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Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase.

ABSTRACT: When the standard procedure for determining antibiotic susceptibility of bacteria is used, the results are delayed, especially for bacteria that grow slowly, such as Helicobacter pylori. Treatment for this bacterium may involve clarithromycin, a compound for which resistance has been associated with point mutations on the 23S rRNA gene. This resistance is currently found in organisms isolated from 0 to 15% of patients and jeopardizes the success of the treatment. We have designed a test involving amplification and colorimetric hybridization in the liquid phase to detect the mutation at the molecular level. First, four reference strains, including the wild type and three strains with the mutations A2143C, A2143G, and A2144G, were used to optimize the method. Amplification was carried out with primers previously published. The amplified products were added to probe-coated microtiter wells. A DNA enzyme immunoassay was used to detect the hybrids. The optimal conditions of the hybridization were defined for each probe. Nineteen H. pylori strains resistant to clarithromycin and 22 susceptible according to phenotypic data were submitted to restriction with BsaI and BbsI, and part of the 23S rRNA gene was sequenced in order to determine the mutation involved for the resistant strains. The new assay showed a complete correlation with the reference methods, except for one strain. Cross-hybridizations as well as application of the reaction to other bacteria did not lead to optical densities higher than the cutoff values chosen with the receiving operating characteristic curve. This method can be easily standardized and gives a result within a day. Its application directly to the biopsy specimens or infected gastric juice is planned in the future.

SUBMITTER: Pina M

PROVIDER: S-EPMC105316 | biostudies-literature | 1998 Nov

REPOSITORIES: biostudies-literature

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Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase.

Pina M M Occhialini A A Monteiro L L Doermann H P HP Mégraud F F

Journal of clinical microbiology 19981101 11

When the standard procedure for determining antibiotic susceptibility of bacteria is used, the results are delayed, especially for bacteria that grow slowly, such as Helicobacter pylori. Treatment for this bacterium may involve clarithromycin, a compound for which resistance has been associated with point mutations on the 23S rRNA gene. This resistance is currently found in organisms isolated from 0 to 15% of patients and jeopardizes the success of the treatment. We have designed a test involvin ...[more]

PMID: 9774580

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Project description:BackgroundHelicobacter pylori is curved Gram negative and microaerophilic bacilli that have infected half of the world's population. It is recognized as the causative agent of duodenal ulcer, gastritis peptic ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma and is associated with gastric adenocarcinoma. Resistance to clarithromycin is related to point mutations in 23SrRNA gene on nt 2143 and 2144, when A turns to G, and A2143G is the most important type. These mutations lead to reduced affinity of antibiotics to their ribosomal target and are considered as the main cause of treatment failure.ObjectivesThe aim of this study was to determine the frequency of A2143G point mutation in 23SrRNA of H. pylori strains isolated from gastric biopsies of patients in Rasht, north of Iran, by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).Patients and methodsA descriptive study was performed on 89 H. pylori strains, which were isolated from gastric biopsies of patients with gastric disorders such as gastritis, peptic ulcer, duodenal ulcer, non-ulcer dyspepsia and gastric adenocarcinoma. Isolated strains were tested for clarithromycin resistance using as breakpoint a minimum inhibitory concentration (MIC) of ≥ 1 mg/L by the E-test. The presence of H. pylori DNA was confirmed by amplifying the ureC (glmM) gene by PCR. Also, point mutation on 23SrRNA gene (A2142G and A2143G) was detected by PCR-RFLP using MboII and BsaI restriction endonucleases in all extracted DNA.ResultsOf the 89 H. pylori isolates, eighty-four were susceptible to clarithromycin, while five (5.6%) were resistant. All DNA samples of resistant strains, which were treated with BsaI had A2143G mutation. There was no point mutation in the sensitive strains of H. pylori. Also, we detected no mutation on nt A2142G of resistant strains.ConclusionsIn the present study, the frequency of clarithromycin resistance was lower than the other studies conducted in Iran. Resistance frequency in samples isolated from gastric ulcer was higher than other gastric disorders. Women and patients aged more than 60 years old showed the most resistance frequency in this study. All resistant strains had the A2143G genotype.

Dataset Information

Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase.

Publications

Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase.

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