Adaptation of an in‐house PCR for the detection of Helicobacter pylori and the mutations associated with macrolide resistance into ready‐to‐use PCR microwell strips

Helicobacter ◽  
2021 ◽  
Author(s):  
Lucie Bénéjat ◽  
Astrid Ducournau ◽  
Chloé Domingues‐Martins ◽  
Mélanie Lecoeur ◽  
Alice Blosse ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Macrolide Resistance

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 Macrolide resistance in Helicobacter pylori: mechanism and stability in strains from clarithromycin-treated patients.

1997 ◽  
Vol 41 (11) ◽  
pp. 2550-2553 ◽  
Author(s):  
K Hultén ◽  
A Gibreel ◽  
O Sköld ◽  
L Engstrand
Keyword(s):  
Helicobacter Pylori ◽  
Genetic Relatedness ◽  
Macrolide Resistance ◽  
Biological Assays ◽  
Clarithromycin Resistance ◽  
Stability Of Resistance ◽  
Development Mechanism

Helicobacter pylori strains from seven patients treated with clarithromycin were investigated for development, mechanism, and stability of resistance. Genetic relatedness between pre- and posttreatment isolates was shown by arbitrary primed PCR. Clarithromycin resistance was associated with A-to-G transitions at either position 2143 or 2144 or at both positions 2116 and 2142. In four cases, the mutations were homozygous. The Cla(r) phenotype was stable after 50 subcultivations in vitro. No erythromycin-modifying enzymes or rRNA methylases were found by biological assays, PCR and sequencing, or cloning methods.

Macrolide resistance in the normal microbiota after Helicobacter pylori treatment

2007 ◽  
Vol 39 (9) ◽  
pp. 757-763 ◽  
Author(s):  
Hedvig Jakobsson ◽  
Karin Wreiber ◽  
Katja Fall ◽  
Björn Fjelstad ◽  
Olof Nyrén ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Macrolide Resistance ◽  
Normal Microbiota ◽  
Helicobacter Pylori Treatment

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 Accurate Prediction of Macrolide Resistance in Helicobacter pylori by a PCR Line Probe Assay for Detection of Mutations in the 23S rRNA Gene: Multicenter Validation Study

2001 ◽  
Vol 45 (5) ◽  
pp. 1500-1504 ◽  
Author(s):  
Leen-Jan van Doorn ◽  
Youri Glupczynski ◽  
Johannes G. Kusters ◽  
Francis Mégraud ◽  
Peter Midolo ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Strong Association ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Line Probe Assay ◽  
Resistant Strains ◽  
Probe Assay ◽  
H Pylori ◽  
Detection Of Mutations

ABSTRACT Helicobacter pylori strains from 299 patients were tested in six laboratories in different countries. Macrolide susceptibility of the strains was determined by agar dilution (17.4%) or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA (rDNA) that are associated with macrolide resistance were analyzed by PCR and reverse hybridization (PCR-line probe assay [LiPA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and A2143C mutations in the 23S rDNA. vacA s-region (s1a, s1b, s1c, and s2) and m-region (m1, m2a, and m2b) genotypes andcagA status were also determined using another PCR-LiPA system. Of the 299 strains investigated by MIC testing, 130 (43.5%) were resistant and 169 (56.5%) were susceptible to clarithromycin. Of the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wild-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutations. Only five resistant strains contained the A2142C mutation (three of these in combination with the A2142G mutation), and the A2115G, G2141A, A2142T, and A2143C mutations were not found. MICs of clarithromycin for the A2142G mutant strains were significantly higher than MICs for the A2143G strains. Although there was no significant association between 23S rDNA mutations and the vacA andcagA status, clarithromycin-susceptible strains more often contained mixed vacA genotypes, indicating the presence of multiple H. pylori strains. In conclusion, our data confirmed the very strong association between 23S rDNA mutations and macrolide resistance and showed that the PCR-LiPA permits accurate and reliable diagnosis of macrolide resistance in H. pylori.

Combination Treatment with Ranitidine Is Highly Efficient Against Helicobacter pylori Despite Negative Impact of Macrolide Resistance

Helicobacter ◽  
1997 ◽  
Vol 2 (4) ◽  
pp. 188-193 ◽  
Author(s):  
Kristina Hultén ◽  
Bernhard Jaup ◽  
Bo Stenquist ◽  
Lars Engstrand
Keyword(s):  
Helicobacter Pylori ◽  
Combination Treatment ◽  
Negative Impact ◽  
Macrolide Resistance ◽  
Highly Efficient

Characteristics and trends in macrolide resistance among Helicobacter pylori strains isolated in Bulgaria over four years

1999 ◽  
Vol 34 (4) ◽  
pp. 309-313 ◽  
Author(s):  
Ludmila Boyanova ◽  
Zoya Spassova ◽  
Zacharii Krastev ◽  
Stephan Petrov ◽  
Irina Stancheva ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Macrolide Resistance

scholarly journals Rapid PCR Detection of Helicobacter pylori-Associated Virulence and Resistance Genes Directly from Gastric Biopsy Material

1998 ◽  
Vol 36 (12) ◽  
pp. 3689-3690 ◽  
Author(s):  
Britta Björkholm ◽  
Ragnar Befrits ◽  
Bernhard Jaup ◽  
Lars Engstrand
Keyword(s):  
Helicobacter Pylori ◽  
Resistance Genes ◽  
Virulence Gene ◽  
Gastric Biopsy ◽  
Macrolide Resistance ◽  
Biopsy Material ◽  
Rapid Pcr ◽  
Biopsy Specimens ◽  
Total Dna ◽  
Lengthy Process

We have developed a PCR-based method to detect macrolide resistance and the virulence gene cagA in Helicobacter pylori within 24 h, thereby improving the lengthy process of culture-based approaches. Total DNA was prepared directly from stomach biopsy specimens. The procedure proved to be rapid and reliable and could be utilized for diagnostic purposes.

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