scholarly journals High Prevalence of Antibiotic Resistance in Iranian Helicobacter pylori Isolates: Importance of Functional and Mutational Analysis of Resistance Genes and Virulence Genotyping

2019 ◽  
Vol 8 (11) ◽  
pp. 2004 ◽  
Author(s):  
Nastaran Farzi ◽  
Abbas Yadegar ◽  
Amir Sadeghi ◽  
Hamid Asadzadeh Aghdaei ◽  
Sinéad Marian Smith ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibiotic Resistance ◽  
Rrna Genes ◽  
23S Rrna ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Gyra Gene ◽  
Therapeutic Regime ◽  
High Prevalence ◽  
H Pylori

The high prevalence of antibiotic resistance in Helicobacter pylori has become a great challenge in Iran. The genetic mutations that contribute to the resistance have yet to be precisely identified. This study aimed to investigate the prevalence of antibiotic resistance and virulence markers in Iranian H. pylori isolates and to analyze if there is any association between resistance and genotype. Antibiotic susceptibility patterns of 68 H. pylori isolates were investigated against metronidazole, clarithromycin, amoxicillin, rifampicin, ciprofloxacin, levofloxacin, and tetracycline by the agar dilution method. The frxA, rdxA, gyrA, gyrB, and 23S rRNA genes of the isolates were sequenced. The virulence genotypes were also determined using PCR. Metronidazole resistance was present in 82.4% of the isolates, followed by clarithromycin (33.8%), ciprofloxacin (33.8%), rifampicin (32.4%), amoxicillin (30.9%), levofloxacin (27.9%), and tetracycline (4.4%). Overall, 75% of the isolates were resistant to at least two antibiotics tested and considered as a multidrug resistance (MDR) phenotype. Most of the metronidazole-resistant isolates carried frameshift mutations in both frxA and rdxA genes, and premature termination occurred in positions Q5Stop and Q50Stop, respectively. Amino acid substitutions M191I, G208E, and V199A were predominantly found in gyrA gene of fluoroquinolone-resistant isolates. A2143G and C2195T mutations of 23S rRNA were found in four clarithromycin-resistant isolates. Interestingly, significant associations were found between resistance to metronidazole (MNZ) and cagA-, sabA-, and dupA-positive genotypes, with p = 0.0002, p = 0.0001, and p = 0.0001, respectively. Furthermore, a significant association was found between oipA “on” status and resistance to amoxicillin (AMX) (p = 0.02). The prevalence of H. pylori antibiotic resistance is high in our region, particularly that of metronidazole, clarithromycin, ciprofloxacin, and MDR. Simultaneous screening of virulence and resistance genotypes can help clinicians to choose the appropriate therapeutic regime against H. pylori infection.

scholarly journals High prevalence of antibiotic resistance inHelicobacter pyloriisolates from Iran: importance of functional and mutational analysis of resistance genes and virulence genotyping

2019 ◽  
Author(s):  
Nastaran Farzi ◽  
Abbas Yadegar ◽  
Hamid Asadzadeh Aghdaei ◽  
Amir Sadeghi ◽  
Mohammad Reza Zali
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Mutational Analysis ◽  
Rrna Genes ◽  
23S Rrna ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Virulence Markers ◽  
High Prevalence ◽  
H Pylori

AbstractThe high prevalence of antibiotic resistance inHelicobacter pylorihas become a great challenge in Iran. The genetic mutations that contribute to the resistance have yet to be precisely identified. This study aimed to investigate the prevalence of antibiotic resistance and virulence markers in IranianH. pyloriisolates and to analyze if there is any association between resistance and genotype. Antibiotic susceptibility patterns of 33H. pyloriisolates were investigated against metronidazole, clarithromycin, amoxicillin, rifampicin, ciprofloxacin, levofloxacin and tetracycline by the agar dilution method. ThefrxA, rdxA, gyrA, gyrBand 23S rRNA genes of the isolates were sequenced. The virulence genotypes were also determined using PCR. Metronidazole resistance was present in 81.8% of the isolates, followed by clarithromycin (36.4%), ciprofloxacin (36.4%), amoxicillin (30.3%), rifampicin (30.3%), levofloxacin (27.3%) and tetracycline (6.1%). Most of the metronidazole-resistant isolates carried frameshift mutations in bothfrxAandrdxAgenes, and premature termination was occurred in positions Q5Stop and Q50Stop, respectively. Amino acid substitutions M191I, G208E, and V199A were predominantly found ingyrAgene of fluoroquinolone-resistant isolates. A2143G and C2195T mutations of 23S rRNA were found in four isolates. Interestingly, significant associations were demonstrated between intactcagPAI and resistance to rifampicin (P= 0.027), and between susceptibility to amoxicillin andcagPAI intactness (P= 0.016). The prevalence ofH. pyloriantibiotic resistance is high in our region, particularly that of metronidazole, clarithromycin, ciprofloxacin and multidrug resistance. Occurrence of mutations in resistance genes were involved in the development of resistance, especially in less virulent isolates.

scholarly journals Genotypic and Phenotypic Resistance to Clarithromycin in Helicobacter pylori Strains

2020 ◽  
Vol 9 (6) ◽  
pp. 1930
Author(s):  
Eun Jeong Gong ◽  
Ji Yong Ahn ◽  
Jung Mogg Kim ◽  
Sun Mi Lee ◽  
Hee Kyong Na ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antimicrobial Resistance ◽  
Rrna Genes ◽  
23S Rrna ◽  
Dilution Method ◽  
Phenotypic Resistance ◽  
Clarithromycin Resistance ◽  
Resistant Phenotype ◽  
23S Rrna Genes ◽  
H Pylori

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 in 9 of 13 subjects before treatment and all subjects after unsuccessful eradication. Whole-genome sequencing of the 23S rRNA genes detected 42 mutations on 40 nonidentical loci, including 2147A>G (formerly 2143A>G) and 2146A>G (formerly 2142A>G). All strains with clarithromycin-resistant phenotype had either 2147A>G or 2146A>G mutation. When comparing genotype and phenotype for clarithromycin resistance, there was a significant association between 2147A>G mutation and clarithromycin-resistant phenotype. Conclusions: All clarithromycin-resistant strains had either 2146A>G or 2147A>G mutation, suggesting that tests targeting these two mutations may be enough for the prediction of clarithromycin resistance in this population.

scholarly journals Detection of Clarithromycin-Resistant Helicobacter pylori Strains by a Preferential Homoduplex Formation Assay

2000 ◽  
Vol 38 (1) ◽  
pp. 210-214
Author(s):  
Shin Maeda ◽  
Haruhiko Yoshida ◽  
Hironari Matsunaga ◽  
Keiji Ogura ◽  
Osamu Kawamata ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Point Mutations ◽  
23S Rrna ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Wild Type ◽  
Urease Test ◽  
Agar Dilution ◽  
Type Gene ◽  
H Pylori

ABSTRACT It has been shown that resistance to clarithromycin, a major cause of failure in Helicobacter pylori eradication therapy, is associated with point mutations in the 23S rRNA gene. We sought to apply the preferential homoduplex formation assay (PHFA), a novel technique for the efficient detection of point mutations, to detection of the mutations. PHFA was performed on streptavidin-coated microtiter plates with biotin- and dinitrophenyl-labeled amplicons to detect the wild-type gene or each mutant gene. DNA samples were extracted from gastric juice specimens of 412 patients with H. pylori infection and were applied to the assay. The detection threshold of PHFA was as few as 10 gene copies. The sensitivity of PHFA for the detection of H. pylori infection was higher than those of culture and the rapid urease test. A total of 337 (81.8%) samples had the wild-type gene, 38 (9.2%) had the A2144G mutation, and 37 (9.0%) contained both the wild type and a mutation (A2144G in 30 samples, A2143G in 5 samples, and A2143G plus A2144G in 2 samples). About half the strains isolated from patients with mixed infection were susceptible by the agar dilution method (MIC, <0.1 mg/liter). Therefore, PHFA can detect clarithromycin-resistant H. pylori strains, even in patients with mixed infections with the wild type, that are not detectable by the agar dilution method.

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 Potential of FTIR-Spectroscopy for Drugs Screening against Helicobacter pylori

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 897
Author(s):  
Pedro Sousa Sampaio ◽  
Cecília R. C. Calado
Keyword(s):  
Helicobacter Pylori ◽  
Ftir Spectroscopy ◽  
Partial Least Square ◽  
Least Square ◽  
New Drugs ◽  
Partial Least Square Regression ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Molecular Fingerprint ◽  
H Pylori

Helicobacter pylori colonizes the human stomach of half of the world’s population. The infection if not treated, persists through life, leading to chronic gastric inflammation, that may progress to severe diseases as peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. The first line of treatment, based on 7 to 21 days of two antibiotics associated with a proton pump inhibitor, is, however, already failing most due to patient non-compliance that leads to antibiotic resistance. It is, therefore, urgent to screen for new and more efficient antimicrobials against this bacterium. In this work, Fourier Transform Infrared (FTIR) spectroscopy was evaluated to screen new drugs against H. pylori, in rapid (between 1 to 6 h), and high-throughput mode and based on a microliter volume processes in relation to the agar dilution method. The reference H. pylori strains 26,695 and J99, were evaluated against a peptide-based antimicrobial and the clinical antibiotic clarithromycin, respectively. After optimization of the assay conditions, as the composition of the incubation mixture, the time of incubation, and spectral pre-processing, it was possible to reproducibly observe the effect of the drug on the bacterial molecular fingerprint as pointed by the spectra principal component analysis. The spectra, obtained from both reference strains, after its incubation with drugs concentrations lower than the MIC, presented peak ratios statistically different (p < 0.05) in relation to the bacteria incubated with drugs concentrations equal or higher to the MIC. It was possible to develop a partial least square regression model, enabling to predict from spectra of both bacteria strains, the drug concentration on the assay, with a high correlation coefficient between predicted and experimental data (0.91) and root square error of 40% of the minimum inhibitory concentration. All this points to the high potential of the technique for drug screening against this fastidious growth bacterium.

scholarly journals A Next-Generation Sequencing-Based Approach to Identify Genetic Determinants of Antibiotic Resistance in Cambodian Helicobacter pylori Clinical Isolates

2019 ◽  
Vol 8 (6) ◽  
pp. 858 ◽  
Author(s):  
Vo Phuoc Tuan ◽  
Dou Narith ◽  
Evariste Tshibangu-Kabamba ◽  
Ho Dang Quy Dung ◽  
Pham Thanh Viet ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibiotic Resistance ◽  
Genetic Resistance ◽  
23S Rrna ◽  
Next Generation ◽  
Genetic Determinants ◽  
Resistance Determinants ◽  
Genotypic Analysis ◽  
H Pylori ◽  
Resistant Genotypes

We evaluated the primary resistance of Helicobacter pylori (H. pylori) to routinely used antibiotics in Cambodia, an unexplored topic in the country, and assessed next-generation sequencing’s (NGS) potential to discover genetic resistance determinants. Fifty-five H. pylori strains were successfully cultured and screened for antibiotic susceptibility using agar dilution. Genotypic analysis was performed using NGS data with a CLC genomic workbench. PlasmidSeeker was used to detect plasmids. The correlation between resistant genotypes and phenotypes was evaluated statistically. Resistances to metronidazole (MTZ), levofloxacin (LVX), clarithromycin (CLR), and amoxicillin (AMX) were 96.4%, 67.3%, 25.5%, and 9.1%, respectively. No resistance to tetracycline (TET) was observed. Multi-drug resistance affected 76.4% of strains. No plasmids were found, but genetic determinants of resistance to CLR, LVX, and AMX were 23S rRNA (A2146G and A2147G), GyrA (N87K and D91Y/N/G), and pbp1 (P473L), respectively. No determinants were genetically linked to MTZ or TET resistance. There was high concordance between resistant genotypes and phenotypes for AMX, LVX, and CLR. We observed high antibiotic resistance rates of CLR, MTZ, and LVX, emphasizing the need for periodic evaluation and alternative therapies in Cambodia. NGS showed high capability for detecting genetic resistance determinants and potential for implementation in local treatment policies.

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

2002 ◽  
Vol 46 (12) ◽  
pp. 3765-3769 ◽  
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.

scholarly journals Antimicrobial Sensitivity Pattern of Helicobacter pylori Isolates among Subgroup of Bangladeshi Patients

2014 ◽  
Vol 8 (2) ◽  
pp. 49-52
Author(s):  
MMSU Islam ◽  
Shamsun Nahar ◽  
Mst Naznin Sarker ◽  
ASM Salimullah ◽  
Mohammad Asadur Rahman ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Diarrhoeal Disease ◽  
Cross Sectional Study ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Cross Sectional ◽  
Antimicrobial Sensitivity ◽  
Sensitivity Pattern ◽  
H Pylori ◽  
Medical University

This cross sectional study was carried out at Bangabandhu Sheikh Mujib Medical University (BSMMU) and International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) from July 2008 to September 2009. Aim of the study was to find out the antimicrobial susceptibility profile of Helicobacter pylori isolates from dyspeptic patients. Total 224 dyspeptic patients from Out Patient Department (OPD) of BSMMU were initially enrolled after informed written consent. After upper GI endoscopy 157 patients were finally included who had erosions, ulcers or atrophic changes in the stomach or duodenum. Two biopsy samples were taken from each of them. Samples were incubated at 37°C in a double gas incubator with 5%O2, 10%CO2 and 85%N2. Total 82 (52.23%) samples were found positive for H. pylori. Isolated organisms were then tested for sensitivity to Amoxicillin, Clarithromycin, Tetracycline, Levofloxacin and Metronidazole by Agar dilution method. Among 82 patients 51(62.2%) were male and 31(37.8) were female with a male:female ratio 1.6:1. Patients were categorized into two groups one having gastric or duodenal ulcer (30.5%) and other having no ulcer (69.5%). Among these isolates 92.7% were sensitive to Amoxicillin, 89% to Clarithromycin, 81.7% to Tetracycline, 80.5% to Levofloxacin and only 26.8% to Metronidazole. Beside these, 81.7% isolates were sensitive to both Amoxicillin and Clarithromycin, 74.4% to Amoxicillin and Tetracycline, 73.2% to Amoxicillin and Levofloxacin, 72% to Clarithromycin and Tetracycline, 59% to Clarithromycin and Levofloxacin and 51% to Tetracycline and Levofloxacin DOI: http://dx.doi.org/10.3329/fmcj.v8i2.20280 Faridpur Med. Coll. J. 2013;8(2): 49-52

scholarly journals Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Yi Song ◽  
Fengna Dou ◽  
Zhe Zhou ◽  
Ningmin Yang ◽  
Jing Zhong ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibiotic Resistance ◽  
Limit Of Detection ◽  
Eradication Therapy ◽  
Gastric Biopsy ◽  
Individual Therapy ◽  
23S Rrna ◽  
Antibiotic Administration ◽  
Cyp2c19 Polymorphism ◽  
H Pylori

Background. Helicobacter pylori (H. pylori) is one of the most frequent and persistent bacterial infections that affect nearly half of the world’s population. Antibiotic resistance is a constantly evolving process and local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy. The aim of this study was to establish a microarray-based detection to identify H. pylori infection, clarithromycin and levofloxacin susceptibility, and CYP2C19 genetic polymorphism and guide to potential choice of proton pump inhibitor (PPI), antibiotic administration for tailored H. pylori eradication therapy. Methods. By analyzing the sequence of human genomic CYP2C19⁎2 and CYP2C19⁎3 and mutations within the 23S rRNA and gyrA gene regions conferring clarithromycin and levofloxacin resistance, respectively, we developed a microarray for individual therapy detection of H. pylori infection. Plasmids were established as positive or limit of detection (LOD) reference materials. The specificity and sensitivity of the microarray had been performed. And a total of 1083 gastric biopsy samples were tested and the Kappa value had been calculated between the array and Sanger sequencing. We also analyzed the resistance to clarithromycin and levofloxacin in China, as well as the CYP2C19 polymorphisms. Results. The LOD of detecting H. pylori was 103 CFU/mL and human genome DNA was 2 ng/μL. The detection results of 1083 gastric biopsy samples showed that 691 (63.80%) were H. pylori positive, of which 266 (38.49%) were resistant to clarithromycin, 192 (27.79%) were resistant to levofloxacin, and 61 (8.83%) were resistant to both of them. For the type of CYP2C19 polymorphism, 412 (38.04%) were homozygous fast type (HomEM), 574 (53%) were heterozygous EM (HetEM), and 97 (8.96%) were poor metabolizer (PM). Conclusions. The proposed microarray-based detection has high specificity, sensitivity, and reproducibility for detecting the resistance of clarithromycin or levofloxacin as well as CYP2C19 polymorphism, which may help to improve the clinical eradication rate of H. pylori.

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