scholarly journals Rapid Detection, by PCR and Reverse Hybridization, of Mutations in the Helicobacter pylori 23S rRNA Gene, Associated with Macrolide Resistance

1999 ◽  
Vol 43 (7) ◽  
pp. 1779-1782 ◽  
Author(s):  
Leen-Jan van Doorn ◽  
Yvette J. Debets-Ossenkopp ◽  
Armelle Marais ◽  
Ricardo Sanna ◽  
Francis Mégraud ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Fragment Length Polymorphism ◽  
Simultaneous Detection ◽  
Point Mutations ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Reverse Hybridization ◽  
23S Rrna Gene ◽  
H Pylori

ABSTRACT A PCR-based reverse hybridization system (research prototype kit INNO-LiPA for H. pylori resistance) was developed and evaluated for simultaneous detection of 23S ribosomal DNA point mutations, associated with macrolide resistance in Helicobacter pylori. Fifty-seven H. pylori strains (51 natural, 6 laboratory-derived artificial, 52 resistant, and 5 susceptible strains) were tested by PCR-LiPA (detecting mutations A2115→G, G2141→A, A2142→G, A2142→C, A2143→G, A2143→C, and A2143→T), DNA sequencing, restriction fragment length polymorphism, and/or hybridization to oligonucleotide probes. Results were highly concordant, but PCR-LiPA appears to be more sensitive for the simultaneous detection of multiple mutants.

scholarly journals Molecular Assessment of Resistance to Clarithromycin in Helicobacter pylori Strains Isolated from Patients with Dyspepsia by Fluorescent In Situ Hybridization in the Center of Iran

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Jina Vazirzadeh ◽  
Jamal Falahi ◽  
Sharareh Moghim ◽  
Tahmineh Narimani ◽  
Rahmatollah Rafiei ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
In Situ Hybridization ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
Genotypic Analysis ◽  
23S Rrna Gene ◽  
H Pylori

Background and Aims. Helicobacter pylori is a common infectious bacterium mostly found in gastroduodenal diseases. The increased prevalence of clarithromycin-resistant H. pylori strains is a major challenge in the successful treatment of infections caused by this organism. The present study is aimed at detecting the clarithromycin resistance pattern of H. pylori strains isolated from gastric biopsies and evaluating point mutations of the 23S rRNA gene. Patients and methods. In the present descriptive cross-sectional study, 165 patients with gastrointestinal disorders, who were referred to the Endoscopy Center of Dr. Shariati Hospital of Isfahan, Iran, were enrolled from April to July 2018. H. pylori infection was diagnosed by culture, and susceptibility of the isolates to clarithromycin was assessed by the E-test. Minimum inhibitory concentration (MIC) values were obtained based on EUCAST recommendations. Also, fluorescence in situ hybridization (FISH) was used to determine point mutations associated with clarithromycin resistance. Results. By using culturing, H. pylori was isolated from 50.3% (83/165) gastric biopsy specimens. The overall frequency of resistance to clarithromycin was 25.3% (21/83) by the E-test. In the resistance genotypic analysis, 19 isolates had mutations. The prevalence of A2143G and A2144G mutations was 68.4% (13/19) and 31.5% (6/19), respectively. A2143C mutation was not tracked in any isolate. Two isolates with MIC>0.5 μg/mL had no mutations that could be related to other mechanisms of resistance. Conclusion. As presented in the study, the high prevalence of clarithromycin-resistant H. pylori due to point mutations of the 23S rRNA gene indicates the necessity of revising the standard treatment regimen based on antibiotic susceptibility pattern of each region.

scholarly journals PCR-RFLP detection of point mutations A2143G and A2142G in 23S rRNA gene conferring resistance to clarithromycin in Helicobacter pylori strains.

2014 ◽  
Vol 61 (2) ◽  
Author(s):  
Karolina Klesiewicz ◽  
Paweł Nowak ◽  
Elżbieta Karczewska ◽  
Iwona Skiba ◽  
Izabela Wojtas-Bonior ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Point Mutations ◽  
Rflp Analysis ◽  
23S Rrna ◽  
Rrna Gene ◽  
Efficient Management ◽  
Clarithromycin Resistance ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
H Pylori

The occurrence of clarithromycin resistance among Helicobacter pylori strains is a major cause of the treatment failure. Resistance to this drug is conferred by point mutations in 23S rRNA gene and the most prevalent mutations are A2143G and A2142G. The aim of the study was to evaluate the occurrence of A2143G and A2142G mutations in a group of H. pylori strains resistant to clarithromycin. The study included 21 clarithromycin-resistant H. pylori strains collected between 2006 and 2009 in southern Poland. Resistance to clarithromycin was quantitatively tested with the E-test to determine the minimal inhibitory concentration (MIC value). The point mutations of H. pylori isolates were detected by PCR followed by RFLP analysis. The MIC values for clarithromycin for the analyzed strains ranged from 1.5 mg/L to 64 mg/L. Nine H. pylori strains exhibited A2143G mutation and A2142G mutation was found in 9 isolates as well. The results of RFLP analysis of 3 clarithromycin-resistant strains were negative for both mutations. The average MIC values for A2143G and A2142G mutants were 6 and 30 mg/L, respectively. Frequencies of A2143G and A2142G mutations were the same in all isolates tested. Strains with A2143G mutation exhibited lower MIC values than A2142G mutants. Application of PCR-RFLP method for detection of clarithromycin resistance allows for better and more efficient management of H. pylori infections.

STUDY ON POINT MUTATIONS AT POSITIONS 2132 AND 2143 IN 23S RRNA GENE OF HELICOBACTER PYLORI AMONG PATIENTS WITH CHRONIC GASTRITIS

2016 ◽  
pp. 12-20
Author(s):  
Thi Minh Thi Ha ◽  
Van Huy Tran ◽  
Viet Nhan Nguyen ◽  
Thanh Hoa Nguyen ◽  
Phan Tuong Quynh Le
Keyword(s):  
Helicobacter Pylori ◽  
Chronic Gastritis ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
History Of ◽  
H Pylori

Background: Clarithromycin resistance in Helicobacter pylori has been found to be associated with point mutations at positions 2142 and 2143 in 23SrRNA gene. The aims of this study were: (1) to determine the rates of point mutations A2143G, A2142G and A2142C in 23SrRNA gene of H. pylori among patients with chronic gastritis by PCR-RFLP technique; and (2) to assessthe association between these mutations and some clinical, endoscopic and histopathological characteristics of chronic gastritis. Patients and methods: two hundreds and twenty six patients with H. pylori-positive chronic gastritis were determined A2143G, A2142G and A2142C mutations by PCR-RFLP technique with DNA extracted from endoscopic biopsy specimens of gastric mucosa. Results: The rate of point mutations at positions 2142 and 2143 in 23S rRNA gene of H. pylori was 35.4% in total, the A2143G and A2142G mutationsaccounted for 92.5% and 7.5% of all point mutations, respectively. No A2142C mutation was found. These mutations were not associated with age, gender,distribution of gastritis, and the presence of atrophic gastritis. The rate of A2143G mutation in groups with and without a history of clarithromycin treatment were 44.9% and 24.8%, respectively (p = 0,0065). The A2142G mutation was associated with intestinal metaplasia and/or dysplasia. Conclusion: The point mutations at positions 2142 and 2143 in 23S rRNA gene were found at a high rate in H. pylori strains amongpatients with chronic gastritis, with the absolute predominance of A2143G mutation. The A2143G mutation was associated with a history of clarithromycin treatment. Key words: 23S rRNA gene, Helicobacter pylori, A2143G, A2142G, A2142C mutation, clarithromycin resistance, chronic gastritis.

scholarly journals Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori

2009 ◽  
Vol 58 (11) ◽  
pp. 1443-1448 ◽  
Author(s):  
Shi-Hai Xuan ◽  
Yu-Gui Zhou ◽  
Bo Shao ◽  
Ya-Lin Cui ◽  
Jian Li ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Dna Sequencing ◽  
Point Mutations ◽  
Eradication Therapy ◽  
Dna Chip ◽  
23S Rrna ◽  
Rrna Gene ◽  
Direct Dna Sequencing ◽  
23S Rrna Gene ◽  
H Pylori

Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53×102 copies μl−1. Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

scholarly journals Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.

1997 ◽  
Vol 41 (12) ◽  
pp. 2724-2728 ◽  
Author(s):  
A Occhialini ◽  
M Urdaci ◽  
F Doucet-Populaire ◽  
C M Bébéar ◽  
H Lamouliatte ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Point Mutations ◽  
Restriction Enzymes ◽  
The United States ◽  
Drug Binding ◽  
Macrolide Resistance ◽  
Rrna Genes ◽  
23S Rrna ◽  
Dependent Manner ◽  
H Pylori

Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes.

scholarly journals New Site of Modification of 23S rRNA Associated with Clarithromycin Resistance of Helicobacter pylori Clinical Isolates

2002 ◽  
Vol 46 (12) ◽  
pp. 3765-3769 ◽  
Author(s):  
Carla Fontana ◽  
Marco Favaro ◽  
Silvia Minelli ◽  
Anna Angela Criscuolo ◽  
Antonio Pietroiusti ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
23S Rrna ◽  
Dilution Method ◽  
Rrna Gene ◽  
Functional Sites ◽  
Biopsy Specimens ◽  
Clarithromycin Resistance ◽  
Pcr Product ◽  
23S Rrna Gene ◽  
H Pylori

ABSTRACT Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 μg/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an HhaI restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates.

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