scholarly journals Insertion of Mini-IS605 and Deletion of Adjacent Sequences in the Nitroreductase (rdxA) Gene Cause Metronidazole Resistance in Helicobacter pyloriNCTC11637

1999 ◽  
Vol 43 (11) ◽  
pp. 2657-2662 ◽  
Author(s):  
Yvette J. Debets-Ossenkopp ◽  
Raymond G. J. Pot ◽  
David J. van Westerloo ◽  
Avery Goodwin ◽  
Christina M. J. E. Vandenbroucke-Grauls ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Restriction Analysis ◽  
Point Mutations ◽  
Full Genome Sequence ◽  
Dna Transformation ◽  
Full Genome ◽  
Ulcer Disease ◽  
Metronidazole Resistance ◽  
H Pylori ◽  
Early Risk Factor

ABSTRACT We found that NCTC11637, the type strain of Helicobacter pylori, the causative agent of peptic ulcer disease and an early risk factor for gastric cancer, is metronidazole resistant. DNA transformation, PCR-based restriction analysis, and DNA sequencing collectively showed that the metronidazole resistance of this strain was due to mutation in rdxA (gene HP0954 in the full genome sequence of H. pylori 26695) and that resistance did not depend on mutation in any of the other genes that had previously been suggested: catalase (katA), ferredoxin (fdx), flavodoxin (fldA), pyruvate:flavodoxin oxidoreductase (porγδαβ), RecA (recA), or superoxide dismutase (sodB). This is in accord with another recent study that attributed metronidazole resistance to point mutations inrdxA. However, the mechanism of rdxAinactivation that we found in NCTC11637 is itself also novel: insertion of mini-IS605, one of the endogenous transposable elements of H. pylori, and deletion of adjacent DNA sequences including 462 bp of the 851-bp-long rdxA gene.

scholarly journals Phenotypic and Genotypic Analysis of Resistant Helicobacter pylori Strains Isolated from Children with Gastrointestinal Diseases

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 759
Author(s):  
Monika Maria Biernat ◽  
Aldona Bińkowska ◽  
Łukasz Łaczmański ◽  
Paweł Biernat ◽  
Paweł Krzyżek ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibiotic Resistance ◽  
Point Mutations ◽  
Gastrointestinal Diseases ◽  
Drug Susceptibility Testing ◽  
Test Method ◽  
Ulcer Disease ◽  
Genotypic Analysis ◽  
Clinical Diagnoses ◽  
H Pylori

Antibiotic resistance of Helicobacter pylori is currently a global issue. The aim of this study was to analyze actual antibiotic resistance rates of H. pylori strains isolated from children with primary infections and to compare the incidence of mutations that determine resistance to clarithromycin (CH) and metronidazole (MET) in children with different clinical diagnoses. A total of 91 H. pylori strains were isolated from 108 children with primary infections. Drug susceptibility testing of the strains was performed using E-test method. Classical sequencing of DNA fragments was used to detect point mutations for CH and MET resistance. Resistance to CH was detected in 31% of isolated strains (28/91), while resistance to MET and CH was detected in 35% (32/91) of strains. A2143G was the most frequently detected mutation and was dominant among strains isolated from children with peptic ulcer disease (80%). Mutations in the rdxA gene were found significantly more frequently among MET-resistant strains than MET-sensitive strains (p = 0.03, Chi2 = 4.3909). In children, a higher frequency of H. pylori multiresistant strains was observed compared with the previous study in the same area. Differences were found in the occurrence of point mutations among H. pylori strains resistant to CH isolated from children with different clinical diagnoses.

The Molecular Genetics of Hemophilia A Stylianos

1987 ◽  
Author(s):  
E Antonarakis
Keyword(s):  
Amino Acids ◽  
Dna Sequences ◽  
Hemophilia A ◽  
Restriction Analysis ◽  
Point Mutations ◽  
Leader Peptide ◽  
Molecular Defect ◽  
Severe Hemophilia ◽  
Cpg Dinucleotides ◽  
Oligonucleotide Hybridization

Hemophilia A is a common X linked hereditary disorder of blood coagulation due to deficiency of factor 8. The gene for factor 8 has been cloned and characterized (Nature 312:326-342, 1984). It is divided into 26 exons and 25 introns and spans 186 kb of DNA. The CGNA is 9 kb and codes for 2351 amino acids. The first 19 amino acids comprise the secretory leader peptide and the mature excreted polypeptide consists of 2332 amino acids. The nucleotide sequence of the exons and the exon-intron junctions is known and the complete amino acid sequence has been deducedSeveral laboratories have used cloned factor 8 DNA sequences as probes to characterized mutations that are responsible for hemophilia A in certain pedigrees. These mutations have been characterized by restriction analysis, oligonucleotide hybridization, cloning and sequencing of DNA from appropriate patientsIn about 500 patients with hemophilia A examined, the molecular defect has been recognized in 39. Both gross alterations (mainly deletions) and point mutations of the factor 8 gene have been found.A total of 19 different deletions have been observed. No two unrelated pedigrees share the same exact deletion.The size of the deleted DNA varies from 1.5 kb to more than 210 kb. All but one of these deletions are associated with severe hemophilia A. A deletion of 6 kb that contains exon 22 only is associated with moderate hemophilia. Some deletions are present in patients with inhibitors to factor 8. No correlation of the size or the position of the deletions can be found with the presence of inhibitors to factor 8.A total of 20 point mutations have been characterized. All are recognized by restriction analysis and involve Taq I sites. All are mutations of CpG dinucleotides and generate nonsense or missence codons. Unrelated pedigrees have the same single nucleotide change because of independent origin of the same mutation. In many instances de novo occurrence of a point mutation has been observed. CpG dinucleotides are hot spots for mutation to TG or CA presumably because of spontaneous deamination of methylcytosine. Some point mutations are present in patients with inhibitors but no correlation of the site of mutation and inhibitor formation has been found. The nonsense mutations are present in patients with severe hemophilia A. A missense mutation (Arg Gin) in exon 26 was found in a patient with mild hemophilia while another Arg Gin mutation in exon 24 has been observed in a patient with severe disease. The creation of a donor splice site in IVS 4 of factor 8 gene has been observed in a patient with mild hemophilia.Few DNA polymorphisms within the factor 8 gene and two other closely linked polymorphisms have been used for carrier detection and prenatal diagnosis of hemophilia A. These DNA markers are useful in more than 90% of families at risk for hemophilia A.The author thanks Drs. Gitschier, Din, Olek, Pirastou, Lawn for communication of their data prior to publication.The hemophilia project at Johns Hopkins was supported by an Institutional grant and NIH grant to S.S.A. and Haig H. Kazazian, Jr.

scholarly journals Helicobacter pyloriResistance to Antibiotics: Prevalence, Mechanism, Detection. What’s New?

2003 ◽  
Vol 17 (suppl b) ◽  
pp. 49B-52B ◽  
Author(s):  
Francis Mégraud
Keyword(s):  
Resistance Mechanism ◽  
Point Mutations ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Current Treatment ◽  
Macrolide Resistance ◽  
Treatment Regimens ◽  
First Line Therapy ◽  
Metronidazole Resistance ◽  
H Pylori

Helicobacter pyloriresistance to antibiotics is increasingly reported and may limit the efficacy of current treatment regimens. Their resistance mechanism has been found to be point mutations for all antibiotics. Macrolide resistance is the most clinically important, but can be detected efficiently by molecular methods. Metronidazole resistance has limited clinical impact but testing methods are not reliable. Seldomly found cases of resistance, such as to amoxicillin and tetracycline, have had their mechanism recently elucidated. The existance of rapid and practical methods for the detection of macrolide resistance (eg, Fluorescence Resonance Energy Transfer assay) should improve management ofH pylori-positive patients in the future, by allowing an adapted first-line therapy.

scholarly journals Random Mutagenesis of Helicobacter pylori vacA To Identify Amino Acids Essential for Vacuolating Cytotoxic Activity

2006 ◽  
Vol 74 (11) ◽  
pp. 6188-6195 ◽  
Author(s):  
Mark S. McClain ◽  
Daniel M. Czajkowsky ◽  
Victor J. Torres ◽  
Gabor Szabo ◽  
Zhifeng Shao ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Cytotoxic Activity ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Single Amino Acid ◽  
Hydrophobic Region ◽  
Ulcer Disease ◽  
Amino Terminal ◽  
Selective Membrane ◽  
H Pylori

ABSTRACT VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. In this study, we analyzed a library of plasmids expressing randomly mutated forms of recombinant VacA and identified 10 mutant VacA proteins that lacked vacuolating cytotoxic activity when added to HeLa cells. The mutations included six single amino acid substitutions within an amino-terminal hydrophobic region and four substitutions outside the amino-terminal hydrophobic region. All 10 mutations mapped within the p33 domain of VacA. By introducing mutations into the H. pylori chromosomal vacA gene, we showed that secreted mutant toxins containing V21L, S25L, G121R, or S246L mutations bound to cells and were internalized but had defects in vacuolating activity. In planar lipid bilayer and membrane depolarization assays, VacA proteins containing V21L and S25L mutations were defective in formation of anion-selective membrane channels, whereas proteins containing G121R or S246L mutations retained channel-forming capacity. These are the first point mutations outside the amino-terminal hydrophobic region that are known to abrogate vacuolating toxin activity. In addition, these are the first examples of mutant VacA proteins that have defects in vacuolating activity despite exhibiting channel activities similar to those of wild-type VacA.

scholarly journals Transformation by iontophoretic microinjection of DNA: multiple integrations without tandem insertions.

1983 ◽  
Vol 3 (10) ◽  
pp. 1803-1814 ◽  
Author(s):  
C W Lo
Keyword(s):  
Dna Sequences ◽  
Restriction Analysis ◽  
Regulation Of Gene Expression ◽  
Mouse Tissue ◽  
Dna Transformation ◽  
Recognition Sequence ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Pressure Injection ◽  
Cell Genome

DNA transformations of mouse tissue culture cells and mouse embryos were carried out by iontophoretic microinjection of DNA. Iontophoresis involves the use of an externally applied electric current to expel DNA molecules from the injection micropipette into the impaled cell (microelectrophoresis). Restriction analysis of transformants obtained by using this procedure demonstrated that this method gave strikingly different results from those previously obtained by others with transformants generated by pressure injection. First, tandem insertions were never observed in any of our transformants. Second, despite the lack of tandem insertions, we have obtained transformants which have integrated many copies of the injected DNA sequences, probably via a comparably large number of independent integration events. And third, in one transformant, an injected BamHI restriction fragment was found to have been integrated in its entirety; this included the preservation of the terminal BamHI recognition sequence. Based on these observations, we discuss the potential usefulness of iontophoretic DNA microinjections in DNA transformation studies that are focused either on the analysis of the regulation of gene expression or on the targeting of DNA sequences into the eucaryotic cell genome.

Transformation by iontophoretic microinjection of DNA: multiple integrations without tandem insertions

1983 ◽  
Vol 3 (10) ◽  
pp. 1803-1814
Author(s):  
C W Lo
Keyword(s):  
Dna Sequences ◽  
Restriction Analysis ◽  
Regulation Of Gene Expression ◽  
Mouse Tissue ◽  
Dna Transformation ◽  
Recognition Sequence ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Pressure Injection ◽  
Cell Genome

DNA transformations of mouse tissue culture cells and mouse embryos were carried out by iontophoretic microinjection of DNA. Iontophoresis involves the use of an externally applied electric current to expel DNA molecules from the injection micropipette into the impaled cell (microelectrophoresis). Restriction analysis of transformants obtained by using this procedure demonstrated that this method gave strikingly different results from those previously obtained by others with transformants generated by pressure injection. First, tandem insertions were never observed in any of our transformants. Second, despite the lack of tandem insertions, we have obtained transformants which have integrated many copies of the injected DNA sequences, probably via a comparably large number of independent integration events. And third, in one transformant, an injected BamHI restriction fragment was found to have been integrated in its entirety; this included the preservation of the terminal BamHI recognition sequence. Based on these observations, we discuss the potential usefulness of iontophoretic DNA microinjections in DNA transformation studies that are focused either on the analysis of the regulation of gene expression or on the targeting of DNA sequences into the eucaryotic cell genome.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Lifestyle and Peptic Ulcer Disease

2018 ◽  
Vol 24 (18) ◽  
pp. 2034-2040 ◽  
Author(s):  
Berrak C. Yegen
Keyword(s):  
Peptic Ulcer ◽  
Peptic Ulcer Disease ◽  
Alcohol Intake ◽  
Night Sleep ◽  
Unhealthy Lifestyle ◽  
Coping With Stress ◽  
Analgesic Drugs ◽  
Ulcer Disease ◽  
Balanced Diet ◽  
H Pylori

The risk of developing Peptic Ulcer Disease (PUD) was shown to be associated with genetic inheritance, lifestyle and social status of the patients. Unhealthy lifestyle habits and failure in coping with stress have been closely associated with the occurrence of PUD. In contrary, limiting the use of analgesic drugs and glucocorticoids, controlling environmental and socioeconomic factors that predispose to H. Pylori infection, having a balanced diet, exercising regularly, coping successfully with stress, avoiding smoking, limiting alcohol intake and getting sufficient night sleep are essential in prevention and healing of PUD.

scholarly journals The number of household members as a risk factor for peptic ulcer disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mi Hong Yim ◽  
Keun Ho Kim ◽  
Bum Ju Lee
Keyword(s):  
Peptic Ulcer ◽  
Peptic Ulcer Disease ◽  
Binary Logistic Regression ◽  
Cross Sectional Study ◽  
Living Alone ◽  
Cross Sectional ◽  
Ulcer Disease ◽  
Logistic Regression Models ◽  
Household Members ◽  
H Pylori

AbstractPeptic ulcer disease (PUD) is caused by many sociodemographic and economic risk factors other than H. pylori infection. However, no studies reported an association between PUD and the number of household members. We showed the number of family members affected by PUD based on sex in a Korean population. This cross-sectional study used 1998–2009 data from the Korea National Health and Nutrition Examination Survey of the Korea Centers for Disease Control and Prevention. Multiple binary logistic regression models adjusted for confounders were constructed to analyze the association of PUD with the number of household members. The number of household members was associated with PUD, age, body mass index (BMI), waist circumference, systolic blood pressure, hemoglobin, glucose, location (urban/rural), income, education level, stress, current drinking, and smoking in both sexes. Men with other household members had a higher PUD risk compared to men or women living alone (reference), and the opposite was observed for women. Men with 4 household members had a higher PUD risk than men living alone in the model adjusted for age, BMI, income, location, education, and stress (OR = 2.04 [95% CI 1.28–3.27], p value = .003). Women with more than 6 household members had a lower PUD risk than women living alone in the adjusted model (OR = 0.50 [0.33–0.75], p value = .001). Women with more household members had a lower PUD risk. However, more men had PUD than women regardless of the number of household members.

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