Mutations of Helicobacter pylori RdxA are mainly related to the phylogenetic origin of the strain and not to metronidazole resistance

Abstract Objectives Drug resistance of Helicobacter pylori is a major clinical problem worldwide. The objective of the present study was to investigate the prevalence of antibiotic-resistant H. pylori in the city of Shenzhen in China, as well as to identify the genetic mutations specifically associated with drug resistance rather than unrelated phylogenetic signals. Methods Antibiotic susceptibility testing was performed on 238 clinical strains successfully isolated from H. pylori-positive dyspeptic patients who underwent gastroscopy at the Department of Gastroenterology in Shenzhen People’s Second Hospital. Following WGS of all strains using Illumina technology, mutation and phylogenetic analyses were performed. Results The resistance rates were 84.9%, 35.3%, 25.2% and 2.1% for metronidazole, clarithromycin, ciprofloxacin and rifampicin, respectively. An A2143G conversion in the 23S rRNA gene was the primary mutation observed in clarithromycin-resistant strains, whilst N87K/I and D91G/N/Y in GyrA were detected in ciprofloxacin-resistant strains. In RdxA, our results demonstrated that only R16H/C and M21A are significant contributors to metronidazole resistance; there were 15 other sites, but these are phylogenetically related and thus unrelated to metronidazole resistance. Conclusions There is a high prevalence of metronidazole, clarithromycin and ciprofloxacin resistance and a low prevalence of rifampicin resistance in H. pylori from Shenzhen, China. Omission of phylogenetically related sites will help to improve identification of sites genuinely related to antibiotic resistance in H. pylori and, we believe, other species.

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Resistotype of Helicobacter pylori isolates: the impact on eradication outcome

Journal of Medical Microbiology ◽

10.1099/jmm.0.069781-0 ◽

2014 ◽

Vol 63 (5) ◽

pp. 703-709 ◽

Cited By ~ 16

Author(s):

Hanafiah Alfizah ◽

Ahmad Norazah ◽

Razlan Hamizah ◽

Mohamed Ramelah

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Eradication Therapy ◽

Fluoroquinolone Resistance ◽

23S Rrna ◽

Nonsense Mutations ◽

Metronidazole Resistance ◽

Resistant Strains ◽

H Pylori ◽

The Impact

Antibiotic resistance is increasing worldwide, and it has been regarded as the main factor reducing the efficacy of Helicobacter pylori therapy. The aim of this study was to determine the phenotype and genotype of antibiotic-resistant strains of H. pylori in the Malaysian population and to evaluate the impact of antibiotic resistance to eradication outcome. One hundred and sixty-one H. pylori isolates were analysed in this study. Metronidazole, clarithromycin, fluoroquinolone, amoxicillin and tetracycline susceptibilities were determined by Etest. PCR followed by DNA sequencing was carried out to determine mutations. The medical records of the patients infected with resistant strains were reviewed to determine the eradication outcome. Metronidazole resistance was encountered in 36.6 % of H. pylori isolates, whereas clarithromycin and fluoroquinolone resistance was observed in 1.2 and 1.9 % of isolates, respectively. All strains tested were susceptible to amoxicillin and tetracycline. Frameshift and nonsense mutations in rdxA and frxA genes resulting in stop codons contributed to metronidazole resistance, which leads to reduced eradication efficacy. A2142G and A2143G mutations of 23S rRNA were identified as causing failure of the eradication therapy. Mutation at either codon 87 or 91 of the gyrA gene was identified in fluoroquinolone-resistant strains. However, the effect of resistance could not be assessed. This study showed that frameshift and nonsense mutations in rdxA or frxA genes and point mutations in the 23S rRNA affected the efficacy of H. pylori eradication therapy.

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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.5.1500-1504.2001 ◽

2001 ◽

Vol 45 (5) ◽

pp. 1500-1504 ◽

Cited By ~ 93

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.

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Distribution of Antibiotic MICs for Helicobacter pylori Strains over a 16-Year Period in Patients from Seoul, South Korea

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4843-4847.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4843-4847 ◽

Cited By ~ 120

Author(s):

Jung Mogg Kim ◽

Joo Sung Kim ◽

Hyun Chae Jung ◽

Nayoung Kim ◽

Young-Jeon Kim ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

South Korea ◽

Clinical Problem ◽

23S Rrna ◽

Rrna Gene ◽

Primary Resistance ◽

The Past ◽

23S Rrna Gene ◽

H Pylori

ABSTRACT Recently, the development of antibiotic resistance emerged as a significant clinical problem in the eradication of Helicobacter pylori. We investigated the MICs of antibiotics for 135 H. pylori isolates from adults in Seoul, South Korea, over the past 16 years. The MICs of amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, and ciprofloxacin increased from 1987 to 2003. Rates of primary resistance to clarithromycin increased from 2.8% in 1994 to 13.8% in 2003. The A2144G mutation was frequently observed in the 23S rRNA gene in clarithromycin-resistant isolates. The increase in resistance to clarithromycin seems to result in a decrease in eradication efficacy for H. pylori. These results suggest that the MICs of several antibiotics for H. pylori have increased over the past 16 years in Seoul.

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Frequency of rdx A Deletion Mutation Conferring Metronidazole Resistance in Helicobacter pylori

10.51429/ejmm29308 ◽

2020 ◽

Vol 29 (3) ◽

pp. 59-64

Author(s):

Hanaa M. El Maghraby ◽

Samar Mohaseb

Keyword(s):

Helicobacter Pylori ◽

Epigastric Pain ◽

Deletion Mutation ◽

Rapid Urease Test ◽

Rrna Gene ◽

University Hospitals ◽

Metronidazole Resistance ◽

Urease Test ◽

Gastric Biopsies ◽

H Pylori

Background: Metronidazole is one of the antimicrobial drugs that can be used in combination with other drugs for eradication of Helicobacter pylori (H. pylori).Unfortunately, metronidazole resistance in H. plori is an increasing health problem which may be attributed to inactivation of many genes as rdx A gene. Objective: To determine the frequency of rdx A deletion mutation in H. pylori detected in infected patients attending at the Gastroenterology Unit, Zagazig University Hospitals. Methodology: Two gastric biopsies were taken from each enrolled patient by endoscopy. H.pylori detection was done by rapid urease test and polymerase chain reaction (PCR) amplification of 16S rRNA gene. Deletion mutation in rdx A gene was detected by conventional PCR. Results: Out of 134 doubled gastric biopsies obtained from 134 patients, 52.2% were positive for H. pylori. Epigastric pain, vomiting and gastritis were significantly associated with detection of H. pylori infection (p˂ 0.05). Deletion mutation of rdx A gene was detected in 28.6% of H. pylori positive specimens obtained from infected patients. Conclusion: Deletion mutation of rdx A gene is a frequent determinant of rdx A inactivation conferring metronidazole resistance among H. pylori.

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Drug Resistance Mechanisms ofMycoplasma pneumoniaeto Macrolide Antibiotics

BioMed Research International ◽

10.1155/2014/320801 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Xijie Liu ◽

Yue Jiang ◽

Xiaogeng Chen ◽

Jing Li ◽

Dawei Shi ◽

...

Keyword(s):

Drug Resistance ◽

Resistance Mechanisms ◽

23S Rrna ◽

Macrolide Antibiotics ◽

Rrna Gene ◽

Site Mutation ◽

23S Rrna Gene ◽

Resistant Strains ◽

Domain V ◽

The 16S Rrna Gene

Throat swabs from children with suspectedMycoplasma pneumoniae(M. pneumoniae) infection were cultured for the presence ofM. pneumoniaeand its species specificity using the 16S rRNA gene. Seventy-sixM. pneumoniaestrains isolated from 580 swabs showed that 70 were erythromycin resistant with minimum inhibitory concentrations (MIC) around 32–512 mg/L. FiftyM. pneumoniaestrains (46 resistant, 4 sensitive) were tested for sensitivity to tetracycline, ciprofloxacin, and gentamicin. Tetracycline and ciprofloxacin had some effect, and gentamicin had an effect on the majority ofM. pneumoniaestrains. Domains II and V of the 23S rRNA gene and the ribosomal protein L4 and L22 genes, both of which are considered to be associated with macrolide resistance, were sequenced and the sequences were compared with the corresponding sequences in M129 registered with NCBI and the FH strain. The 70 resistant strains all showed a 2063 or 2064 site mutation in domain V of the 23S rRNA but no mutations in domain II. Site mutations of L4 or L22 can be observed in either resistant or sensitive strains, although it is not known whether this is associated with drug resistance.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.12.3765-3769.2002 ◽

2002 ◽

Vol 46 (12) ◽

pp. 3765-3769 ◽

Cited By ~ 52

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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Analysis of rdxA and Involvement of Additional Genes Encoding NAD(P)H Flavin Oxidoreductase (FrxA) and Ferredoxin-Like Protein (FdxB) in Metronidazole Resistance of Helicobacter pylori

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.8.2133-2142.2000 ◽

2000 ◽

Vol 44 (8) ◽

pp. 2133-2142 ◽

Cited By ~ 78

Author(s):

Dong-Hyeon Kwon ◽

Fouad A. K. El-Zaatari ◽

Mototsugu Kato ◽

Michael S. Osato ◽

Rita Reddy ◽

...

Keyword(s):

Helicobacter Pylori ◽

Resistant Strain ◽

Resistance Mechanisms ◽

Sensitive Strain ◽

Metronidazole Resistance ◽

Genes Encoding ◽

Resistant Strains ◽

Flavin Oxidoreductase ◽

H Pylori

ABSTRACT Metronidazole (Mtz) is a critical ingredient of modern multidrug therapies for Helicobacter pylori infection. Mtz resistance reduces the effectiveness of these combinations. Although null mutations in a rdxA gene that encodes oxygen-insensitive NAD(P)H nitroreductase was reported in Mtz-resistant H. pylori, an intact rdxA gene has also been reported in Mtz-resistant H. pylori, suggesting that additional Mtz resistance mechanisms exist in H. pylori. We explored the nature of Mtz resistance among 544 clinical H. pyloriisolates to clarify the role of rdxA inactivation in Mtz resistance and to identify another gene(s) responsible for Mtz resistance in H. pylori. Mtz resistance was present in 33% (181 of 544) of the clinical isolates. There was marked heterogeneity of resistance, with Mtz MICs ranging from 8 to ≥256 μg/ml.rdxA inactivation resulted in Mtz MICs of up to 32 μg/ml for 6 Mtz-sensitive H. pylori strains and 128 μg/ml for one Mtz-sensitive strain. Single or dual (with rdxA) inactivation of genes that encode ferredoxin-like protein (designatedfdxB) and NAD(P)H flavin oxidoreductase (frxA) also increased the MICs of Mtz for sensitive and resistant strains with low to moderate levels of Mtz resistance. fdxB inactivation resulted in a lower level of resistance than that from rdxAinactivation, whereas frxA inactivation resulted in MICs similar to those seen with rdxA inactivation. Further evidence for involvement of the frxA gene in Mtz resistance included the finding of a naturally inactivated frxA but an intact rdxA in an Mtz-resistant strain, complementation of Mtz sensitivity from an Mtz-sensitive strain to an Mtz-resistant strain or vice versa by use of naturally inactivated or functionalfrxA genes, respectively, and transformation of an Mtz-resistant Escherichia coli strain to an Mtz sensitive strain by a naturally functional frxA gene but not an inactivated frxA gene. These results are consistent with the hypothesis that null mutations in fdxB,frxA, or rdxA may be involved in Mtz resistance.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1779 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1779-1782 ◽

Cited By ~ 51

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.

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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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Antibiotic Susceptibilities ofHelicobacter pyloriStrains Isolated in the Province of Alberta

Canadian Journal of Gastroenterology ◽

10.1155/1998/672746 ◽

1998 ◽

Vol 12 (4) ◽

pp. 295-298 ◽

Cited By ~ 12

Author(s):

Diane E Taylor ◽

Qin Jiang ◽

Richard N Fedorak

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Gastric Biopsy ◽

Minimal Inhibitory Concentrations ◽

Antibiotic Susceptibilities ◽

Biopsy Specimens ◽

Metronidazole Resistance ◽

Agar Dilution ◽

Resistant Strains ◽

H Pylori

The incidence of antibiotic resistance to amoxicillin, clarithromycin, erythromycin, metronidazole and tetracycline inHelicobacter pyloristrains isolated from gastric biopsy specimens obtained in Alberta was investigated. Results for all antibiotics were obtained using agar dilution, and in addition to metronidazole, the E test was used. Resistance to amoxicillin and tetracycline was not detected. Metronidazole resistance determined using agar dilution was approximately 12% (95% CI 4% to 26%) when minimal inhibitory concentrations (MICs) were at least 8 µg/mL, but fell to 2% (95% CI 0.1% to 13%) when MICs were set at 32 µg/mL or greater. The E test for metronidazole resistance (MIC 8 µg/mL or greater) yielded a slightly higher percentage of resistant strains compared with agar dilution tests (14%, 95% CI 5% to 29%). One of the 31 strains was resistant to clarithromycin (MIC 8 µg/mL) and erythromycin (MIC 16 µg/mL). Thus, the incidence of resistance to clarithromycin, part of the currently used triple therapy for eradication ofH pylori, was 3% (95% CI 0.1% to 17%).

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