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Mechanism of metronidazole resistance in Helicobacter pylori: comparison of the rdxA gene sequences in 30 strains.

ABSTRACT: The rdxA gene of 30 independently isolated Helicobacter pylori strains was sequenced. A comparison of the rdxA sequences revealed a higher percentage of amino acid substitutions in the corresponding protein than in other housekeeping genes. Out of 122 point mutations, 41 were missense and 4 were nonsense. A resistant strain with a nucleotide insertion in the rdxA sequence was also found. With the exception of the point mutations and the insertion generating a stop signal, no particular nucleotide mutation or amino acid substitution could be associated to metronidazole resistance. Moreover, phylogenetic analysis of the 30 nucleotide sequences did not demonstrate specific clusters associated with the resistance phenotype.

SUBMITTER: Solca NM

PROVIDER: S-EPMC90043 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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Mechanism of metronidazole resistance in Helicobacter pylori: comparison of the rdxA gene sequences in 30 strains.

Solcà N M NM Bernasconi M V MV Piffaretti J C JC

Antimicrobial agents and chemotherapy 20000801 8

The rdxA gene of 30 independently isolated Helicobacter pylori strains was sequenced. A comparison of the rdxA sequences revealed a higher percentage of amino acid substitutions in the corresponding protein than in other housekeeping genes. Out of 122 point mutations, 41 were missense and 4 were nonsense. A resistant strain with a nucleotide insertion in the rdxA sequence was also found. With the exception of the point mutations and the insertion generating a stop signal, no particular nucleotid ...[more]

PMID: 10898705

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Project description:Background:The aim of this study was to evaluate the antimicrobial resistance and genetic basis for metronidazole (Mtz) and clarithromycin (Cla) resistance in strains of Helicobacter pylori, isolated from patients with gastroduodenal disorders. Patients and methods:A total of 157 H. pylori isolates (from 22 gastric cancer, 38 peptic ulcer disease, and 97 non-ulcer dyspepsia patients) were analyzed for drug susceptibility to Mtz and Cla, by gradient diffusion test (E-test, MAST). The PCR and sequence analysis of the rdxA and frxA for Mtz-resistant strains and the 23S rRNA for Cla-resistant strains were used to determine the genetic basis of drug resistance in H. pylori strains. Increased expression of TolC homologous genes (hefA) that upregulates efflux pump activity was determined in multidrug-resistant (MDR) strain of H. pylori by real-time PCR technique. Results:Among 157 H. pylori isolates, 32 (20.4%) strains were resistant to at least one of the antimicrobial agents. The highest resistance rate was attributed to Mtz (n=69, 43.94%). Among the resistant strains of H. pylori, 15 cases (9.55%) were detected as MDR. Mutations in the rdxA (85.5%) and A2143G point mutations (63.1%) in the 23S rRNA were the most common cause of resistance to Mtz and Cla in strains of H. pylori, respectively. In MDR strains, the rdxA mutation and A2143G-point mutation in the 23S rRNA were the most abundant mutations responsible for drug resistance. The relative expression of hefA in MDR strains (mean 3.706) was higher than the susceptible strains (mean 1.07). Conclusion:Mutational inactivation and efflux pump overexpression are two mechanisms that increase the resistance to H. pylori antimicrobial agents and the rate of MDR strains. In Iran, the mutations of rdxA and frxA in Mtz-resistant strains and A2143G and A2142G of the 23S rRNA in Cla-resistant strains were significant. The screening for these mutations could help to prevent antibiotic resistance, and to determine the most effective anti-H. pylori drugs.

Project description:Metronidazole (Mtz) is a critical ingredient of modern multidrug therapies for Helicobacter pylori infection. Mtz resistance reduces the effectiveness of these combinations. Although null mutations in a rdxA gene that encodes oxygen-insensitive NAD(P)H nitroreductase was reported in Mtz-resistant H. pylori, an intact rdxA gene has also been reported in Mtz-resistant H. pylori, suggesting that additional Mtz resistance mechanisms exist in H. pylori. We explored the nature of Mtz resistance among 544 clinical H. pylori isolates to clarify the role of rdxA inactivation in Mtz resistance and to identify another gene(s) responsible for Mtz resistance in H. pylori. Mtz resistance was present in 33% (181 of 544) of the clinical isolates. There was marked heterogeneity of resistance, with Mtz MICs ranging from 8 to >/=256 microg/ml. rdxA inactivation resulted in Mtz MICs of up to 32 microg/ml for 6 Mtz-sensitive H. pylori strains and 128 microg/ml for one Mtz-sensitive strain. Single or dual (with rdxA) inactivation of genes that encode ferredoxin-like protein (designated fdxB) and NAD(P)H flavin oxidoreductase (frxA) also increased the MICs of Mtz for sensitive and resistant strains with low to moderate levels of Mtz resistance. fdxB inactivation resulted in a lower level of resistance than that from rdxA inactivation, whereas frxA inactivation resulted in MICs similar to those seen with rdxA inactivation. Further evidence for involvement of the frxA gene in Mtz resistance included the finding of a naturally inactivated frxA but an intact rdxA in an Mtz-resistant strain, complementation of Mtz sensitivity from an Mtz-sensitive strain to an Mtz-resistant strain or vice versa by use of naturally inactivated or functional frxA genes, respectively, and transformation of an Mtz-resistant Escherichia coli strain to an Mtz sensitive strain by a naturally functional frxA gene but not an inactivated frxA gene. These results are consistent with the hypothesis that null mutations in fdxB, frxA, or rdxA may be involved in Mtz resistance.

Project description:Efficacy of Helicobacter pylori (H. pylori) eradication therapy has declined due to rapid rises in antibiotic resistance. We investigated how increased temperature affected H. pylori (NCTC 11637) growth and its sensitivity to metronidazole in vitro. We performed transcriptomic profiling using RNA-sequencing to identify differentially expressed genes (DEGs) associated with increased temperature. Transcriptional pathways involved in temperature-driven metronidazole resistance changes were analyzed through bioinformatic and literature curation approaches. We showed that H. pylori growth was inhibited at 41°C and inhibition was more apparent with prolonged incubation. Resistance to metronidazole was also reduced-minimum inhibitory concentration for metronidazole decreased from > 256 μg/ml at 37°C to 8 μg/ml at 41°C after culturing for 3 days. RNA-sequencing results, which were highly concordant within treatment conditions, revealed more than one third of genes (583/1,552) to be differentially expressed at increased temperatures with similar proportions up and down-regulated. Quantitative real-time PCR validation for 8 out of 10 DEGs tested gave consistent direction in gene expression changes. We found enrichment for redox and oxygen radical pathways, highlighting a mechanistic pathway driving temperature-related metronidazole resistance. Independent literature review of published genes associated with metronidazole resistance revealed 46 gene candidates, 21 of which showed differential expression and 7 out of 9 DEGs associated with "redox" resistance pathways. Sanger sequencing did not detect any changes in genetic sequences for known resistance genes rdxA, frxA nor fdxB. Our findings suggest that temperature increase can inhibit the growth and reduce H. pylori resistance to metronidazole. Redox pathways are possible potential drivers in metronidazole resistance change induced by temperature. Our study provides insight into potential novel approaches in treating antibiotic resistant H. pylori.

Dataset Information

Mechanism of metronidazole resistance in Helicobacter pylori: comparison of the rdxA gene sequences in 30 strains.

Publications

Mechanism of metronidazole resistance in Helicobacter pylori: comparison of the rdxA gene sequences in 30 strains.

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