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

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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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

BioMed Research International ◽

10.1155/2020/2304173 ◽

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.

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

Acta Biochimica Polonica ◽

10.18388/abp.2014_1901 ◽

2014 ◽

Vol 61 (2) ◽

Cited By ~ 22

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.

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Antimicrobial susceptibility and clarithromycin resistance patterns of Helicobacter pylori clinical isolates in Vietnam

F1000Research ◽

10.12688/f1000research.8239.1 ◽

2016 ◽

Vol 5 ◽

pp. 671 ◽

Cited By ~ 16

Author(s):

Camelia Quek ◽

Son T. Pham ◽

Kieu T. Tran ◽

Binh T. Pham ◽

Loc V. Huynh ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antimicrobial Resistance ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

23S Rrna ◽

Rrna Gene ◽

Clarithromycin Resistance ◽

Resistance Patterns ◽

23S Rrna Gene ◽

H Pylori

Helicobacter pylori is a gastric pathogen that causes several gastroduodenal disorders such as peptic ulcer disease and gastric cancer. Eradication efforts of H. pylori are often hampered by antimicrobial resistance in many countries, including Vietnam. Here, the study aimed to investigate the occurrence of antimicrobial resistance among H. pylori clinical isolates across 13 hospitals in Vietnam. The study further evaluated the clarithromycin resistance patterns of H. pylori strains. In order to address the study interests, antimicrobial susceptibility testing, epsilometer test and PCR-based sequencing were performed on a total of 193 strains isolated from patients, including 136 children (3–15 years of age) and 57 adults (19–69 years of age). Antimicrobial susceptibility testing showed that the overall resistance to amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline was 10.4%, 85.5%, 24.4%, 37.8%, and 23.8% respectively. The distribution of minimum inhibitory concentrations (MICs) of clarithromycin-resistant strains was 85.5% with MIC >0.5 μg/mL. The majority of the clarithromycin resistant isolates (135 of 165 subjects) have MICs ranging from 2 μg/mL to 16 μg/mL. Furthermore, sequencing detection of mutations in 23S rRNA gene revealed that strains resistant and susceptible to clarithromycin contained both A2143G and T2182C mutations. Of all isolates, eight clarithromycin-resistant isolates (MIC >0.5 μg/mL) had no mutations in the 23S rRNA gene. Collectively, these results demonstrated that a proportion of clarithromycin-resistant H. pylori strains, which are not related to the 23S rRNA gene mutations, could be potentially related to other mechanisms such as the presence of an efflux pump or polymorphisms in the CYP2C19 gene. Therefore, the present study suggests that providing susceptibility testing prior to treatment or alternative screening strategies for antimicrobial resistance is important for future clinical practice. Further studies on clinical guidelines and treatment efficacy are pivotal for successful eradication of H. pylori infection.

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Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.12.2621 ◽

1997 ◽

Vol 41 (12) ◽

pp. 2621-2628 ◽

Cited By ~ 141

Author(s):

D E Taylor ◽

Z Ge ◽

D Purych ◽

T Lo ◽

K Hiratsuka

Keyword(s):

Helicobacter Pylori ◽

Natural Transformation ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Donor Strain ◽

Clarithromycin Resistance ◽

23S Rrna Gene ◽

23S Rrna Genes ◽

H Pylori

In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer.

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

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2015.6.2 ◽

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.

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Molecular detection and characterization of mutation on 23S rRNA gene associated with clarithromycin resistant in Helicobacter pylori

10.1101/650432 ◽

2019 ◽

Author(s):

Sahar Obi Abd albagi ◽

Hisham Nour aldayem Altayeb ◽

Nadir Musa Khalil Abuzeid

Keyword(s):

Helicobacter Pylori ◽

Gastric Carcinoma ◽

Reference Sequence ◽

23S Rrna ◽

Rrna Gene ◽

Target Sequence ◽

Clarithromycin Resistance ◽

Two Samples ◽

23S Rrna Gene ◽

H Pylori

AbstractBackgroundHelicobacter pylori consider as pathogenic resistant bacterium was colonized mainly in stomach and causing a prolonged gastritis with gastric ulcers were progressing to gastric carcinoma. Also its resistance to antibiotic considered as the main reason for failure to eradicate of this pathogen has been difficult when this resistance occurred as mutant on protein binding site in 23s ribosomal RNA. The highest cure rates have required multidrug antimicrobial therapies including combinations of omeprazole, clarithromycin and metronidazole.ResultBacterial DNA sequence from gastritis patients with confirmed previous positive ICT samples (Stool and Bloo) were used to obtain co-related between phenotypic & genotypic variant outcome have been observed as SNPs carried on nucleotides which could be altered protein prediction as result of that caused chronic gastritis incline to gastric carcinoma due to abnormal consequence on genetic level in H. species (23s rRNA) was referred to clarithromycin resistance, was achieved on this cross-sectional studies by running two different primers were amplify in PCR machine, first one for urease producing gene (Glm as universal primer) and second one for 23s rRNA as specific primer (rp1/fp1). Two samples out of Four samples were amplified as final isolate in the first cycle and have a specific band in 23s rRNA (NO.11, NO.24) as further DNA samples investigation were sent to get our target sequence.ConclusionBioinformatics tools used to confirm a specific types of mutations give specific position responsible for bacteriostatic activity of macrolides such as clarithromycin depends on capacity to inhibit protein synthesis by binding to the 23S ribosomal subunit (23S rRNA) as resistant gene. The detection tools as MSA (multiple sequence alignment) for our nucleotides sequence to (11&24) samples with Genbank accession number 24_MK208582 and 11_MK208583. One type of mutation has been observed in nucleotide sequence (sample-24) in position 2516 (1344 _complementary) sequence result compared with reference sequence standard reference strain (H. pylori U27270) was confirmed which consider it as novel mutation in database for 23S rRNA Gene of H. pylori associated with Clarithromycin Resistance gene in Sudanese patients.

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Helicobacter pylori isolated from patients who later failed H. pylori eradication triple therapy readily develop resistance to clarithromycin

Journal of Medical Microbiology ◽

10.1099/jmm.0.46316-0 ◽

2006 ◽

Vol 55 (6) ◽

pp. 737-740 ◽

Cited By ~ 17

Author(s):

Intetsu Kobayashi ◽

Takeshi Saika ◽

Hiroe Muraoka ◽

Kazunari Murakami ◽

Toshio Fujioka

Keyword(s):

Helicobacter Pylori ◽

Proton Pump Inhibitor ◽

Eradication Therapy ◽

23S Rrna ◽

Rrna Gene ◽

Triple Combination ◽

Clarithromycin Resistance ◽

23S Rrna Gene ◽

H Pylori ◽

Selection Of

In this study, the ease of selection of clarithromycin resistance was investigated in clarithromycin-susceptible Helicobacter pylori strains isolated from patients with H. pylori infection prior to the administration of triple-combination eradication therapy (clarithromycin plus amoxicillin plus a proton pump inhibitor). Clarithromycin-susceptible strains isolated from ten patients in whom the eradication therapy was successful and from six patients in whom the eradication therapy was unsuccessful were exposed serially to subinhibitory concentrations of clarithromycin. The number of transfers required for the MICs of the strains to increase by 8- and 32-fold were 6.6 and 7.2, respectively, in the successful eradication group, and as few as 2.4 and 1.5, respectively, in the unsuccessful eradication group. The number of transfers required for the A2142G or A2143G point mutation of the 23S rRNA gene to be detected in the strains were 5 and 8, respectively, for the strains in the successful eradication group, and 1 and 2, respectively, for the strains in the unsuccessful eradication group. These results suggest that patients in the unsuccessful eradication group were infected with strains of H. pylori that readily became resistant to clarithromycin on exposure to the drug.

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DETERMINATION OF MUTATIONS IN 23S RRNA GENE OF HELICOBACTER PYLORI AND THE ASSOCIATION OF THESE GENE MUTATIONS WITH CLARITHROMYCIN-RESISTANCE IN CHRONIC GASTRITIS

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2015.4_5.5 ◽

2015 ◽

pp. 36-46

Author(s):

Thi Minh Thi Ha ◽

Van Huy Tran ◽

Viet Nhan Nguyen ◽

Phan Tuong Quynh Le ◽

Trung Nam Phan ◽

...

Keyword(s):

Helicobacter Pylori ◽

Chronic Gastritis ◽

Point Mutations ◽

23S Rrna ◽

Rrna Gene ◽

Biopsy Specimens ◽

Clarithromycin Resistance ◽

Resistant Phenotype ◽

23S Rrna Gene ◽

Domain V

Background: Resistance of Helicobacter pylori to clarithromycin is mostly due to the point mutations in the 23S rRNA. The aims of the present study were to determine mutations in domain V of 23S rRNA gene of Helicobacter pylori in chronic gastritis patients by sequencing; and to assess the association of these mutations with clarithromycin-resistant phenotype determined by E-test. Patients and methods: Gastric mucosal biopsy specimens from 170 patients with chronic gastritis were entered in the study. DNA was extracted from biopsy specimens obtained by endoscopy and prepared for sequencing domain V of gene 23S rRNA. 80 biopsy specimens were determined MIC by E-test. Results: Eight point mutations were detected. At 2142 and 2143 sites of gene, A2143G and A2142G mutations were detected in 35.3% and 3.5%, respectively. Six remaining mutations were G2172T (0.6%), T2182C (86.5%), C2195T (5.3%), A2223G (42.9%), T2244C (98.2%) và A2302G (8.8%). A2143G mutation was associated with clarithromycin-resistant phenotype, OR = 368.58 (95%CI: 34.53 – 3934.12). The rate of clarithromycin-resistance in the group with A2143G mutation was 96.7%, while in the group without A2143G mutation was 10%. Predicted probabilities for clarithromycin-resistance were calculated by logistic regression model with the accuracy rate of 96.1%. Conclusions: There was the association between A2143G mutation and clarithromycin-resistant phenotype. Genotypes determined by sequencing domain V of 23S rRNA gene could be used to predict the probabilities of clarithromycin-resistance. Key words: 23S rRNA gene, clarithromycin-resistance, Helicobacter pylori

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Helicobacter pylori specific nested PCR assay for the detection of 23S rRNA mutation associated with clarithromycin resistance

Gut ◽

10.1136/gut.43.3.317 ◽

1998 ◽

Vol 43 (3) ◽

pp. 317-321 ◽

Cited By ~ 36

Author(s):

S Maeda ◽

H Yoshida ◽

K Ogura ◽

F Kanai ◽

Y Shiratori ◽

...

Keyword(s):

Helicobacter Pylori ◽

Gastric Juice ◽

23S Rrna ◽

Rrna Gene ◽

Sequence Specificity ◽

The Novel ◽

Non Invasive ◽

Clarithromycin Resistance ◽

23S Rrna Gene ◽

H Pylori

Background—Clarithromycin is one of the most important antibiotics for Helicobacter pylori eradication. However, 5–10% of strains are reported to be resistant. It has been shown that one point mutation in the 23S rRNA gene is associated with resistance to clarithromycin.Aims—To establish a polymerase chain reaction (PCR) system which amplifies a segment of the 23S rRNA gene containing the mutation points with primers specific forH pylori, so that H pylori infection and the mutation associated with clarithromycin resistance can be examined simultaneously.Methods—To detectH pylori infection and the mutation simultaneously, primers specific for the H pylori 23S rRNA gene were designed based on sequence conservation among H pylori strains and sequence specificity as compared with other bacteria. DNA from 57 cultured strains and from 39 gastric juice samples was amplified in the seminested 23S rRNA PCR. Clinical applicability was evaluated in 85 patients.Results—DNA samples from 57 cultured strains were all amplified. The novel assay and the urease A PCR agreed in 37/39 gastric juice samples with no false positives. The assay did not amplify the DNA of bacteria other thanH pylori. Eight of 85 samples had the mutation before treatment. In clarithromycin based treatment, eradication was achieved in 2/5 (40%) with the mutation and 29/34 (85%) without the mutation.Conclusion—The assay using gastric juice is quick (within 12 hours) and non-invasive (endoscopy not required), enabling rapid initiation of appropriate antibiotic treatment.

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