Modified allele‐specific primer–polymerase chain reaction method for analysis of susceptibility of <i>Helicobacter pylori</i> strains to clarithromycin

Furuta, Takahisa; Soya, Yoshihiro; Sugimoto, Mitsushige; Shirai, Naohito; Nakamura, Akiko; Kodaira, Chise; Nishino, Masafumi; Okuda, Masumi; Okimoto, Tadayoshi; Murakami, Kazunari; Fujioka, Toshio; Hishida, Akira

doi:10.1111/j.1440-1746.2007.04919.x

Cited by 32 publications

(39 citation statements)

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“…AS-PCR utilizes allele-specific primers such that the second nucleotide from the 3′ end is designed to match the site of the point mutation [30]. This AS-PCR assay is well-established to definitely identify whether a H. pylori strain is sensitive or resistant to CLR [31,32]. Our analysis is a successful comparison between point mutations detected by both AS-PCR and short-read sequencing.…”

Section: Discussionmentioning

confidence: 99%

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Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes

et al. 2014

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BackgroundClarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. Using the MiSeq platform, whole-genome sequencing of the 19 clinical strains and the reference strain ATCC26695 was performed to identify single nucleotide variants (SNVs) of multi-drug resistant efflux pump genes in the CLR-resistant phenotype.ResultsBased on sequencing data of ATCC26695, over one million sequencing reads with over 50-fold coverage were sufficient to detect SNVs, but not indels in the bacterial genome. Sequencing reads of the clinical isolates ranged from 1.82 to 10.8 million, and average coverage ranged from 90.9- to 686.3-fold, which were acceptable criteria for detecting SNVs. Utilizing the conventional approach of allele-specific PCR, point mutations in the 23S rRNA gene were detected in 12 clinical resistant isolates, but not in 7 clinical susceptible isolates. All sequencing reads of CLR-resistant strains had a G mutation in an identical position of the 23S rRNA gene. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of TolC homologues, which have been implicated in multi-drug resistance, were examined. Specific SNVs were dominantly found in resistant strains.ConclusionsGene clusters of TolC homologues are involved in CLR susceptibility profiles in individual H. pylori strains. Whole-genome sequencing has yielded novel understanding of genotype-phenotype relationships.

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Section: Discussionmentioning

confidence: 99%

“…Analysis of alleles with point mutations in 23S rRNA (A2146G and A2147G) was performed according to previous reports [31,32]. The allele-specific primer sequences are listed in Additional file 4: Table S1.…”

Section: Methodsmentioning

confidence: 99%

Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes

et al. 2014

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“…Primer HPrpoB-IscrX, specific for the Rif resistance mediating point mutation in strain J99-R3, was designed according to the method described by Furuta et al . [35]. PCR positive clones were subsequently used for sequencing as described above.…”

Section: Methodsmentioning

confidence: 99%

Mosaic DNA Imports with Interspersions of Recipient Sequence after Natural Transformation of Helicobacter pylori

et al. 2008

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Helicobacter pylori colonizes the gastric mucosa of half of the human population, causing gastritis, ulcers, and cancer. H. pylori is naturally competent for transformation by exogenous DNA, and recombination during mixed infections of one stomach with multiple H. pylori strains generates extensive allelic diversity. We developed an in vitro transformation protocol to study genomic imports after natural transformation of H. pylori. The mean length of imported fragments was dependent on the combination of donor and recipient strain and varied between 1294 bp and 3853 bp. In about 10% of recombinant clones, the imported fragments of donor DNA were interrupted by short interspersed sequences of the recipient (ISR) with a mean length of 82 bp. 18 candidate genes were inactivated in order to identify genes involved in the control of import length and generation of ISR. Inactivation of the antimutator glycosylase MutY increased the length of imports, but did not have a significant effect on ISR frequency. Overexpression of mutY strongly increased the frequency of ISR, indicating that MutY, while not indispensable for ISR formation, is part of at least one ISR-generating pathway. The formation of ISR in H. pylori increases allelic diversity, and contributes to the uniquely low linkage disequilibrium characteristic of this pathogen.

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“…Susceptibility to clarithromycin was assessed by either or both culture and measurement of A to G or C mutations at positions 2142 and 2143 of the 23S rRNA gene of H. pylori (17). Strains were considered resistant when the mean inhibitory concentration (MIC) for clarithromycin was ?…”

Section: Eradication Regimens and Determination Of Success Or Failurementioning

confidence: 99%

Triple Therapy with Ecabet Sodium, Amoxicillin and Lansoprazole for 2 Weeks as the Rescue Regimen for H. pylori Infection

et al. 2011

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Background/Aim Ecabet sodium has an anti-H. pylori effect. We assessed the efficacy of ecabet sodium in the rescue therapy for the eradication of H. pylori. Methods A total of 74 patients with failed eradication of H. pylori after triple therapy with lansoprazole 30 mg bid, amoxicillin 750 mg bid and clarithromycin 200 mg bid were enrolled. They were randomly assigned to the three treatment groups as follows: LAC, lansoprazole 30 mg + amoxicillin 750 mg + clarithromycin 200 mg bid for 1 week; LAC2E, lansoprazole 30 mg bid + amoxicillin 750 mg bid + clarithromycin 200 mg bid + ecabet sodium 2 g bid for 1 week; and LA2E, lansoprazole 30 mg bid + amoxicillin 750 mg bid + ecabet sodium 2 g bid for 2 weeks. Eradication of H. pylori was assessed by the 13C-urea breath test after treatment. Results Eradication rates in intention-to-treat and per-protocol analyses were 20.0% (95% CI: 6.8-40.7) and 20.0% (6.8-40.7) with LAC, respectively, and 16.0% (4.5-36.1) and 17.4% (5.0-38.8) with LAC2E. In contrast, respective rates with LA2E were 75% (53.3-90.2) and 85.7% (63.7-97.0), which were significantly higher than those with LAC (p<0.001 for both ITT and PP) and LAC2E (p<0.001 for both ITT and PP). Conclusion Triple therapy with ecabet sodium, lansoprazole and amoxicillin for 2 weeks was effective as the rescue therapy after failure of the standard clarithromycin-based regimen.

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Modified allele‐specific primer–polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin

Abstract: Our new PCR-based assay for 23S rRNA mutations of H. pylori is a useful method for detecting clarithromycin-resistant strains of H. pylori easily.

Cited by 32 publications

References 35 publications

Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes

Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes

Mosaic DNA Imports with Interspersions of Recipient Sequence after Natural Transformation of Helicobacter pylori

Triple Therapy with Ecabet Sodium, Amoxicillin and Lansoprazole for 2 Weeks as the Rescue Regimen for H. pylori Infection

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