PCR-RFLP detection of point mutations A2143G and A2142G in 23S rRNA gene conferring resistance to clarithromycin in Helicobacter pylori strains (original) (raw)
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Clarithromycin Resistance and 23S rRNA Mutations in Helicobacter pylori
Gastrointestinal Endoscopy, 2011
To determine the 23S rRNA point mutations in clarithromycin resistance of Helicobacter pylori strains isolated from southwest, Iran. This was a cross-sectional survey, which was done on 263 patients who referred to endoscopy department of Shehrekord university of medical sciences. According to gram stain, urease, catalase, oxidase and polymerase chain reaction (PCR) H. pylori identified. Standard National Committee for Clinical Laboratory Standard (NCCLS) method used for assessment of clarithromycin resistance. Specific primers and restriction enzymes BsaI and MboII by PCR-RFLP were used for analysis of A2143G and A2142G mutations. So for the detection of A2142C, specific primers and PCR method were used. 84 strains of H. pylori (31.94%) determined by PCR method. Of 19 (22.62%) clarithromycin resistant strains 13 (68.40%), 3 (15.78%), 2 (10.52%) had A2143G, A2142G, A2142C respectively and one unknown mutation in 23S rRNA gene. Because of considerable resistance to clarithromycin, direct diagnosis of this mutation by molecular approach in other parts of the country is necessary.
Russian Journal of Genetics, 2005
To detect point mutations A2115C, A2143G/C, and A2144G in the 23S rRNA gene of Helicobacter pylori associated with resistance of the microorganism to clarithromycin, a new powerful way of analysis was used. This method involved the reaction of minisequencing followed by MALDI-TOF mass spectrometry of reaction products. In ten analyzed clarithromycin-resistant clinical isolates of H. pylori obtained in Russia, the resistance was found to be mediated only by mutation A2144G in the 23S rRNA gene.
Characterization of clarithromycin resistance in isolates of Helicobacter pylori from the UAE
Indian Journal of Gastroenterology, 2010
Background Clarithromycin therapy is effective in eradicating Helicobacter pylori. However, the resistance of H. pylori to clarithromycin is increasingly reported. The present study aimed to characterize the types of mutations present in the 23S rRNA genes of isolates of clarithromycin-resistant H. pylori from the UAE. Methods Clarithromycin susceptibility of H. pylori isolates (n=26) was determined by E tests. Analyses for point mutations in domain V of the 23S rRNA genes in clarithromycin-resistant and-sensitive strains were performed by sequence analysis of amplified PCR products. Results Out of 100 gastric antral biopsy samples, 26 were positive for H. pylori by culture, and 29 were positive by PCR. Of the 26 culture isolates, five (19.2%) were resistant to clarithromycin and 24 were sensitive. The MIC of the resistant strains ranged from 3 to 24 μg/mL (median 24). All of the clarithromycin-resistant isolates had point mutations in the 23S rRNA gene. Two isolates had an A2142G 23S rRNA mutation, and three had A2143G mutations. Conclusion Clarithromycin resistance was common in this small collection of H. pylori isolates from the UAE. The A2142G and A2143G mutations were associated with clarithromycin resistance.
Journal of Digestive Diseases, 2010
OBJECTIVE: To determine the prevalence of primary clarithromycin resistance amongst Helicobacter pylori (H. pylori) strains in Malaysian patients with gastroduodenal diseases, by using restriction fragment length polymorphism (RFLP) in domain V of 23S rRNA.METHODS: Gastric biopsies were obtained from H. pylori positive patients undergoing gastroscopy. DNA extraction was followed by PCR amplification using the primers Hp23-1 and Hp23-2 flanking a region of 425bp within the bacterial 23S rRNA peptidyltranferase (Hp23S fragment). Analysis of the 23S rRNA gene mutations is based on the generation of restriction sites for two restriction enzymes: BbsI and BsaI, which correspond to the base substitutions characteristic of clarithromycin resistance from A to G at positions 2142 and 2143, respectively.RESULTS: Gastric biopsy samples were obtained from 107 patients. A fragment of size 425bp corresponding to that expected from amplification of domain V of 23S rRNA was PCR-amplified from only 105 samples. The amplicon was subsequently subjected to restriction by BbsI and BsaI. Only 1 sample (0.95%) had the BbsI mutation (base substitution at A2142G) and 2 samples (1.90%) the BsaI mutation (base substitution at A2143G). Thus 3 of 105 (2.9%) samples harbored clarithromycin resistant strains.CONCLUSION: In our experience, PCR-RFLP is a rapid and precise method to detect the resistance of H. pylori to clarithromycin. Using this method, a low prevalence of clarithromycin resistance was detected in our local Malaysian strains. This augurs well for the continued use of clarithromycin as a first line drug in the treatment and eradication of H. pylori infection.
Iranian journal of medical sciences, 2011
Clarithromycin resistance in Helicbacter pylori has been found to be associated with point mutations in 23s rRNA gene leads to reduced affinity of the antibiotic to its ribosomal target or changing the site of methylation. The aim of this study was to determine the most important point mutations in 23s rRNA gene in H. pylori that are closely related to clarithromycin resistance among such isolates. Sixty three H. pylori isolates, obtained from gastric biopsy speciemens in Kerman, Iran, were used to evaluate their susceptibility to clarithromycin by disk diffusion test, and to detect the most common point mutations in 23s rRNA gene associated with clarithromycin resistance by Polymerase chain reaction-amplification and restriction fragment length polymorphism (PCR-RFLP) and 3'-mismatch PCR. 31.7% of the H. pylori isolates were resistant to clarithromycin, and each of the resistant isolate had at least one of the most common point mutations in 23s rRNA gene associated with calrith...
Genotypic and Phenotypic Resistance to Clarithromycin in Helicobacter pylori Strains
Journal of Clinical Medicine
Background: The increasing prevalence of antimicrobial resistance, together with the lack of novel treatment options, negatively affects successful eradication of Helicobacter pylori. The aim of this study was to investigate genetic mutations in the 23S rRNA genes, which is associated with clarithromycin resistance, and to determine the clinical impact of genotype on phenotypic antimicrobial resistance. Methods: A total of 46 H. pylori strains were obtained from 13 patients, before and after unsuccessful eradication with clarithromycin-based triple therapy. The phenotypic resistance of each H. pylori strain was determined by minimum inhibitory concentration against clarithromycin using the serial two-fold agar dilution method. The genomic sequences of 23S rRNA genes were identified through next-generation sequencing, and nucleotide variants were determined based on comparison with genome sequences of the reference strain H. pylori 26695. Results: Clarithromycin resistance was found ...
Mutations in the 23S rRNA gene of clarithromycin-resistant Helicobacter pylori from Japan
International Journal of Antimicrobial Agents, 2007
The 23S rRNA gene in clinical isolates of Helicobacter pylori isolated between 1995 and 2004 from Japan was investigated and the relationship between mutations in this gene and clarithromycin susceptibility was studied. Among nine mutations that have previously been reported to confer clarithromycin resistance, an adenine → guanine transition at position 2142 (A2142G) or 2143 (A2143G) was detected in all clarithromycin-resistant strains (n = 67) but not in any clarithromycin-susceptible strains (n = 17). Mutations at positions 2182, 2223, 2244 and 2288 have previously been reported to confer clarithromycin resistance in H. pylori isolates from Bangladesh, China and Brazil. However, these mutations were not associated with clarithromycin resistance in H. pylori isolates from Japan in this study. Other mutations at positions 2115, 2144 and 2711, which have also been reported to confer clarithromycin resistance in H. pylori from Sweden and Italy, were not detected in the strains in this study. Our results suggest that susceptibility to clarithromycin is predicted by detection of mutations at positions 2142 and 2143 of the 23S rRNA gene in H. pylori isolates in Japan.
Comparative Clinical Pathology, 2012
Currently, a seven-day, triple-drug regimen has been recommended as one of the first-line therapies for Helicobacter pylori management in which clarithromycin is a key component. Development of clarithromycin resistance leads to the long term assessment of the efficacy of clarithromycin in the triple-drug regimen. The aim of this study was to rapidly and directly assess clarithromycin resistance point mutations on gastric biopsy specimens by using PCR-RFLP method. Biopsy samples were obtained over a 6-months period of 2009, from 200 dyspeptic patients referred to Shahrekord University of Medical Sciences, Iran. Initially, rapid urease test was performed and then DNA was isolated from each tissue and used for molecular analysis such as PCR (for H. pylori diagnostic) and PCR-RFLP (for Cla resistance determination). RUT and PCR results showed that 164 (82%) of the patients were H. pylori-positive. Resistance was evaluated in 164 samples by using enzymes BsaI and MboII. Thirty nine (39) (23/78%) clarithromycin-resistant strains were detected which were identified as 15 (9.15%) A2143G, 15 (9.15%) A2142G and 9 (5.49%) mix strains. The results showed that PCR-RFLP method had a high accuracy to detect A2142G and A2143G mutations associated with resistance to clarithromycin in the minimum possible time.