatment and matched MtzR strains post-treatment. Sequences of frxA contained frameshift mutations that led to premature protein truncation in at least one strain from most (17/18) patient sets. These mutations were present in all strains, irrespective of Mtz resistotype in 13/18 patients. Frameshift due to a single adenine deletion at nucleotide 53 was the most common mutation and was present in isolates from 11/18 patients. A novel real-time (LightCycler) PCR-based probe hybridization melting-point assay applied to a further 119 isolates confirmed that the frameshift-53 mutation occurred frequently, in 20% of isolates, and could be present in MtzS as well as MtzR strains (42% vs 58%). This study demonstrates that frameshift mutations occur in MtzS strains as well as in MtzR strains, and are thus unlikely to cause Mtz resistance.

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.

DNA sequence analysis of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant clinical Helicobacter pylori isolates.

We previously reported that inactivation of rdxA and/or frxA converted Helicobacter pylori from metronidazole sensitive to metronidazole resistant. To examine the individual roles of rdxA and frxA in the development of metronidazole resistance in H. pylori, we examined the status of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant H. pylori isolates obtained following unsuccessful therapy containing metronidazole. Arbitrary primed fingerprinting analyses revealed that the genotypes of 11 sensitive and resistant pairs of strains were essentially identical. Amino acid sequence identities of RdxA and FrxA from the 14 metronidazole-sensitive isolates ranged from 92 to 98% and 95 to 98%, respectively, compared to that of H. pylori J99 (MIC, 1 microg/ml). All strains with high-level metronidazole resistance (MICs, 128 microg/ml) contained premature truncation of both RdxA and FrxA caused by nonsense and/or frameshift mutations. Strains with intermediate resistance to metronidazole (MICs, 32 to 64 microg/ml) contained a single premature truncation and/or altered RdxA and FrxA caused by nonsense, frameshift, and unique missense mutations. The low-level metronidazole-resistant strains (MICs, 8 microg/ml) contained unique missense mutations in FrxA but no specific changes in RdxA. The results demonstrate that alterations in both the rdxA and frxA genes are required for moderate and high-level metronidazole resistance and that metronidazole resistance that develops during anti-H. pylori therapy containing metronidazole is most likely to involve a single sensitive strain infection rather than a coinfection with a metronidazole-resistant strain.

Sequential inactivation of rdxA (HP0954) and frxA (HP0642) nitroreductase genes causes moderate and high-level metronidazole resistance in Helicobacter pylori.

Helicobacter pylori is a human-pathogenic bacterial species that is subdivided geographically, with different genotypes predominating in different parts of the world. Here we test and extend an earlier conclusion that metronidazole (Mtz) resistance is due to mutation in rdxA (HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We found that (i) rdxA genes PCR amplified from 50 representative Mtz(r) strains from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtz(s) H. pylori to Mtz(r); (ii) Mtz(r) mutant derivatives of a cultured Mtz(s) strain resulted from mutation in rdxA; and (iii) transformation of Mtz(s) strains with rdxA-null alleles usually resulted in moderate level Mtz resistance (16 microg/ml). However, resistance to higher Mtz levels was common among clinical isolates, a result that implicates at least one additional gene. Expression in Escherichia coli of frxA (HP0642; flavin oxidoreductase), an rdxA paralog, made this normally resistant species Mtz(s), and frxA inactivation enhanced Mtz resistance in rdxA-deficient cells but had little effect on the Mtz susceptibility of rdxA(+) cells. Strains carrying frxA-null and rdxA-null alleles could mutate to even higher resistance, a result implicating one or more additional genes in residual Mtz susceptibility and hyperresistance. We conclude that most Mtz resistance in H. pylori depends on rdxA inactivation, that mutations in frxA can enhance resistance, and that genes that confer Mtz resistance without rdxA inactivation are rare or nonexistent in H. pylori populations.